LU504853B1 - Sample carrier for use in a rotation-based method for amplification of DNA and/or determination of nucleic acids - Google Patents

Abstract

The invention relates to a sample carrier (1) for use in a rotation-based method for amplifying DNA and/or determining nucleic acids, with a base body (2) and at least one amplification chamber (3) formed in the base body (3). The sample carrier is characterized in that the at least one amplification chamber (3) has a thickness (d) of less than 1 mm, in particular in a range between 10 μm and 0.9 mm, preferably between 10 μm and 0.5 mm.

Description

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Sample carrier for use in a rotation-based process for the replication of

DNA and/or determination of nucleic acids

The invention relates to a sample carrier for use in a rotation-based method for

Replication of DNA and/or determination of nucleic acids. In addition, the

The invention relates to a system with such a sample carrier and a method for operating a

Rotation device. The invention also relates to the use of such a sample carrier for amplifying DNA and/or determining nucleic acids.

Rotation-based methods are used, for example, in the medical field. They are usually used to isolate nucleic acids, such as DNA (deoxyribonucleic acid) or

RNA (ribonucleic acid). In addition, the methods for amplifying

DNA is used.

The polymerase chain reaction (PCR) is usually used to replicate DNA. PCR is the standard method for the specific replication of DNA and is essential for many modern biological and biomedical in-vitro processes. In particular, real-time

PCR, in which the progressive amplification of the target DNA is continuously monitored by fluorescent

Probes are widely used and can be used, for example, for the specific

Identification of infectious pathogens from patient samples. The miniaturization of the PCR method by microfluidic techniques offers several advantages compared to conventional, macroscopic PCR.

Advantages in terms of cost, speed, transportability and possible complete integration and automation of several consecutive processes.

Essential for the PCR method is thermal cycling, in which the

Liquid temperature in the amplification chamber containing the relevant biological components is adjusted to the temperatures required for the biological processes. The speed at which this temperature change can be carried out has a direct influence on the

Overall effectiveness of the PCR and the duration of the overall cycle, with faster PCRs becoming increasingly important, for example for point-of-care diagnostics.

That is why there are a variety of techniques that are specifically designed to

to accelerate temperature change. Examples include shunting microfluidic PCR, in which the liquid is moved back and forth between different heating zones,

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LU504853 the continuous-flow PCR technique, in which the liquid flows through different temperature zones and methods that are not based on contact heating, such as heating by laser or

Microwave radiation. The most common and dominant method for commercial use is still temperature control by contact heating/cooling. The speed of the

Temperature change The decisive factor here is the volume of the liquid, which is directly proportional to the thickness of the chamber in relation to its contact surface with the contact heater.

For thinner chambers, the temperature of the PCR liquid can be adjusted between the relevant temperatures more quickly than for chambers with a greater thickness. This makes it desirable to perform PCR in very thin chambers.

However, the smaller the size scales become, the greater the relative effects of

Surface tension and wetting. As the chamber size decreases, it becomes increasingly difficult to remove air bubbles from a readout area of the amplification chamber. Air bubbles can be created in various ways, for example by repeated, cyclic

Heating to temperatures close to the boiling point of water can lead to degassing of

Gases dissolved in the reaction volume or the dissolution of lyophilisates can generate or promote bubbles. However, bubbles are a known disruptive factor for the optical readout of DNA amplification and/or detection in the microliter range. In addition, flat amplification chambers are difficult to fill without bubbles. This requires special geometries — which complicate the production process of the sample carrier. The formation of bubbles in very thin chambers with a small liquid volume can have a significant influence on the correct signal detection, especially if bubbles are in the direct readout path of the optical readout device.

The object of the invention is therefore to provide a sample carrier by means of which the

time of the PCR process is reduced.

The task is solved by a sample carrier for use in a rotation-based

Method for amplifying DNA and/or determining nucleic acids, with a

Base body and at least one amplification chamber formed in the base body, characterized in that the at least one amplification chamber has a thickness of less than 1 mm (millimeter), in particular in a range between 10um (micrometer) and 0.9 mm, preferably between 10um and 0.5mm.

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Furthermore, it is an object of the invention to provide a method by means of which the duration of the PCR process is reduced.

The object is achieved by a method for operating a rotary device with a

Carrier holder carrying at least one sample carrier according to the invention, wherein the

Rotating device is operated in a reading mode or a venting mode, wherein the speed of the rotating device is lower in the reading mode than in the venting mode.

The sample carrier according to the invention has the advantage that the effectiveness of the overall PCR cycle is increased because the duration of the overall PCR cycle is reduced. This is possible because the selected thickness of the amplification chamber allows the temperature change of the

The liquid in the amplification chamber is rapidly transferred. In other words, the

The liquid in the amplification chamber can be heated or cooled very quickly to the target temperature. This is because thin amplification chambers can operate in small absolute spatial temperature differences and/or small temperature gradients within the

liquid. The resulting more even temperature distribution allows homogeneous

reaction conditions in the amplification chamber, which reduces the time of the PCR

is beneficial to the overall cycle.

The sample carrier according to the invention thus enables the duration of the above-described

PCR process is reduced. At the same time, when the sample carrier is rotated, the

Rotation device allows the amplification chamber to be read well by the optical reading device without gas bubbles, especially air bubbles, affecting the reading process. This is explained in more detail below.

For the purposes of the invention, “thickness” is understood to mean the extent of the amplification chamber in a direction that is parallel to the axis of rotation and/or normal to the base body. The “thickness” can correspond to an “average thickness” of the amplification chamber.

The average thickness is the quotient between the, in particular heated, liquid volume within the amplification chamber divided by the heated base area or the heated

Base areas of the amplification chamber. The at least one heated base area corresponds to the

Base area of the amplification chamber, which is wetted with liquid during PCR operation. The heated base area allows the essential part of the heat to be transferred to the

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The heated base area of the amplification chamber may correspond to at least a part of a base area of the

amplification chamber.

The term “reading operation” is understood to mean an operation of the rotation device in which a reading of the at least one amplification chamber takes place. The term “reading” is understood to mean a process in which it is determined using the optical reading device whether nucleic acids are present in the

amplification chamber is present. A data processing device of the rotation device can determine on the basis of the read data whether nucleic acid is present in the amplification chamber. Alternatively or additionally, the data processing device can determine on the basis of the

The data obtained during the reading process determine the replication of the DNA in the amplification chamber.

The electrical or electronic data processing device can have at least one processor or can be a processor. The data processing device can be part of a

circuit board. In addition, the data processing device can be installed in an interior of the

Rotating device or be arranged outside the interior of the rotating device.

In venting mode, the sample carrier is rotated at at least a predetermined speed.

In venting mode, the amplification chamber is not read by the

Reading device. The speed in venting mode is higher than in reading mode. Alternatively, the speed in venting mode can be the same as the speed in reading mode.

During venting operation, the speed of the carrier holder is so high that gas bubbles are released from the

amplification chamber.

A rotation device can be operated in a rotation mode. In rotation mode, the sample carrier is rotated at at least one predetermined speed.

Rotation mode no reading of the amplification chamber by the reading device. In

In rotation mode, the added sample, especially biological sample, is processed by the reagents in the sample carrier. The speed in rotation mode can be higher than the speed in readout mode. This is necessary because a minimum speed is required to process the sample and the speed of the detector limits the maximum speed during readout. However, it cannot be ruled out that in rotation mode the

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Speed is equal to or less than the speed in readout mode.

Of particular advantage is also a rotation device with at least one sample carrier according to the invention and a rotation device which has a rotatable carrier holder which holds the sample carrier. The rotation device has the advantage that

By rotating the sample carrier, the interfering gas bubbles can be removed from the amplification chamber. This is possible because the forces generated by the rotation on the air bubbles mean that the gas bubbles are no longer located in the optical path of the reading device.

More specifically, the rotational forces enable a constant removal of gas bubbles from the

Readout section of the amplification chamber. In particular, the displacement of the gas bubbles is achieved by a higher speed of the carrier holder in venting mode than in readout mode, through which the liquid in the amplification chamber is pressed radially outwards and thus displaces the lighter gas bubbles radially inwards. With “radial in” or “radial out”, the

Radial direction relative to the axis of rotation of the sample carrier and/or the carrier holder.

The sample carrier according to the invention enables reliable real-time PCR for thin

Amplification chambers, especially for amplification chambers with a thickness in the range of 10pm and 0.5mm. Due to the thin design of the amplification chamber, a rapid adjustment of the liquid in the amplification chamber to a target temperature is possible. This enables rapid thermal cycling and thus fast, real-time PCR.

The determination of nucleic acids can involve detection of nucleic acids and/or

Quantification of nucleic acids. The nucleic acid can be detected in the entire

Amplification chamber, provided that the optical readout device covers the entire

amplification chamber. Alternatively, a readout section of the amplification chamber can be read, whereby the remaining section of the amplification chamber is not read.

In one embodiment, a reading device for reading the amplification chamber can be present. The part of the amplification chamber that is filled with liquid can be read. The reading device can be suitable for determining nucleic acids, in particular DNA and/or RNA. The reading device can be set up to

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Amplification chamber optically. The readout device can have an objective and at least one optical element, such as at least one deflection mirror and/or optical filter. The readout device can be a fluorescence detector. In this

In this case, the readout device is configured to receive signals from the amplification chamber

To capture and/or process fluorescence signals. For this purpose, fluorescent probes located in the amplification chamber can be read using the reading device and a

Conclusions can be drawn about the replication of the DNA in the amplification chamber.

The at least one amplification chamber of the sample carrier can have sections with different

thicknesses. The at least one amplification chamber can have two sections with different thicknesses, with the deeper of the two sections being readable. An amplification chamber designed in this way has the advantage that good readability is ensured.

This means that the reading device cannot read the entire amplification chamber, but only a

Readout section of the amplification chamber. Preferably, in particular only one

The readout section of the amplification chamber is read out, whereby the readout section is deeper than a

remaining section of the amplification chamber. A deeper chamber increases the probability that more target material, such as DNA and/or RNA, is located in the readout section of the amplification chamber. With a higher amount of target material in the readout section, a stronger

Signal can be received by the reading device.

The target material, in particular the nucleic acid, can be determined directly using the reading device. Alternatively, it is possible to determine another material from which the

Target material, especially nucleic acid, can be determined. This way, a by-product produced during a reaction can be determined and the amount of nucleic acid in the

Amplification chamber. In a fluorescent measurement method, the

By-product and can be determined by the optical reading device.

The readout section can be arranged radially between at least two remaining sections of the amplification chamber with respect to the axis of rotation of the sample carrier. This means that there can be a first remaining section which is arranged radially closer to the axis of rotation than the

In addition, a second residual section may exist which is radially from the

Rotation axis is further away than the readout section. The readout in the deeper

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The advantage of reading out the section is that more material can be read out compared to reading out the remaining section. This improves the signal to noise ratio.

The readout section of the at least one amplification chamber can have a thickness of at least 1 mm. In particular, the readout section can have a thickness in a range between 1 mm and 2 mm. The remaining section of the amplification chamber can have a thickness of less than 1 mm. In particular, the amplification chamber can have a thickness in a range between 10 pm and 0.9 mm, preferably between 10 pm and 0.5 mm.

In particular, the sample carrier can have an amplification chamber with a readout section that has an average thickness of less than 1 mm. In such an embodiment, the thickness can vary in the radial and/or azimuthal direction.

A further advantage of an amplification chamber with sections of different depths is that liquid can be pumped out of the amplification chamber through the deep amplification chamber section, i.e. the amplification chamber section with a thickness of at least 1 mm.

The flat amplification chamber section, i.e. the amplification chamber section with a thickness of less than 1 mm, has a high resistance that counteracts rapid and reproducible pumping of liquid from the amplification chamber.

The readout section may comprise at least 10% of the amplification chamber, in particular an area between 10-50% of the amplification chamber. The readout section includes the

Part of the amplification chamber that is filled with liquid. In particular, the

Readout section a bottom surface of the amplification chamber that is wetted with liquid.

In contrast, a section of the amplification chamber that does not contain liquid cannot be read.

A readout section designed in this way enables good readout of the readout section and detection of target material located in the readout section, in particular fluorophores.

At the same time, the readout section is not too large, so that a temperature change of the

The liquid in the amplification chamber can be quickly removed. The rotation of the

Sample carrier results in thermal convection which quickly distributes heat evenly throughout the

amplification chamber. Therefore, the deeper readout section does not have a strong adverse effect

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LU504853 on the uniform and rapid temperature change of the liquid in the amplification chamber.

In a special embodiment, the average thickness, i.e. the quotient of the liquid volume of the liquid in the amplification chamber divided by a base area wetted with the liquid, in particular the bottom area, of the amplification chamber can be less than 1 mm.

The liquid-wetted base area of the amplification chamber can correspond to the entire base area of the amplification chamber, in particular the entire bottom area of the amplification chamber, through which heat is introduced into the amplification chamber or removed from the

amplification chamber. Alternatively, the liquid-wetted base of the

Amplification chamber must be smaller than the total base area of the sample carrier, in particular the total bottom area of the amplification chamber through which heat is introduced into or removed from the amplification chamber.

Designs are possible in which the liquid does not cover the entire bottom surface of the

Amplification chamber wetted. In these versions, the liquid wetted bottom surface of the amplification chamber differs from the support surface of the sample carrier above the

Heat is introduced into or removed from the amplification chamber.

The amplification chamber, especially the main amplification chamber, can be in radial

Direction in a range between 1mm and 10mm, in particular 5mm, and/or can extend in an azimuthal direction in a range between 1mm and 6mm, in particular 4mm. An amplification chamber designed in this way has a sufficiently large volume so that there is a high probability that a large amount of target material is arranged in the amplification chamber. At the same time, the amplification chamber is not too large so that a

Temperature change of the liquid in the amplification chamber can occur quickly.

A pre-amplification chamber can be designed differently from the main amplification chamber. In particular, the pre-amplification chamber can extend 20 mm in the radial direction and/or 20 mm in the azimuthal direction.

The amplification chamber can be a main amplification chamber or a

Pre-amplification chamber. The sample carrier can contain one or more

Main amplification chambers. In addition, the sample carrier can have one or more

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pre-amplification chambers. The sample carrier may also have at least one

Main amplification chamber and at least one pre-amplification chamber. The sample introduced into the sample carrier, in particular a biological sample, can first be

Pre-amplification chamber = processed before processing in the

main amplification chamber.

By providing at least one pre-amplification chamber, the amount of target material, such as nucleic acid, that enters the main amplification chamber can be increased by a factor of 100 or more. This can ensure that enough target material is in the

Main amplification chamber is present so that the target material is detected by the readout device. This avoids the problem of no

target material is present. This problem arises because the main amplification chamber is flat.

In addition, the provision of at least one pre-amplification chamber has the advantage of achieving a high level of robustness against inhibitors. In a PCR pre-amplification with 5-20 cycles, typically not comparable amounts of DNA copies are produced compared to a 1-step PCR with 30-50 cycles. As a result, biomolecules such as primers or polymerase are usually not limited and requirements for their processivity and activity in reaction environments are less critical. Furthermore, typical inhibition mechanisms of a real-time PCR, in which fluorescence generation is disrupted, do not apply to the pre-amplification. The disruptions only play a role from the second amplification in the main amplification chamber, where there is then a dilution factor of typically 5-40.

A sample carrier can be designed in such a way that, in the case of a pre-amplification of the nucleic acid in the

pre-amplification chamber before the nucleic acid is transferred into the

Main amplification chamber. The pre-amplification and main amplification take place after the sample carrier is inserted into the rotation device. In particular, the

Pre-amplification and main amplification due to the rotation of the sample carrier by the

carrier holder.

The main amplification chamber is the chamber in which the DNA is amplified and/or in which the nucleic acid, in particular DNA and/or RNA, is determined. The

The reading device is designed and arranged such that it reads the main amplification chamber.

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In a particular embodiment, the main amplification chamber may have at least one section with a thickness, in particular an average thickness, of less than 1 mm. If the

If the embodiment has a pre-amplification chamber, the pre-amplification chamber can have a thickness, in particular an average thickness, of less than 1 mm or of at least 1 mm. It is advantageous that the pre-amplification chamber has a thickness of less than 1 mm so that the amplification process can be carried out quickly.

In one embodiment, the base body can be disk-shaped and/or circular segment-shaped and/or a microfluidic channel and/or chamber structure can be provided in the

The amplification chamber is part of the microfluidic channel and/or chamber structure. A “microfluidic channel structure” is understood to mean a channel structure whose dimensions in at least one spatial direction, in particular in the depth direction and/or

Tangential direction, in a range between 30 to 700 pm (micrometers).

The rotatable carrier holder of the rotation device can be designed in such a way that it accommodates one or more sample carriers. In the case that the sample carrier is designed in the shape of a circular segment, the rotatable carrier holder can accommodate at least two sample carriers. The

Sample carriers can be rotated simultaneously by the carrier holder.

The sample carrier can be designed and configured such that at least one of the following process steps can be carried out using the sample carrier. The sample carrier can have an access line into which the sample to be processed is introduced. In addition, thermal and/or chemical and/or mechanical lysis can be carried out. In addition, the sample can be mixed with a pre-amplification buffer. The mixture can be mixed with a first lyophilisate which contains enzymes for a

Amplification reaction. A pre-amplification can be carried out, e.g. as PCR in 50-500 pl with 5-20 cycles. A part of the pre-amplifier can be measured and mixed with

Main amplification buffer and lyophilisate, which contains enzymes such as polymerase for

The mixture of main amplification buffer,

Lyophilisate and pre-amplifier can be divided into one or more main amplification chambers.

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The sample carrier can have a foil element which is attached to the base body and/or forms a bottom of the amplification chamber. The amplification chamber can thus be limited by the foil element and base body walls. In particular, the

The foil element can be attached, in particular directly, to a base body side and/or attached to the base body in such a way that the base body is sealed. The foil element can be elastic. In particular, the foil element can have a smaller modulus of elasticity than the base body. The foil can have a thickness in a range between 30 µm and 200 µm, in particular 90 µm. The foil element can rest on the carrier holder, in particular directly. In this case, heat input from the carrier holder can be transferred through the foil element into the

Amplification chamber to heat the liquid in the amplification chamber. An almost exclusively conductive heat input from the carrier holder into the

The foil element can provide convective heat input into the

amplification chamber. Alternatively, convective heat transfer from the

Amplification chamber into the foil element.

In one embodiment, the base body may have an inlet for admitting liquid into the

Amplification chamber. The liquid can contain the nucleic acid. A gas bubble located in the amplification chamber can be discharged through the inlet from the amplification chamber when the rotation device is in rotation mode. This means that the gas bubble can be discharged through the same inlet from the amplification chamber through which the

Liquid was introduced into the amplification chamber. This means that no gas bubble chambers need to be provided in the amplification chamber, which facilitates the formation of the

Amplification chamber simplified.

In one embodiment, the reading device and/or the sample carrier can be designed such that the amplification chamber is read out in a reading operation of the rotation device. The reading device and the rotatable carrier holder can be opposite one another with respect to the sample carrier. In particular, the side of the

sample carrier towards the reading device.

The rotation device may comprise a device for heating or cooling the amplification chamber. The device may be a Peltier element and/or resistance heating wire and/or arranged to heat or cool the carrier holder. The device is used to adjust the temperature of the amplification chamber.

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The sample carrier can be arranged on the carrier holder in such a way that the temperature change of the liquid is carried out, in particular exclusively, by the carrier holder. In particular, the

Sample carriers should be arranged, preferably directly, on the carrier holder so that the

Amplification chamber is heated or cooled by the carrier holder. In other words, the

Heat input into the amplification device occurs only from one side of the sample carrier.

This allows a simple construction of the rotation device because it is not necessary

Devices for heating or cooling can be provided at different points of the rotating device. Alternatively, several devices for heating or cooling the

The liquid present in the amplification chamber must be provided.

The rotation device may comprise a vacuum device for applying a vacuum to the

Sample carrier, in particular the foil element. Negative pressure is understood to be a pressure that is lower than atmospheric pressure. By applying a negative pressure, the

The foil element and the base body are held in place on the carrier holder. This means that when negative pressure is applied, the carrier holder and the sample carrier cannot be moved relative to each other. Another advantage of applying negative pressure is that the

The tightening is necessary because the film element is otherwise wavy due to its elastic design and the volume of the amplification chamber fluctuates and/or is undefined, which makes it difficult to read the amplification chamber.

Need to use a foil element as thin as possible in order to achieve the fastest possible

Temperature change in the amplification chamber. The speed of the

Temperature change depends on the thickness of the film element.

In a special embodiment, the data processing device can have a rotary drive for driving the carrier holder. The data processing device can be connected to the

Rotary drive must be connected to the data technology. The data technology connection is electrical

Connection by means of which data can be exchanged between components.

The data processing device can control the rotary drive in such a way that the

Rotation device is operated, in particular selectively, in a rotation mode or a readout mode. For this purpose, a corresponding control command can be transmitted from the data processing device to the rotary drive.

In venting mode, the speed of the carrier holder is sufficiently high that the forces acting on the gas bubbles and the liquid are sufficiently high that the gas bubbles move radially inwards

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LU504853 are pushed, i.e. move in the direction of the rotation axis. The rotation axis is positioned normally on the base body. The gas bubbles can be released from the amplification chamber via the inlet.

The speed of the rotation device in readout mode can be lower than in

Ventilation operation. The data processing device can cause the

Venting operation is carried out before a readout operation. This offers the advantage of ensuring that most or all of the gas bubbles are removed from the amplification chamber before the readout device optically reads the amplification chamber in the readout operation.

The data processing device can cause the rotation device to

The system is operated in the venting mode at the start of operation and the rotation device is then operated only in the readout mode or rotation mode. This makes use of the fact that most gas bubbles are created when the liquid is filled into the sample carrier and during the

During rotational operation, no or almost no gas bubbles are formed. In particular,

No gas bubbles are generated during reading operation because the temperature of the liquid in the

Amplification chamber in readout mode is lower than in rotation mode.

The data processing device can be configured such that the rotation device is operated selectively in venting mode or in rotation mode or in readout mode.

The speed of the sample carrier in readout mode can be between 1Hz and 20Hz, in particular 2Hz to 5Hz. In contrast, the speed in rotation mode can be between 2Hz and 120Hz.

The reading time can be between 1 second and 6 seconds. The reading time is the

Time in which the amplification chamber is read. In particular, the readout time can be 1 to 2 seconds if the sample carrier is rotated at a speed of 5 Hz.

Alternatively, the readout time can be 4 to 6 seconds if the sample carrier is rotated at a speed of 2 Hz.

In ventilation mode, the speed can be between 16 Hz and 120 Hz. The

The rotating device may be operated in venting mode for a maximum period of 2 minutes, in particular a period of between 1 minute and 2 minutes.

Venting operation must be carried out first after switching on the rotation device.

In particular, the venting operation can be carried out for a predetermined period of time.

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After the venting operation, the rotation device can be operated in the readout operation or in the rotation operation for at least one cycle, in particular for all cycles. The rotational speed of the carrier holder in the readout operation and/or in the rotation operation can be lower than in the venting operation.

In a special design, the amplification chamber can be alternately heated and cooled. In particular, the temperature of the liquid in the amplification chamber can be changed cyclically between at least two temperatures. A cycle includes the

A plurality of heating or cooling processes in which the liquid is brought to different temperatures. Several cycles can be carried out, each of which involves changing the liquid temperature to at least two temperatures. The cycles can last for a predetermined period of time and/or can be repeated at predetermined intervals.

The individual cycles can differ in their duration and/or the

temperatures differ from each other.

The sample carrier can be connected to the rotation device in a rotationally fixed manner.

Heat input into the amplification chamber by means of the device for heating or cooling or a

Heat can be removed from the amplification chamber by means of the device for heating or cooling through the same sample carrier section, in particular the film element section. This enables a simply constructed sample carrier and/or a simply constructed rotation device.

It is particularly advantageous if a sample carrier according to the invention is used for the amplification of DNA and/or determination of nucleic acids.

The subject matter of the invention is shown schematically in the figures, with identical or equivalent elements mostly being provided with the same reference numerals.

Fig. 1 is a representation of a base body with amplification chambers of an inventive

sample carrier.

Fig. 2 is an exploded view of a sample carrier according to the invention.

Fig. 3 is a plan view of a rotatable carrier holder of a rotation device.

Fig. 4 is a side view of the carrier holder.

Fig. 5 is a plan view of the carrier holder on which the base body of the inventive

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sample carrier.

Fig. 6 is a plan view of the carrier holder on which the sample carrier according to the invention is mounted.

Fig. 7 is a side sectional view of a portion of the sample carrier according to a first

Version with one amplification chamber.

Fig. 8 is a side sectional view of a portion of the sample carrier according to a second

Version with one amplification chamber.

Fig.9 is a side sectional view of a portion of the sample carrier according to a third

Version with one amplification chamber.

Fig. 10a a sectional view from above of a base body section to a state in which the carrier holder does not rotate.

Fig. 10b is a top sectional view of a base body portion to a state where the carrier holder rotates.

Fig. 11 is a schematic representation of the rotation device.

Fig. 1 shows a simplified representation of a base body 2 of a

Sample carrier 1, which is used in a rotation-based process for the amplification of DNA and/or

Determination, in particular for detection and/or quantification, of nucleic acids. In the base carrier 2 shown in Figure 1, in comparison to the base carrier shown in Figure 2,

In the case of the base body 2, a plurality of channels and/or chambers formed in the base body 2 are omitted. Thus, in Figure 1, only several amplification chambers 3 are shown as examples, which are formed in the base body 3. In particular, two pre-amplification chambers 3b and five main amplification chambers 3a are shown. However, sample carriers 1 are conceivable which have a different number of pre-amplification chambers 3b and main amplification chambers 3a. The base body 2 is disk-shaped and circular segment-shaped.

The amplification chamber 3, in particular the main amplification chamber 3a, has a thickness d of less than 1mm (millimeter), in particular in a range between 10um (micrometer) and 0.9 mm, preferably between 10um and 0.5mm. The main amplification chambers 3a and the

Pre-amplification chambers 3b are fluidically connected to one another. The amplification chambers 3 are part of a microfluidic channel and chamber structure shown in Figure 2.

Fig. 2 shows an exploded view of a sample carrier 1 according to the invention. The channel and

Chamber structure 8, which is formed in the base body 2, has a plurality of channels and

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chambers that are fluidically connected to each other. In the state shown in Figure 2, a

Side of the channel and chamber structure 8 is open.

The sample carrier 1 has a foil element 6, by means of which the open side of the channel and

The film element 6 acts as a base for the open channels and chambers in the base body 2 when the sample carrier 1 is placed on the

Carrier holder 10 is arranged. The film element 6 is attached directly to one side of the base body. The film element 6 is thus heat-sealed onto the base body 2. As explained in more detail below, heat is introduced into the channel and chamber structure 4 or dissipated from it via the film element 6.

The base body 2 has on its chord side an access section 17 of an access line 32 shown in Figure 5, which protrudes from the base body 2 in the radial direction and which is connected to the

Channel and chamber structure 4 is fluidically connected. A sample, in particular a biological sample, can be introduced into the access line and processed from there in individual chambers of the sample carrier. The access section 17 can be reversibly closed by means of a capsule 31 in order to enable the introduction of the sample and subsequent re-closure. The

Access section 17 and the access line 32 are designed such that a swab (not shown in the figures) on which the sample to be processed is located can be introduced into the access line.

In an initial state, i.e. in a state in which no sample has yet been introduced into the access 10, the sample carrier 1 contains several upstream liquid reagents in various chambers.

In addition, primers are stored in the pre-amplification chambers 3b. Further primers and

Probes in the form of poly- or oligonucleotides are the

The primer pairs in the pre-amplification chambers 3b are identical or different, depending on the specific aim of the analysis method and/or the specific procedure. The primers in the main amplification chambers 3a are identical to the primers in the

Pre-amplification chambers 3b are identical in pairs or, for example, are intended for a "nested PCR" known in the state of the art and are thus designed differently.

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In other chambers, lyophilisates are stored, which contain, for example, enzymes, polymerase, dNTPs {deoxynucleoside triphosphates), salts and/or other upstream reagents {e.g. PCR additives,

Nuclease inhibitors, co-factors of the enzymes involved, etc.). The access line can contain lysis and agents for process control, e.g. spores, fungi, phages or artificially produced

Targets may be included. A lysis chamber connected to the access line contains a

Lysis pellet as well as a magnet and a grinding medium. The latter consists of glass and/or zirconia particles, for example.

The sample body 1 also has a cover body 18, which is attached to the edge of the

The cover body 18 is attached to the side of the base body 1 facing away from the foil element 6. The cover body 18, with the exception of the edge, does not lie directly on the base body 2, but there is a

Air layer between the cover body 18 and the base body 2. This is shown in Figures 7 to 9. The cover body 18 can be fixed to the base body 2 by means of a locking hook in corresponding recesses 19. The cover body 18 has a readout window 20. The readout window 20 is transparent and arranged in such a way that the content of the

Main amplification chambers 3a can be read through the readout window 20. In addition, the cover body 18 has a recess 21 which is used to receive the

access line attached to base body 2.

The base body 2 has several openings 22 which allow for clear alignment and

Positioning of the sample carrier on a carrier holder 10 shown in Figure 3 of the

rotation device 9. Positioning pins 23 of the

carrier holder 10.

Fig. 3 shows a plan view of a rotatable carrier holder 10 and Fig. 4 shows a side view of the

Carrier holder 10. The carrier holder 10 is circular and serves to rotate the

Sample carrier 1 about a rotation axis R. The rotation axis R is perpendicular to the disk-shaped base body 2. The carrier holder 10 is designed such that it can carry two base bodies 2 shown in Figures 1 and 2.

The carrier holder 10 has several heating sections 30. A device 12 for heating or cooling the sample carrier 1 is arranged below the heating section 30 as seen along the axis of rotation R, which is shown in Figures 6 and 7. The devices 12 can each have a

020A0001LU 03.08.2023 18

LU504853

Peltier element or resistance heating plates. The heating sections 30 are arranged offset from one another and can be designed as plates that cover the device 12. In addition, the carrier holder 10 has a carrier base 32. The heating sections 30 and devices 12 are arranged in recesses in the carrier base 32.

In addition, the carrier holder 10 has a seal 24 which seals one or more of the

Heating sections 30 are completely enclosed. The seals 24 each have a plurality of holes (not shown) which are arranged offset from one another along the seal 24.

To fix the sample carrier 1 on the carrier holder 10, a

A vacuum is applied to the holes using the vacuum device 13. In the fixed state, the sample carrier 1, in particular the film element 6, rests against the seals 24.

Fig. 5 shows a top view of the carrier holder 10 on which the base body 2 of the inventive

Sample carrier 1. A swab with the sample to be processed is placed in the

Access line 32 is arranged. The base body 2 rests on the carrier holder 10 and is connected to the carrier holder 10 in a rotationally fixed manner. As can be seen from Figure 5, the base body 2 only covers half of the carrier holder 10. Another base body 2 not shown in the figures can be arranged on the remaining section of the carrier holder 10. In the embodiment shown in Figure 5, the sample carrier 1 is shown without a cover body 18. In contrast, in

Figure 6 shows the sample carrier 1 with cover body 18.

Fig. 7 shows a side sectional view of an amplification chamber 3 according to a first

Execution. In particular, Figure 7 shows a side sectional view of a portion of a

Base body 2, in which the amplification chamber 3 is arranged and a part of the carrier holder 10, in particular the heating section 30 and the device 12. The amplification chamber 3 is the main amplification chamber 3a in the present embodiment.

Main amplification chamber 3a in the example shown is only partially filled with liquid 33. In Figure 7, a boundary surface 35 between an air section and a liquid section is shown in the

Amplification chamber 3 is shown. In an embodiment not shown, the

Amplification chamber 3 may additionally or alternatively be the pre-amplification chamber 3b.

From Figure 7 it can be seen that the film element 6 rests on the carrier holder 10. The carrier holder 10 has the device 12 for heating or cooling the sample carrier 1, which is located below the

heating section 30. The device 12 allows the amplification chamber 3

020A0001LU 03.08.2023 19

LU504853 liquid can be brought to different temperatures.

The amplification chamber 3 has a thickness d. The amplification chamber 3 is formed by the

Foil element 6 and walls of the base body 2. The rotation device 9 also has a reading device 11 which is arranged along the rotation axis R above the

Amplification chamber 3 is arranged. The reading device 11 can optically read the amplification chamber 3 and thus determine nucleic acids, in particular DNA, in the amplification chamber 3. A data-technically connected to the reading device 11

Data processing device 14 can determine, on the basis of the received data, how the DNA has been replicated in the amplification chamber 3.

The carrier holder 10 has a temperature sensor 26. The temperature sensor 26 measures the

Temperature of the heating section 30. Since the foil element 6 is thin, the temperature of the

Liquid inside the amplification chamber 3 has almost the same temperature as the

heating section 30.

In Figure 7, a base area of the amplification chamber 29 is shown, by means of which heat is introduced from the foil element 6 into the amplification chamber 3. The base area of the

Amplification chamber 3 corresponds to a bottom surface of the amplification chamber 3. In Figure 7, the liquid 33 wets only a part of the base surface of the amplification chamber 29, whereby the

The liquid-wetted part of the base area is hereinafter referred to as the heated base area 34.

An average thickness of the amplification chamber 3 corresponds to the quotient of the volume of the

Amplification chamber 3 divided by the heated base area 34, i.e. the part of the base area of the amplification chamber 3 that is wetted with liquid 33. It is advantageous that the average thickness of the amplification chamber 3 is less than 1 mm, in particular between 10 µm and 0.9 mm.

Fig. 8 shows a side sectional view of an amplification chamber 3 according to a second

The second embodiment differs from the embodiment shown in Figure 7 in the design of the amplification chamber 3. The amplification chamber 3 shown in Figure 8 has two sections with different thicknesses. The amplification chamber 3 has a

Reading section 4, which is used by the reading device 11 in the reading mode of the

rotation device 9. In addition, the amplification chamber 3 has a

Remaining section 5, which is not read by the reading device 11. The reading section 4

020A0001LU 03.08.2023 20

LU504853 is thicker than the remaining section 5. The remaining section 5 is arranged closer to a rotation axis (not shown in Figure 8) than the readout section 4, as seen in the radial direction.

The readout section 5 can be thicker than the remaining section 4 of the amplification chamber 3. The

Readout section 5 can have a thickness of at least 1 mm. In order to obtain the advantages of the invention, the average thickness of the readout section should be less than 1 mm, ideally between pm and 0.9 mm. The readout section 5 is shown at the edge of the amplification chamber 4, but in embodiments it can be located at any location within the

amplification chamber 3. 10

Fig. 9 shows a side sectional view of a portion of the sample carrier according to a third

Version with an amplification chamber 3. The third version differs from the one in

Figure 8 shows the second embodiment in the design of the amplification chamber 3. Thus, the

The remaining section 5 is transferred into the reading section 4 by means of a slope 36.

Fig. 10a shows a sectional view from above of a section of the base body 2, in which the

Amplification chamber 3 is formed, to a state in which the carrier holder 10 does not rotate. The carrier holder 10 is not shown in Figure 10a. In the state shown,

Gas bubbles 27 are arranged in the amplification chamber 3. In addition, in the

Amplification chamber 3 liquid is arranged, whereby the amplification chamber 3 is only partially filled with

filled with liquid. In particular, the amplification chamber 3 is 90% filled with liquid.

Fig. 10b shows a sectional view from above of the base body section shown in Fig. 10a in a state in which the carrier holder 10 (not shown) rotates. In particular, the

Rotation device 9 is operated in a venting mode. In the venting mode, the carrier holder 10 rotates about the rotation axis R and the carrier holder 10 is operated at a speed at which the forces acting on the liquid and the gas bubbles cause the

Gas bubbles are displaced radially inwards. The pressure on the liquid and the gas bubbles

The direction of force Z is shown in Figure 10b. The gas bubbles can be discharged through an inlet 28 from the

amplification chamber 3. The inlet 28 also serves to introduce

liquid into the amplification chamber 3.

Fig. 11 shows a schematic representation of the rotation device 9. The rotation device 9 has a rotary drive 16 for driving the carrier holders 10. The rotary drive 16 is

020A0001LU 03.08.2023 21

LU504853 is coupled to the carrier holder 10 and/or has a not shown

Drive motor. The sample carrier 1 is arranged on the carrier holder 10. The

Reading device 11 is arranged above the sample carrier 1, viewed along the rotation axis R.

The rotation device 9 also has the data processing device 14. The

Data processing device 14 is connected to the reading device 11, a

Vacuum device 13 and the rotary drive 16. The data processing device 14 can transmit control commands to the rotary drive 16 so that the rotation device 9 in the

rotation mode or in readout mode.

The vacuum device 13 can be a pump that creates a vacuum in the holes in order to fix the sample carrier 1 to the carrier holder 10. The data connection is shown in dashed lines in Figure 11. The rotation device 9 has a housing 15 that encloses an interior space. The above-mentioned components are arranged inside the housing 15.

020A0001LU 03.08.2023 22

LU504853

List of reference symbols 1 Sample carrier 2 Base body 3 Amplification chamber 3a Main amplification chamber 3b Pre-amplification chamber 4 Readout section 5 Remaining section 6 Foil element 8 Channel and chamber structure 9 Rotation device 10 Carrier holder 11 Readout device 12 Device for heating or cooling 13 Vacuum device 14 Data processing device 15 Housing 16 Rotary drive 17 Access section 18 Cover body 19 Recess 20 Readout window 21 Recess 22 Opening 23 Positioning pin 24 Seal 26 Temperature sensor 27 Gas bubbles 28 Inlet 29 Sample carrier section 30 Heating section 31 Capsule 32 Carrier base 33 Liquid 34 Base of the amplification chamber 35 Interface 36 Slope

R rotation axis

Z force direction

Claims (25)

020A0001LU August 3, 2023 23 LU504853 patent claims 1. Sample carrier (1) for use in a rotation-based method for amplifying DNA and/or determining nucleic acids, with a base body (2) and at least one amplification chamber (3) formed in the base body (3), characterized in that the at least one amplification chamber (3) has a thickness (d) of less than 1 mm, in particular in a range between 10 μm and 0.9 mm, preferably between 10 μm and 0.5 mm. 2. Sample carrier (1) according to claim 1, characterized in that a. the at least one amplification chamber (3) has sections with different thicknesses or that b. the at least one amplification chamber (3) has two sections with different thicknesses, wherein the deeper of the two sections can be read by a reading device (11). 3. Sample carrier (1) according to claim 1 or 2, characterized in that a. a readout section (4) of the at least one amplification chamber (3) has a thickness (d) of at least 1 mm, in particular in a range between 1 mm and 2 mm, and/or a remaining section of the amplification chamber (3) has a thickness of less than 1 mm, in particular in a range between 10 µm and 0.9 mm, preferably between 10 µm and 0.5 mm, and/or that b. the thickness (d) is an average thickness of the amplification chamber (3). 4, Sample carrier (1) according to claim 3, characterized in that the readout section (4) comprises at least 10% of the amplification chamber (3), in particular an area between 10-50% of the amplification chamber (3). 5. Sample carrier according to one of claims 1 to 4, characterized in that the amplification chamber, in particular the main amplification chamber, extends in the radial direction in a range between 1 mm and 10 mm, in particular 5 mm, and/or extends in the azimuthal direction in a range between 1 mm and 6 mm, in particular 4 mm. 020A0001LU 03.08.2023 24 LU504853 6. Sample carrier (1) according to one of claims 1 to 5, characterized in that the amplification chamber (3) is a main amplification chamber (3a) or a pre-amplification chamber (3b). 7. Sample carrier (1) according to one of claims 1 to 6, characterized in that a. the base body (2) is disk-shaped and/or circular segment-shaped and/or that b. a microfluidic channel and/or chamber structure is present in the base body (2). 8. Sample carrier (1) according to one of claims 1 to 7, characterized in that the sample carrier (1) has a film element (6) which is attached to the base body (2) and/or forms a bottom of the amplification chamber (3). 9. Sample carrier according to one of claims 1 to 8, characterized in that the base body (2) has an inlet (28) for admitting liquid into the amplification chamber (3), wherein a gas bubble (27) located in the amplification chamber (3) can be discharged from the amplification chamber (3) through the inlet (28). 10. Rotation device (9) with at least one sample carrier (1) according to one of claims 1 to 9 and a rotatable carrier holder (10) which holds the sample carrier (1). 11. Rotation device (9) according to claim 10, characterized in that the rotation device (9) has a read-out device (11) for reading the amplification chamber (3), in particular the read-out section (4). 12. Rotation device (9) according to claim 11, characterized in that the reading device (11) and/or the sample carrier (1) is designed such that the amplification chamber (3) can be read in a reading operation of the rotation device (9). 13. Rotation device (9) according to one of claims 10 to 12, characterized in that the rotation device (9) has a device (12) for heating or cooling the amplification chamber (3). 020A0001LU 03.08.2023 25 LU504853 14. Rotation device (9) according to one of claims 10 to 13, characterized in that the rotation device (9) has a negative pressure device (13) for applying a negative pressure to the sample carrier (1), in particular the film element (6). 15. Rotation device (9) according to one of claims 10 to 14, characterized in that the rotation device (9) has a data processing device (14) which is connected for data purposes to the reading device (11) and/or a rotary drive (16) for the carrier holder (10). 16. Rotation device (9) according to claim 15, characterized in that the data processing device (14) causes the rotation device (9) to be operated in a read-out mode or a venting mode, wherein the rotational speed of the carrier holder (10) is lower in the read-out mode than in the venting mode. 17. Rotation device (9) according to claim 10 or 16, characterized in that the data processing device (14) causes a venting operation to be carried out before a reading operation. 18. Rotation device (9) according to one of claims 10 to 17, characterized in that a. the rotational speed of the carrier holder (10) in the reading mode is between 1 Hz to 6 Hz, in particular 2 Hz to 5 Hz, and/or that b. the rotational speed of the carrier holder (10) in the venting mode is between 16 Hz to 120 Hz. 19. Rotation device (9) according to one of claims 10 to 18, characterized in that a readout time is between 1 second and 6 seconds. 20. Rotation device (9) according to one of claims 15 to 19, characterized in that the sample carrier (1) is connected to the carrier holder (10) in a rotationally fixed manner, wherein heat is introduced into the amplification chamber (3) by means of the device (12) for heating or cooling or heat is removed from the amplification chamber (3) by means of the device (12) for heating or cooling through the same sample carrier section (29), in particular film element section. 020A0001LU 03.08.2023 26 LU504853 21. Method for operating a rotation device (9), in particular according to one of claims 10 to 20, with a carrier holder (10) which carries at least one sample carrier (1) according to one of claims 1 to 9, wherein the rotation device (9) is operated in a read-out mode or a venting mode, wherein the rotational speed of the rotation device (9) is lower in the read-out mode than in the venting mode. 22. Method according to claim 21, characterized in that the rotation operation is carried out before the reading operation. 23. Method according to claim 21 or 22, characterized in that a. the rotational speed of the sample carrier (1) in the readout mode is between 1 Hz and 6 Hz and/or that b. the rotational speed of the sample carrier (1) in the rotation mode is between 16 Hz and 120 Hz. 24. Method according to one of claims 21 to 23, characterized in that a. the amplification chamber (3) is alternately heated and cooled and/or that b. the temperature of the amplification chamber (3) is cyclically changed between at least two temperatures. 25. Method according to one of claims 21 to 24, characterized in that a pre-amplification of the nucleic acid is carried out in a pre-amplification chamber (3b) before the nucleic acid is amplified in the main amplification chamber (3a). 25. Use of a sample carrier (1) according to one of claims 1 to 9 for the amplification of DNA and/or determination of nucleic acids.