WO2015003722A1 - Single-use device with a reaction chamber and a method for controlling the temperature in the device and uses thereof - Google Patents

Single-use device with a reaction chamber and a method for controlling the temperature in the device and uses thereof Download PDF

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Publication number
WO2015003722A1
WO2015003722A1 PCT/DK2014/050216 DK2014050216W WO2015003722A1 WO 2015003722 A1 WO2015003722 A1 WO 2015003722A1 DK 2014050216 W DK2014050216 W DK 2014050216W WO 2015003722 A1 WO2015003722 A1 WO 2015003722A1
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WIPO (PCT)
Prior art keywords
reaction chamber
layer
temperature
use device
layers
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PCT/DK2014/050216
Other languages
French (fr)
Inventor
Kenneth William Harlow
Ivan R. PERCH-NIELSEN
Morten Bech
Jan Harries Hansen
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Delta Dansk Elektronik, Lys & Akustik
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Publication of WO2015003722A1 publication Critical patent/WO2015003722A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B01L2200/147—Employing temperature sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium

Definitions

  • Focke, et al. discloses a roll-to-roll process in which a flat stamping tool or a roller with projecting mould elements is used to form micro structures in a polymer material. It mentions that laser cutting can be used for cutting slits in an intermediate layer in the laminate. However as mentioned, the laser cutting is not suitable for large batch productions and is only suitable for forming micro structures having a height less than 40 ⁇ .
  • US 6369893 B l discloses a device, termed vessel, comprising a square-shaped reaction chamber formed by a rigid frame closed off by a top and bottom layer wherein the three layers may be made of polypropylene.
  • At least one electrical heating element is integrated in the device and arranged on at least one of the outer surfaces, wherein the heating element is configured to be cou- pled to an external power source for producing and transferring heat to the reaction chamber.
  • the reaction chamber is configured to form a closed-off chamber having a predetermined volume.
  • the inner surfaces of the first and second layers define two heat exchanging surfaces of the device which are related to the volume of the chamber.
  • the reaction chamber may be configured to have a characteristic length, L c , between the volume of the chamber and the surface area of either one of the heat exchanging surfaces of 0.05 to 0.5 mm.
  • the volume of the chamber may advantageously be between 5-300 ⁇ . or ⁇ - ⁇ This configuration allows a very fast heating and/or cooling of the test sample inside the chamber, since the device has an appropriate heat exchange surface in relation to the volume of the test sample inside the chamber.
  • the configuration of the single-use device described above allows it to be manufactured using an industrial standard roll-to-roll manufacturing process, such as those used in the RFID-tag industries, which allows for a cheap and cost-effective mass- production of the single-use device.
  • the roll-to-roll process is defined as a process in which one or more large continuous sheets or rolls of a thin flexible film are fed through a series of units, such as rollers, punch presses, laser cutters and other units, arranged along a production line, wherein each unit is configured to convert the sheet into an intermediate form as it moves through the unit or perform any other known roll-to-roll process step.
  • the third layer is then fed to a second unit, such as a set of rollers, at which the first and second layers are introduced on both sides of the third layer.
  • the layers may then be adhered together using an adhesive layer integrated into one or more of the three layers.
  • an adhesive layer may be introduced between the two layers or may be applied to at least one of the surfaces of these two layers.
  • the adhesive has adhesive capabilities to the thin flexible material of the laminate.
  • the laminate may then be fed to a third unit in which the reagent is applied to the reaction chamber or a storage chamber connected to the reaction chamber.
  • the first and second layers may be introduced in separate units.
  • the reagent may instead be applied to the reaction chamber in the third layer after the manufacturing process.
  • the three layers may be bonded together by applying heat and pressure to the laminate so a heat seal is formed between the layers.
  • the temperature is increased from the first temperature level towards the second temperature level at a rate of at least 10 / s and/or decreased from the second temperature level towards the first or third temperature level at a rate of at least 5 / s .
  • the reaction chamber 2 may have at least one heat exchanging surface formed on the inner surface 10, 12 and defined by the sidewalls 13 of the third layer 6.
  • the laminate 3 may be made of thin flexible layers of a polymer material, such as PET.
  • the reaction chamber 2 may be shaped as a planar chamber having a predetermined length and width or as an oval or ellipse with one or more foci.
  • the height of the reaction chamber 2 may be defined by the thickness of the third layer 6 and optionally the adhesive layers 7, 8.
  • the reaction chamber 2 is configured so that the ratio between the area of the heat exchanging surface and the thermal mass defined by the volume is at least 2: 1.
  • a second protective layer 26 may be applied over the heating element 23 and tempera- ture sensor 24.
  • the protective layer 26 may be configured to protect the electrical components from environmental impact or shocks that might damage the electrical components or cause it to corrode.
  • the protective layer 26 may be made of the same material as the first protective layer 22 and/or have a thickness between 100 ⁇ and 200 ⁇ .
  • a window 25 may be arranged in the protective layer 26 and aligned with the window 25 located in the first layer 4.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The present invention relates to a single-use device comprising a laminate having at least three layers of a thin flexible material. A reaction chamber is formed in a middle layer which is then closed off by the two other layers. An inlet is connected to the reaction chamber for leading a test sample into the reaction chamber. A heating element for heating the test sample is contained on at least one of the outer surfaces of the upper and lower layers of the laminate. A temperature sensor is arranged inside the laminate or on one of the outer surfaces for measuring the temperature inside the reaction chamber. This provides a simple and cheap single-use device with an optimal ratio between the heat exchanging surfaces and the volume of the reaction chamber that allows for an extremely rapid heating and cooling of the test sample inside the reaction chamber. The single-use device is manufactured according to a roll-to-roll process where the structure of the reaction chamber and the assembled device itself are punched, cut or machined out. This allows the single-use device to be mass-produced using a cost-effective manufacturing process.

Description

Single-use device with a reaction chamber and a method for controlling the temperature in the device and uses thereof
Field of the Invention
The present invention relates to a single-use device for diagnostic testing comprising:
- at least one reaction chamber formed by a laminate of at least three layers, wherein the reaction chamber is configured to receive a test sample and a reagent for generating a chemical reaction with a test sample, wherein the reaction chamber is connected to at least one inlet via a fluid channel for receiving the test sample,
- wherein the reaction chamber is defined by a first inner surface of a first layer connected to a second inner surface of a second layer via one or more sidewalls formed in at least a third layer, wherein the first and second layers both comprise an outer surface facing away from the reaction chamber. The present invention also relates to a method of controlling a process in a single-use device, where the method comprises the steps of:
- transferring a test sample into a reaction chamber formed inside the single-use device as described above,
- applying a reagent to the reaction chamber for influencing a chemical process inside the reaction chamber when energy from an energy source is transmitted to the reagent;
- measuring the temperature inside the reaction chamber utilizing a temperature sensor arranged relative to the reaction chamber, and
- transferring the measured temperature to a control unit which in turn controls the transfer of energy to the reaction chamber by means of at least one heating element located in the device.
The present invention also relates to a method of manufacturing a single-use device as described above, the device comprising a laminate of at least a first layer, a second layer and a third layer, where the method comprises the steps of:
- forming at least one reaction chamber connected to at least one inlet via a fluid channel in the third layer of a laminate which has a predetermined length and width, - arranging the first layer of the laminate onto the third layer and coupling the first layer to the third layer, e.g. by means of an adhesive, and
- arranging the second layer of the laminate onto the third layer and coupling the second layer to the third layer, e.g. by means of an adhesive.
The present invention also relates to particular uses of the device as described above. Background of the Invention
Currently traditional thermocyclers and related instruments meant for use in the polymerase chain reaction (PCR) are tabletop instruments of varying size meant for run- ning multiple amplification reactions in small test tubes. Such an arrangement is well suited for laboratory use, however, this requires that the test sample has to be transported to a centralized laboratory for further analyses. This adds to the total analysis time and adds to the logistic complexity for such tests. These tabletop thermocyclers are relatively slow, often with an amplification time of from at least an hour to several hours.
There is presently a need for handheld point-of-care equipment with a fast analysis time that can provide a significant improvement of efficiency for field operations in the event of an epidemic, a catastrophe, or at a point of rescue.
The slowness of current systems is mainly due both to the thermal mass of the combined system, and the unfavourable surface to volume ratios of traditional PCR tubes. This latter fact results in poor heat transfer and ineffective warming and cooling of the PCR sample solutions.
The article "Genotyping of single nucleotide polymorphisms by melting curve analysis using thin film semi-transparentheaters integrated in a lab-on-foil system" by Ohlander, et al., published in Lab on a Chip, 13, pg. 2075-2082 in 2013 discloses a device configured to precisely control temperature within a microfluidic device in order to perform melting curve analysis, the utility of which is dependent upon the temperature accuracy, precision and resolution of the thermal system used to produce the time-temperature gradient. The microfluidic device described comprises a spacer layer made of a pressure sensitive double-sided adhesive tape closed off by top and bottom layers of a PEN foil. A mesh heating element and a PtlOOO temperature sensor are arranged on the inner surface of a bottom layer so that the heating element is in direct contact with the reaction chamber. Furthermore, the heater is chemically coated with a 2 μηι parylene C layer to electrically isolate the heaters from the solution and to provide a substrate for further chemical modification of the heater surface for further attachment of complementary and mismatched capture probes. The article states that a mesh shaped heater provides a better heat distribution over the heat transferring surface than a meander shaped heater, and that the best result is achieved by having a mesh with a line width of 5 μιη and spacing width of 50 μιη. The pressure sensitive double-sided adhesive tape of 50 μιη makes it difficult to obtain a uniform height of the chamber, since the adhesive is squeezed together during lamination. The structure of the reaction chamber and fluid channels are cut using a laser cutting tool. Fabrication of the Ohlander device thus requires a relatively complex manufacturing process including a combination of physical and chemical processing steps not conducive to high volume low cost production techniques.
The article "Lab-on-a-foil: Micro fluidics on thin and flexible films" by Focke, et al., published in Lab on a Chip, 10, pg. 1365-1386 in 2010 is an review article describing different manufacturing processes for forming microfludic features in thin polymer films and their applications. The article, does not mention integration of non-fluidic process control features, such as heaters and sensors directly into or onto the polymer foils. It discloses a device having a laminate consisting of a PP layer, a PC layer and an aluminium (Al) layer wherein a thermocouple is arranged between the PP and PC layers and the PC and Al layers respectively. As mentioned in the article, the alumini- um layer is used as a passive heat conductor for transferring heat from an external heater to the fluid chamber. This configuration further requires external pumps for fluid transport thereby increasing the risk of unwanted dead volume in the device.
Focke, et al. discloses a roll-to-roll process in which a flat stamping tool or a roller with projecting mould elements is used to form micro structures in a polymer material. It mentions that laser cutting can be used for cutting slits in an intermediate layer in the laminate. However as mentioned, the laser cutting is not suitable for large batch productions and is only suitable for forming micro structures having a height less than 40 μηι. US 6369893 B l discloses a device, termed vessel, comprising a square-shaped reaction chamber formed by a rigid frame closed off by a top and bottom layer wherein the three layers may be made of polypropylene. The frame comprises two transparent side walls for optical detection by allowing transmittance of light from a light source in the reader unit through one sidewall and detection of the transmitted or emitted light via a light sensing unit through the other sidewall. The rigid frame is manufactured in an injection moulding process while the other two layers are cast or extruded and then cut into the desired size and shape and laminated to the frame. The device is placed in a reader unit which comprises two heating plates with an integrated temperature sensor for heating the chamber. The reader unit includes a fan which cools the chamber by blowing cold air along the side walls. It is not suggested that the heating element or the temperature sensor can be placed on the device. The device is manufactured in a time-consuming and relatively complex process, requiring initial frame and layer pro- duction followed by a final assembly step where side walls are laminated to the frame.
Object of the Invention
An object of this invention is to provide a single-use device that has a small volume and a relative large heat exchanging surface allowing for a rapid thermal cycling of a sample.
An object of this invention is to provide a simple and cost-effective manufacturing process of a single-use device.
An object of this invention is to provide a method for controlling the temperature in a process in a single-use device.
An object of this invention is to provide a single-use device that allows an optical identification of analyte components in a sample.
Description of the Invention
An object of the invention is achieved by a single-use device characterised in that
- the at least three layers forming the laminate are made of a polymer material, and
- at least one electrical heating element is integrated in the device and arranged on at least one of the outer surfaces, wherein the heating element is configured to be cou- pled to an external power source for producing and transferring heat to the reaction chamber.
This provides a simple and cheap single-use device that is suitable for mass- production using a cost-effective manufacturing process. The reaction chamber is configured to form a closed-off chamber having a predetermined volume. The inner surfaces of the first and second layers define two heat exchanging surfaces of the device which are related to the volume of the chamber. The reaction chamber may be configured to have a characteristic length, Lc, between the volume of the chamber and the surface area of either one of the heat exchanging surfaces of 0.05 to 0.5 mm. The volume of the chamber may advantageously be between 5-300μΙ. or ΙΟ-ΙΟΟμ This configuration allows a very fast heating and/or cooling of the test sample inside the chamber, since the device has an appropriate heat exchange surface in relation to the volume of the test sample inside the chamber. The single-use device is well suited for various applications where a thermal treatment of the test sample located inside the reaction chamber is needed. The thermal treatment is preferably performed using two, three or more temperature levels, which are preferably repeated in a number of cycles. The chamber is thus particularly well suited for performing various tests involving a PCR amplification process, e.g. for detection of one or more specific nucleic acid se- quences in a test sample, for identifying and/or diagnosing certain diseases or disease agents, such as neoplastic diseases or certain infections, such as bacterial, fungal or viral infections, or other diagnostic procedures or tests requiring constant temperatures above ambient or variation of the temperature during the course of the diagnostic test. The test sample is defined as any liquid sample, such as a solution or suspension, which may or may not comprise one or more compounds of interest. The liquid sample may e.g. be a biological sample or a non-biological sample. A biological sample comprises biological material and is selected from dermal swabs, blood samples, tissue samples, urine, faeces, or sputum. In addition, the test sample may relate to liquid suspensions of other test samples such as air, water or earth samples containing one or more particles, or powders.
According to the present invention, the biological material present in the test sample is made available for further analysis in the liquid sample by releasing biological materi- al of one or more biological cells present in the test sample, e.g. by opening and/or rupturing the cell wall or cell barrier of the biological cell sufficiently to allow the biological material to escape into the surrounding liquid, in the following also called extraction. This may be performed prior to transferring the test sample to the reaction chamber or in the reaction chamber.
In the present context the term "biological cell" is related to a particle comprising e.g., a microorganism, a virus, a fungus, a eukaryote cell or a fragment thereof. The eukar- yote cell may e.g. be a plant cell, a plant spore, an animal cell such as a mammal cell. The mammal cell may e.g. be a human or animal cell, such as a white blood cell or a nucleated red blood cell of a human or animal. The microorganism may e.g. be selected from the group consisting of an archeal microorganism, a eubacterial microorganism or a eukaryotic microorganism. The microorganism may be selected from the group consisting of a bacterium, a bacterial spore, a virus, a fungus, and a fungal spore.
It is preferred that the biological cell may be a vegetative bacterium, or a spore, preferably selected from Staphylococcus or Pneumococcus strains, such as Staphylococcus aureus and in particular methicillin resistant S. aureus (MRS A) and/or pneumo- coccus genus. The biological material extracted from the biological cell typically comprises a component selected from the group consisting of a cell organelle, a genetic material, and a protein. The genetic material may e.g. comprise chromosomal DNA and/or plasmid DNA and/or any type of RNA. The protein may be selected from the group consisting of enzymes, structural proteins, transport proteins, ion channels, tox- ins, hormones, and receptors.
The reaction chamber is preferably used for testing for presence of any of the above mentioned biological cells based on existing test methods therefore using assays which preferably involves PCR amplification or adaption of such test methods in order to be able to run in the reaction chamber according to the present invention. Additionally the liquid sample may comprise one or more reagents or additives required to perform a nucleic acid amplification or PCR amplification. Such reagents or additives are well known to the person skilled in the art. The reagents may be added prior to transferring the test sample to the reaction chamber, or they may be added to the reac- tion chamber separately, either prior to, during or after transferring the test sample to the reaction chamber.
Preferably, the biological material comprises DNA and/or RNA. The term "nucleic acid", "nucleic acid sequence" or "nucleic acid molecule" should be interpreted broadly and may for example be an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes molecules composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as molecules having non-naturally occurring nucleobases, sug- ars and covalent internucleoside (backbone) linkages which function similarly or combinations thereof. Such modified or substituted nucleic acids may be preferred over native forms because of desirable properties such as, for example, enhanced affinity for nucleic acid target molecule and increased stability in the presence of nucleases and other enzymes, and are in the present context described by the terms "nucleic acid analogues" or "nucleic acid mimics". Preferred examples of nucleic acid mimetics are peptide nucleic acid (PNA-), Locked Nucleic Acid (LNA-), xylo-LNA-, phos- phorothioate-, 2'-methoxy-, 2'-methoxyethoxy-, morpholino- and phosphoramidate- comprising molecules or functionally similar nucleic acid derivatives. The term "primer" relates to a nucleic acid molecule, which typically comprises in the range 5-100 nucleotides, such as 5-20, 20-50 and 50-100 nucleotides. In a preferred embodiment a primer comprises in the range 5-40 nucleotides, such as 5-10, 10-20, 20-30 and 30-40 nucleotides. A primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. The primer is preferably single- stranded for maximum efficiency in amplification, but the primer can be double-stranded.
The term "nucleic acid polymerase" relates to a DNA- or RNA-dependent DNA polymerase enzyme that preferably is heat stable, i.e. the enzyme catalyses the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double- stranded template nucleic acids. Generally, the synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from thermophilic or caldoactive strains such as Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Thermococcus litoralis, Pyrococcus furiosus, Bacillus stearother- mophilus and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable can also be employed in nucleic acid amplification provided the enzyme is replenished.
The reaction chamber is formed by a laminate made of thin flexible layers of a polymer material, such as homo- or copolymers of polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polyimide (PI) and/or combination hereof. The laminate is defined as two or more pre-formed layers (manufactured in a separate process) of either an unmodified or modified sheet or roll material. A "modified material" is a material where its initial form is chemically or physically modified/altered while an unmodified material is a material maintaining its initial form. In addition, one or more of the layers may itself be a laminate comprising two or more layers of at least one of the above mentioned polymers. In addition, one or more of the layers in the laminate may made of another suitable thermoplastic or thermoset material, preferably a material suitable for medical or diagnostic applications. The flexible material is defined as a material with a tensile modulus of between 0.2 to 20 GPa, preferably 2 to 12 GPa. According to one embodiment, at least one of the first and second layers has a thickness between 10-100 μιη, or that the third layer has a thickness of 100-500 μιη
The reaction chamber may be a planar shaped chamber having a circular, elliptic or rectangular heat exchanging surface or a combination hereof. In one embodiment, the chamber has a rectangular shape having a width or diameter of 1-5 mm or 2-3 mm and a length of 10-20 mm or 14-16 mm. The reaction chamber may have a height of 100- 500 μιη, 150-350 μιη, 225-275 μιη or 250 μιη measured between the two heat exchanging surfaces. The height also defines the thickness of the third layer. The layers may be coupled together via an adhesive layer with adhesive capabilities to the thin flexible material or bonded together via a heat seal. The layers closing off the reaction chamber, i.e. the first and second layers, and/or the adhesive layers may have a thickness between 10-100 μιη, 20-80 μιη or 30-60 μιη. One or more structures, i.e. microstructures, like chambers and/or interconnecting channels may be formed in one or more of the three layers, e.g. the third layer, if further processing is needed. The structures are configured to guide a fluid between the inlet and the outlet or between various chambers. The additional chambers may be configured for different purposes or applications. The reaction chamber may alternatively extend into the first and/or second layer so that the inner surface of the chamber is retracted relative to the remaining inner surface of the first or second layer.
The heating element may be applied, i.e. deposited, onto the outer surface of the first and/or second layer and distributed over the heat exchanging/transferring surface. Heat is then transferred in and/or out of the chamber through the first and/or second layer. A protective layer may advantagely be coupled to at least part of the outer surface for protecting the heating element from external impacts from other objects and environmental impacts. This allows a more even distribution of heat over the entire heat exchanging surface in this invention. In one embodiment, the heating element may be arranged on the inner surface of the first and/or second layer, these being the outer layers of the device in this embodiment. In this embodiment, the protective layer may advantagely be arranged between the heating element and the chamber as an intermediate layer for protecting the heating element from reacting with the reagent or test sample, e.g. by corrosion, oxidation or leaching. The intermediate layer may be another thin layer of the same material as the first and/or second layer or of different suitable material as mentioned above. The intermediate and protective layer(s) may have thickness of 10-100 μιη, 20-80 μιη or 30-60 μιη or even less than 10 μιη. This layer is preferably a prefabricated layer suitable for application in a low volume low cost roll-to-roll step unlike the layer of Ohlander et al. which is applied directly onto the heater in a complex chemical vapour deposition process. In Ohlander et al., the temperature in the chamber is used for analytical purposes in a melting curve analysis which not only requires a very precise determination of the temperature in the chamber but also requires the heat to be distributed in the chamber in a very uniform man- ner in order to be able to use the device for analytical purposes. This results in a rather expensive device which is less preferred because of cost considerations in single use point-of-care applications. The heating element may be configured as a meander shaped wire having a line width of 100-300 μι¾ 150-250 μιη or 200 μιη. The spacing width between the lines may correspond to the line width, i.e. 100-300 μιη, 150-250 μιη or 200 μιη. The height or thickness of the meander structure may be 5-20 μιη or 9-18 μιη. In one embodiment, the heating element may be configured as one or more plate members, a mesh or as one or more coils arranged relative to the heat exchanging surface. The heating element is preferably made of metal, such as copper, metal oxides, or another suitable material. For some applications, the heating element may be made of different alloys, such as a nickel alloy. The line spacing between two or more lines may be greater than the line width to allow optical detection between these lines.
The heating element may have a size that is equal to or advantageously greater than the size of the reaction chamber. The heating element may extend further outwards from the entire periphery of the chamber or only a part thereof. The heating element may extend further outwards in a distance of 1 to 10 mm, e.g. 5 mm. This configuration allows the chamber to be heated evenly over the entire volume.
The heating element may be coupled to the power source through at least two electrical terminals for transmitting a current through the heating element. Alternatively, the heating element may instead be heated inductively via an electromagnetic field generated in the power source which then generates a current in the heating element which in turn heats up the reaction chamber. The heating element may be made of a transparent conductive material in the form of conductive oxides, like indium tin oxide (ITO) or aluminum/gallium/indium doped zinc oxide (AZO, GZO or IZO), or conduc- tive polymers, like polyaniline (PANI), poly(3,4-ethylenedioxythiophene) (PEDOT), poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) or another suitable material for allowing a visual or optical detection of the reaction, e.g. the temperature inside the chamber. According to one embodiment, the device comprises one or more sensor units, such as at least one temperature sensor configured to sense the temperature inside the reaction chamber, arranged relative to the reaction chamber. The temperature may be measured electronically using a temperature sensor arranged on or inside the laminate which may be coupled to the control unit, e.g. a microprocessor unit, configured to regulate the energy supplied by the external energy source. In one embodiment, the temperature is sensed by one or more temperature sensors in the form of a bolometer, thermopile or a low cost thermal infrared sensor which may be arranged relative to the reaction chamber.
Other sensor units may be arranged in the device, such as chemical sensor electrodes for sensing an electrochemical activity inside the reaction chamber. These sensor elec- trades may be arranged on the inner surface of the first and/or second layers. In one embodiment, the sensor unit may be applied, e.g. deposited, on an intermediate layer located between the third layer and the first and/or second layer. The sensor units, as well as the temperature sensor, may be connected to two or more electrical terminals for coupling to the reader unit or coupled directly to the microprocessor unit arranged in the device.
According to a special embodiment, the sensor unit is arranged on at least one of the outer surfaces of the first and second layers or arranged inside the laminate between the first and second layers.
The temperature sensor may be configured as a resistance temperature detector (thermistor) or a thermocouple. The temperature sensor may be integrated into the heating element on the same layer. This allows the temperature to be sensed at the heat source for a better control of the applied heat. In one embodiment, the temperature may be a separate temperature sensor which may be applied, e.g. deposited, on the inner surface of the first and/or second layer or at the other outer surface relative to the heating element. In one embodiment, the temperature sensor may be arranged on an intermediate layer located between the third layer and the first and/or second layer or be placed directly inside the chamber. This allows for a better control of the actual temperature inside the chamber. The temperature sensor may be made of any suitable materials or compounds, such as copper, gold, platinum, nickel, metal oxide, or polymer.
In one embodiment, one or more thermal sensors in the form of a bolometer, thermopile or a low cost thermal infrared sensor may be used to sense the temperature. The thermal sensor may be arranged in a short distance from the reaction chamber, e.g. on one of the layers forming the laminate, such as on the first and/or second layer.
According to one embodiment, at least one window, such as a transparent window, is arranged in at least one of the outer surfaces.
The window may be configured as one or more transparent windows arranged in the first and/or second layer over at least a part of the heat exchanging surface. In one embodiment, the window more or less corresponds to the entire heat exchanging sur- face of the chamber.
In one embodiment the temperature inside the chamber may be measured using a sensor unit located in the device and/or a receiver module located in the reader unit which is configured to sense the temperature inside the chamber, e.g. through one of the windows. In one embodiment, a fluorescent temperature indicator can be used to measure the temperature assisted by a sensor unit located in the reader unit.
In another embodiment, important control and status parameters, such as the progress of a PCR amplification process or DNA identification, can be monitored, e.g. using fluorescent indicators inside the chamber, and monitored through the window by a receiver module located in the reader unit. The window may be used to trigger and control photochemical reactions in chamber. Photons can be used to activate, maintain and inhibit chemical reactions, including enzymatic reactions, in the chamber. In one embodiment, the test sample may be optically heated, when the device is placed in or coupled to the reader unit, using an energy source in the reader unit. The energy source may be a light source configured to transmit a light beam (e.g. infrared light) or a laser beam into the sample through at least one of the windows, wherein the reagent and/or test sample is configured to absorb at least a portion of the energy in the beam. In one embodiment, the test sample may be inductively heated by transmitting microwaves or radio waves (RF signals) into the reaction chamber, e.g. through one of the windows, from an electromagnetic energy source, wherein the reagent and/or test sample is configured to absorb at least a portion of the energy in the field or RF signal. The heating and temperature sensing may be done through the same window or through different windows.
According to one embodiment, the reaction chamber is an amplification chamber, wherein the reagent is configured to copy a genetic sequence in the test sample into a plurality of copies and for detecting one or more specific gene sequences in the copied genetic sequences.
The reaction chamber is configured as a PCR amplification chamber for amplifying DNA or RNA sequences. In one embodiment, the reaction chamber may be configured for detecting infectious deceases or disease agents, such as bacteria, fungus, virus, parasites, and spores of bacteria or fungus, or cancer cells, or other relevant diseases. The single-use device may in one particularly preferred embodiment be configured for detecting methicillin resistant S. aureus (MRS A) and/or pneumococcus spe- cies and/or presence of indicators in a test sample indicating infection of humans or animals caused by these species as defined above for diagnostic purposes.
According to one embodiment, a second heating element or cooling means for cooling the reaction chamber are arranged on the other outer surface and relative to the reac- tion chamber.
The reaction chamber may be cooled by using natural convection from the heat exchanging surfaces. The cooling may be improved by using forced convection where a coolant, such as air or water, from a fan or pump in the reader unit is lead over at least one of the heat exchanging surfaces. In one embodiment, one or more cooling fins or channels may be arranged on or in the outer surface of the first and/or second layer which may be coupled to the fan or pump via at least one fluid coupling. In one embodiment, a Peltier element or another thermoelectric element may be coupled to the device when it is placed/coupled to the reader unit. The thermoelectric element may be configured to actively draw heat away from the reaction chamber which allows for faster cooling than by natural convection. The thermoelectric element may also be used as a heating element for heating the test sample in the reaction chamber. In one embodiment, the thermoelectric element is not directly coupled with the device, but used to further cool the coolant from the fan or pump before it passes over the heat exchanging surface. This allows the cooling to be further improved.
In one embodiment, a heating element may be arranged relative to both heat exchang- ing surfaces. The two heating elements may be arranged on the outer or inner surfaces of the first and second layers. In one embodiment, one heating element may be arranged on the inner surface and the other heating element may be arranged on the outer surface of first and/or second layers. This allows the heating elements to be oriented in the same direction which allows for easier coupling to the reader unit and reduced the risk of short-circuiting. This allows the test sample to be heated from two sides and allows the characteristic length, Lc, of the chamber to be reduced by half, since the surface area is doubled. The heat applied from the two heating elements may be controlled individually or synchronised according to a common set-point temperature depending on the tolerances of the heating elements. In addition, the presence of two heating elements provides a more homogeneous heating, makes the system less sensitive to outside temperature, reduces thermal gradients and allows temperatures to equilibrate to the set-point temperature more rapidly.
According to one embodiment, the device comprises at least one electronic circuit, e.g. a microprocessor unit, configured to control or monitor at least one operation of the device, e.g. the temperature control, wherein the electronic circuit is arranged on at least one of the outer surfaces of the first and second layers or on at least a fourth layer of the laminate. The use of a standard roll-to-roll manufacturing process allows the amount of electrical components needed for a desired application to be applied to the device in a cost- effective manner. In one embodiment, the electronic circuits may be arranged on the same surface, e.g. at least one of the outer surfaces, as one of the heating elements or the sensor unit. The electronic circuits may be offset relative to the reaction chamber so that they are not located above the heat exchanging surfaces. In one embodiment, the electronic circuits may be arranged on a fourth and/or fifth layer of the laminate which are located on the outer surface of the first three layers. One or more electrical through-holes deposited in one or more of the first three layers may be used to interconnect the electrical components in the laminate. In one embodiment, the electronic circuit comprises a data processing unit, such as an ASIC (Application Specific Integrated Circuit) chip. The ASIC chip can be configured to form a calibrated temperature sensor. In another embodiment of the ASIC, it can be configured to hold calibration and identification information to support the functionality and versatility of the system. In general terms, an ASIC can be a combination of several electronic circuits with a very broad range of tasks on the same chip. The combination of sensing electronics, data processing and communication units are common applications. Most ASIC functionality can be combined by using "off-the- shelf electronics. The ASIC solution may be adapted to a desired high-volume and low cost application which is advantageous both in price, power consumption, and complexity during manufacturing. By integrating electronics, the versatility of the system is enhanced and a number of useful advantages can be applied. In one embodiment, the electronic circuit comprises a communication module configured to com- municate with the reader unit via a wireless communication. This allows the number of electrical terminals in the device to be reduced, and thus reducing the production cost. Further functionality like preamplifiers and data processing for other sensing elements can further enhance the functionality and reduce the total cost. The data processing unit may further be configured to control one or more operations of the single-use device, such as the operation of the heating elements and/or cooling means. The operation may be controlled according to at least one set of parameters which may define a temperature cycle. In one embodiment, the electronic circuit may be coupled to one or more electrically activated fluid valves or other flow regulating means located in the fluid channels in the device. This allows the fluid flow between the different chambers to be controlled, if needed. In one embodiment, the electronic circuit may be coupled to means for facilitating the lysis of cells in the test sample. The lysis of cells is defined as any chem- ical, mechanical, electrical or temperature means causing rupture of the cells, phages or spores of the test sample. Any suitable lytic enzyme or mixture of enzymes, such as lysozyme or a detergent, may be used to at least partially break down the cells, spores or phages and release the DNA or RNA present in the cells, spores, or phages. According to one embodiment, the reaction chamber and the reagents are configured to detect nucleic acids or pathogens, such as microorganisms, bacteria, vira, fungus, parasites, spores of bacteria or fungus, or malign tumour cells and/or neoplastic cells. The single-use device is particularly suitable for testing biologic samples in point-of- care applications where lateral flow assays are often used today. In one embodiment, the reaction chamber is configured for nucleic acid targeting where the reagent is selected to react with one or more specified nucleic acid sequence(s). In another embodiment, the reaction chamber is configured for pathogen detection where the reagent is selected to react with the desired pathogen such as bacteria, vira, parasites, fungus, spores of bacteria or fungus, or malign tumour cells and/or neoplastic cells. The reagent may be any primer, nucleic acids, nucleic acid polymerases, nucleotide triphosphates or another suitable reagent as defined above. An object of the invention is achieved by a manufacturing method characterised in that
- the material of the third layer defining the structures of the reaction chamber, the inlet and the fluid channels are removed in a first unit, and then fed to at least a second unit wherein at least one of the first and second layer is laminated to the third layer.
The configuration of the single-use device described above allows it to be manufactured using an industrial standard roll-to-roll manufacturing process, such as those used in the RFID-tag industries, which allows for a cheap and cost-effective mass- production of the single-use device. The roll-to-roll process is defined as a process in which one or more large continuous sheets or rolls of a thin flexible film are fed through a series of units, such as rollers, punch presses, laser cutters and other units, arranged along a production line, wherein each unit is configured to convert the sheet into an intermediate form as it moves through the unit or perform any other known roll-to-roll process step.
By cutting out the fluid structures prior to laminating the polymer layers, the height of the reaction chamber can be increased to more than 40 μιη compared to the laser cutting of Focke, et al. Furthermore, it allows other faster and more effective cutting techniques to be used which are well-suited for the roll-to-roll process. According to one embodiment, the third layer is fed through a punch press wherein the material of the third layer is mechanically removed by moving a first cutting tool, such as a punch, through the third layer.
The third layer of a thin flexible material is in one embodiment passed through a punch press which punches out the structure of the reaction chamber and associated fluid channels and inlets. The punch press may be driven by hydraulics, pneumatics or an electrical power source which drives one or more rams on which the punches are located through the third layer using a predetermined pressure. Alternatively, a rotating punch press may be used instead which may be arranged along the production line. This provides a faster and cheaper way of forming the fluid structures of the device compared to the laser cutting tool used in Ohlander, et al. According to one embodiment, the third layer is fed through a laser cutter wherein the material of the third layer is cut away by means of at least one laser beam.
The third layer is alternatively passed through a laser cutter, e.g. a laser micromachin- ing unit, comprising one or more moveable laser cutting tools capable of being moved along at least an X-axis and a Y-axis in a horizontal plane. The third layer may be placed in a stationary position or moved forward at a predetermined speed as the laser cutting tools are moved relative to the third layer. Alternatively the laser cutting tools may be stationary and the third layer may be moved relative to the tools instead. This provides an alternative way of cutting out the desired fluid structure. The use of laser beams also allows for a more complex and detailed structure to be cut out if needed. Since the structures is cut out prior to laminating the two other layers, a laser cutting tool suitable for industrial roll-to-roll processes can be used.
The third layer is then fed to a second unit, such as a set of rollers, at which the first and second layers are introduced on both sides of the third layer. The layers may then be adhered together using an adhesive layer integrated into one or more of the three layers. In one embodiment, an adhesive layer may be introduced between the two layers or may be applied to at least one of the surfaces of these two layers. The adhesive has adhesive capabilities to the thin flexible material of the laminate. The laminate may then be fed to a third unit in which the reagent is applied to the reaction chamber or a storage chamber connected to the reaction chamber. In one embodiment, the first and second layers may be introduced in separate units. The reagent may instead be applied to the reaction chamber in the third layer after the manufacturing process. In one embodiment, the three layers may be bonded together by applying heat and pressure to the laminate so a heat seal is formed between the layers.
The electrical components, such as the heating element, the sensor units and/or the electronic circuits, may be applied, e.g. deposited, to the outer surface and/or inner surface of the first and/or second layer before it is introduced into the rollers. A photolithographic process may be used to deposit the electrical components. In one embodiment, one or more of the electrical components, such as the temperature sensor and/or other sensor units, may be applied to one or more intermediate layers, e.g. of a thin flexible material, as described above. The intermediate layer may then be introduced between and coupled to the first layer and the second and/or the third layer at the second and/or the fourth unit. The intermediate layers may be coupled to the first and/or the second layer by means of an adhesive or a heat seal. In one embodiment, the intermediate layers may be coupled to the first and the second layers before they are coupled to the third layer.
The use of a polymer material for the third layer allows a more precise control of the height of the reaction chamber comparing to the teachings of Ohlander et al. The height of the reaction chamber may be adapted to a desired application, e.g. by simply using another layer having a different thickness or by passing the layer through a pair of rollers, e.g. a set of hot or cold rollers, which presses it into a thinner layer.
In one embodiment, the inlet and/or outlet is located in the edge of the third layer. This allows the entire fluid structure or micro structure to be punched out in the third layer or cut out using lasers. In one embodiment, the inlet and/or outlet is located in the outer surface of the first and/or second layer. The first and/or second layer may then be fed through the first unit or another punch press having a different punch for that layer. The inlets and outlets may be etched into the first and/or the second layer instead. The inlet can be configured to partly receive a needle or a syringe for transferring a test sample to the reaction chamber. According to one embodiment, the method further comprises the steps of:
- feeding the laminate into a third unit, such as cutting tool, wherein the excess material of the three layers is cut away.
This allows the layers forming the laminate to be laminated together before being led into a second punching unit or cutting tool which cuts the laminate into the desired size and shape. This eliminates the need for cutting each of the three layers into the desired size and then assembling them afterwards which is a time consuming process and require a more precise handling of the layers. This also allows multiple devices to be punched/cut out in a single step. The structure of the reaction chamber and connecting channels and inlets/outlets and/or the size and shape of the devices may be cut out using two or more cutting tools or punches. This allows devices with different configurations and sizes to be produced in a single step.
Alternatively one or more laser cutters may be used to cut out the size and shape of the devices. Optionally a laser cutter may be used for cutting out the fluid structures and a cutting tool having a punch or knife may be used for cutting out the devices, or vice versa.
An object of the invention is achieved by a control method characterised in that
- the temperature is thermally cycled between at least a first temperature level and a second temperature level in a predetermined maximum number of cycles, wherein a full cycle is performed within a time period of less than 30 sec.
The high ratio of the heat exchanging surface to the volume in the single-use device allows an extremely fast thermal cycling of the temperature inside the reaction chamber. The temperature change between a first temperature level and a second temperature level may be done by selectively activating the heating element and/or cooling means in accordance with at least one predetermined set of parameters. The thermal cycling is defined as a full thermal cycle where the temperature is initially raised from a first temperature and finally lowered to the first temperature again, including dwell times for each temperature level in the cycle. When performing PCR amplification in the reaction chamber, the nucleic acid sequences present in the test sample are copied in each run, i.e. the amount of genetic material is in theory doubled in each temperature cycle electronic circuit. In order to obtain enough genetic material to ensure a certain test assay provides a test result with a low number of false positive or false negative results, the thermal cycles are repeated a number of times. The temperature is cycled in a number of cycles up to maximum 50 cycles, e.g. between 10-40 cycles or up to 20 or up to 30 cycles. The temperature is cycled until a positive result is obtained which usually occurs after up to 10-20 cycles. It is preferred that the number of cycles is less than 40 since a false positive result may be a result of more than 40 cycles. The necessary number of cycles for obtaining a positive result depends on the amount of DNA present in the test and on the sensitivity of the relevant test assay.
According to one embodiment, a full cycle is performed within a time period of less than 20 sec. The configuration of the single-use device allows each thermal cycle, including dwell times at each temperature level, to be performed over a time period of up to 30 sec, up to 20 sec, or between 10 and 15 sec.
According to one embodiment, the first temperature level is selected between 45 and 55°C and the second temperature level is selected between 90 and 100°C and a third temperature level is selected between 65 and 80°C.
In one embodiment, the temperature is cycled between a first temperature range of 35 -55°C or about 50°C and a second temperature of 90-100°C or about 95°C followed by a third temperature of 65-80°C or preferably 70-74 °C. The temperature levels may be selected according to the desired application in which a thermal cycling of a sample is needed, such as a PCR amplification process, in particular used for detecting one or more specific nucleic acid sequences which are specific for one of the biological cells mentioned above. The specific nucleic acid sequence may e.g. be a gene, or a frag- ment thereof. The detection of the specific nucleic acid sequence is preferably used for analytical or diagnostic purposes. Polymerase chain reaction (PCR) is one of the most commonly used nucleic acid amplification techniques. US 4683202 B l, US 4683195 B l, US 4800159 A, and US 4965188 A all disclose embodiments of the PCR technique. The DNA melts or denatures at temperatures of 90-100°C when performing PCR amplification of the genetic material present in the test sample. This causes the double stranded DNA, or fragments of DNA, to separate into two single strands. In some situations the temperature may even be above 100 °C, but such high temperatures may cause enzymes or other ingredients of the relevant assay to decompose and may thus be less preferred. Each single strand present in the test sample is then copied using enzymes, such as those mentioned above, for synthesizing the second strand of the DNA from each single strand.
PCR typically employs two oligonucleotide primers, as defined above, that bind to a selected area of the specific nucleic acid sequence of the single strands of the denatured DNA or of the RNA of interest. In order to obtain maximum efficiency in amplification, the primer is preferably single- stranded, but the primer can also be double- stranded. If double stranded primers are used, they are first denatured, i.e., treated to separate the strands, e.g. by heating.
When the double- stranded nucleic acid is denatured by heat, the primer is allowed to attach or hybridize to the selected area of the specific nucleic acid sequence by cooling the reaction mixture. The temperature is usually from about 35°C to about 55°C or preferably about 50 °C.
In order to copy the nucleic acid sequences of the two denatured single strands of DNA (or denatured fragments of DNA) present in the reaction mixture, the temperature is adjusted to a temperature at which the activity of the polymerase is promoted or preferably optimized, i.e. a temperature sufficient for extension to occur from the pri- mers to generate products complementary to the template nucleic acid. The temperature for the copying step is preferably 65-80°C, or preferably 70-74°C, which is sufficient to synthesize an extension product from each primer, but not so high that the copied nucleic acid sequence strand denatures from its complementary template. When RNA is copied, reverse transcriptase is used in the assay for transcribing the single stranded RNA into a complementary DNA (cDNA) strand prior to copying the DNA sequence. Subsequently, the newly synthesized cDNA is amplified using traditional PCR as described above. The temperature cycle is then repeated and for each cycle the genetic material present in the test sample is copied. The temperature may vary slightly within the intervals depending on the relevant assays used.
According to a special embodiment, the temperature is further cycled to and from a third temperature level located between the first and second temperature level, wherein the third temperature level is selected between 70-74°C.
According to one embodiment, the temperature is increased from the first temperature level towards the second temperature level at a rate of at least 10 /s and/or decreased from the second temperature level towards the first or third temperature level at a rate of at least 5 /s.
The integrated heating elements located on at least one side relative to the reaction chamber allows for a fast increase in the temperature inside the reaction chamber due to the large heat exchanging surface. Heat may also be quickly removed from the reaction chamber since the heat exchanging surface forms a large surface area through which the heat is able to dissipate due to natural convection.
According to one embodiment, the temperature is increased at a rate of 20 /s and/or decreased at a rate of 10 /s.
The rate of the heating may be increased by arranging a second heating element at the other side of the reaction chamber. This allows heat to be transferred to the chamber through both surfaces which in turn decreases the time needed to heat the test sample from one level to another level. The heating element and/or the activation (e.g. the duty cycles) of the heating element may be configured to minimize the settling time within the chamber by briefly over- or undershoot the heaters. The tolerance at each temperature level may be up to +1°C in order to control the chemical process. The rate of the cooling may be increased by using cooling means coupled to one of the heat exchanging surfaces or by arranging cooling means on one of the outer surfaces of the device. A coolant, such as air or water, is led past the heat exchanging surface where the heat from the chamber is transferred to the coolant. This allows heat to be removed faster through the use of forced convection.
The rate of the heating and cooling may be determined according to the configuration of the heating element and the cooling means. In one embodiment, the temperature may be increased at a rate of at least 5 °c /s, about 20 °c /s or up to 40 °c /s. In one embodi- ment, the temperature may be decreased at a rate of at least 5 /s, about 20 /s or up to
40°c/,
The invention also relates to the particular use of the claimed single-use device to control thermal cycling of a sample located inside a reaction chamber in the single-use device, preferably during a PCR process.
The invention also relates to the particular use of the claimed single-use device or the claimed control method in a diagnostic test for infectious diseases in mammals, in particular humans or animals, caused by staphylococcus and/or pneumococcus species, such as S. aureus or methicillin resistant S. aureus (MRS A).
The invention is not limited to the embodiments described herein and may be modified or adapted without departing from the scope of the present invention as described in the patent claims below. Description of the Drawing
The invention is described by example only and with reference to the drawings, wherein:
Fig. 1 shows a first exemplary embodiment of a single-use device according to the invention,
Fig. 2 shows a second exemplary embodiment of the single-use device,
Fig. 3 shows a third exemplary embodiment of the single-use device,
Fig. 4 shows an exemplary configuration of the single-use device for testing, and Fig. 5 shows an exemplary temperature plot for heating a test sample located in the device shown in fig. 4 in an optimized situation.
In the following text, the figures will be described one by one and the different parts and positions seen in the figures will be numbered with the same numbers in the different figures. Not all parts and positions indicated in a specific figure will necessarily be discussed together with that figure.
Detailed Description of the Invention
Fig. 1 shows a first exemplary embodiment of a single-use device 1 according to the invention. The device 1 may comprise at least one chamber 2 in the form of a reaction chamber formed in a laminate 3. The laminate 3 may comprise at least three layers 4, 5, 6 coupled to each other via at least two adhesive layers 7, 8 arranged between the three layers. A first layer 4 may have an outer surface 9 and an inner surface 10 facing the other two layers 5, 6. A second layer 5 may have an outer surface 11 and an inner surface 12 facing the other two layers 4, 6. A third layer 6 may be arranged between the two other layers 4, 5 in which the reaction chamber 2 is formed. The reaction chamber 2 may be defined by the inner surface 10, at least one sidewall 13 of the third layer 6, and the inner surface 12. The reaction chamber 2 may be configured to receive an applied test sample, such as a biologic test sample, and an applied reagent. The reagent may be selected to generate a chemical reaction with the test sample for a selected application. At least one inlet 14 may be connected to the reaction chamber 2 by means of a channel 15, such as a fluid channel, configured to lead the test sample from the inlet 12 and into the cham- ber 2. At least one outlet 16 may be connected to the reaction chamber 2 by means of a channel 17, such as a fluid channel, configured to serve as a breather to let the air initially located in the inlet channel 15 and in the reaction chamber 2 out, or to lead the test sample from the chamber 2 and out of the device 1 if relevant. The reaction chamber 2 may have at least one heat exchanging surface formed on the inner surface 10, 12 and defined by the sidewalls 13 of the third layer 6. The laminate 3 may be made of thin flexible layers of a polymer material, such as PET. The reaction chamber 2 may be shaped as a planar chamber having a predetermined length and width or as an oval or ellipse with one or more foci. The height of the reaction chamber 2 may be defined by the thickness of the third layer 6 and optionally the adhesive layers 7, 8. The reaction chamber 2 is configured so that the ratio between the area of the heat exchanging surface and the thermal mass defined by the volume is at least 2: 1. The width of the chamber 2 may be in the range of 1-5 mm and the length may be in the range of 10-20 mm or the radius may be in the range of 2-7 mm. The height of the reaction chamber 2 may be between 150 μιη and 350 μιη measured between the inner surfaces of the two heat exchanging surfaces. This allows for an extremely fast thermal cycling of the temperature inside the reaction chamber 2. At least one electrical heating element 18 may be applied to the outer surface 11 of the second layer 5 and arranged relative to the heat exchanging surface of the reaction chamber 2. The heating element 18 is in figs. 1-3 shown separately from the respective outer layer 4, 5. The heating element 18 may be configured to be coupled to an external power source (not shown) via at least one electrical terminal 19 for transfer- ring heat to the reaction chamber 2. The heating element may be shaped as a meander having a predetermined line width and predetermined spacing width between the lines. The line width may be between 100 μιη and 300 μιη and the spacing width may be between 100 μιη and 600 μιη. The heating element 18 may be made of a suitable material, such as copper, or an alloy, such as a nickel alloy.
At least one temperature sensor 20 may be arranged on the outer surface 11 of the second layer 5. The temperature sensor 20 is in figs. 1-3 shown separately from the respective outer layer 4, 5. The temperature sensor 20 may be configured to measure the temperature inside the reaction chamber 2. The temperature sensor 20 may be in- tegrated into the heating element 18, as shown in fig. 1, for better control of the applied heat to the reaction chamber 2. The temperature sensor 20 may be configured as a resistive temperature detector and may be coupled to at least two electrical terminals 21 on the layer 5. The terminals 21 may be coupled to a microprocessor unit, e.g. in an external reader unit which may be configured to control the power source supplying power to the heating element 18. The temperature sensor 20 may be made of any suitable materials, such as copper, or any alloys, such as a nickel alloy.
A protective layer 22 may be coupled to the second layer 5, e.g. to the outer surface 11 on which the heating element 18 and temperature sensor 20 are located. The protective layer 22 may be coupled to the second layer 5 via an adhesive layer (not shown), e.g. integrated into the protective layer 22. The protective layer 22 may be configured to protect the electrical components from environmental impact or shocks that might damage the electrical components or cause it to corrode. The protective layer 22 may be made of the same material as the laminate 3 and/or have a thickness between 100 μηι and 200 μιη.
Fig. 2 shows a second exemplary embodiment of the single-use device 1 where the temperature sensor 20 may be configured as a separate temperature sensor 20'. The temperature sensor 20' may be applied to the outer surface 9 of the first layer 4 for better measurement of the temperature inside the reaction chamber 2. The temperature sensor 20' may be arranged relative to the heat exchanging surface of the reaction chamber 2. The temperature sensor 20' may be aligned with the heating element 18 or offset relative to the heating element 18.
Fig. 3 shows a third exemplary embodiment of the single-use device 1 where a second heating element 23 may be arranged relative to the reaction chamber 2. The heating element 23 may be applied to the outer surface 9 of the first layer 4. The second heating element 23 may have same height and/or configuration, i.e. shaped as a meander, as the first heating element 18 or have a different configuration depending on the desired application. The heating element 23 may be made of the same material as the heating element 18.
A second temperature sensor 24 may be applied to the outer surface 9 of the first layer 4 and arranged relative to the heat exchanging surface of the reaction chamber 2. The temperature sensor 24 may be integrated into the heating element 23, as shown in fig. 1. The temperature sensor 24 is made of the same material as the temperature sensor 20. One or more windows 25, such as a transparent window, may be arranged in the first layer 4. The window 25 may be configured to have a surface area that more or less corresponds to the area of the heat exchanging surface of the reaction chamber 2. The window 25 may be configured for optical detection of the temperature inside the reac- tion chamber 2 via an external temperature sensor module, e.g. located in the reader unit.
A second protective layer 26 may be applied over the heating element 23 and tempera- ture sensor 24. The protective layer 26 may be configured to protect the electrical components from environmental impact or shocks that might damage the electrical components or cause it to corrode. The protective layer 26 may be made of the same material as the first protective layer 22 and/or have a thickness between 100 μηι and 200 μηι. A window 25 may be arranged in the protective layer 26 and aligned with the window 25 located in the first layer 4.
Fig. 4 shows an exemplary configuration of the single-use device 1 used for thermal cycling of a test sample inside the reaction chamber 2. In this configuration, the device 1 may comprise a transparent window 25 arranged in the second layer 5 for optical detection. The heating element 18 may be arranged on the outer layer of the laminate 3, i.e. on the outer surface of the first layer 4. A protective layer 22 may be applied over the heating element 18 for protection. The temperature sensor 20 may be integrated into the heating element 18. Terminals 21 may be located at two intermediate points along the meander shaped heating element 18 where the terminals 19 define the ends of the meander. This provides a configuration of the device 1 in which the heating and temperature measurement are done from the same side. Cooling is done by natural convection.
Fig. 5 shows an exemplary temperature plot 27 for heating a test sample located in the single-use device 1 shown in fig. 4. The heating element 18 and integrated temperature sensor 20 may be made of cupper with thermal properties of 0.393% change in resistance per degree Kelvin. A one-point calibration may be conducted at room temperature before the measurement. The measurements may be carried out by periodically switching the heater off at every 20 ms after which the temperature may be measured for 0.1 ms. The temperature may be measured by applying a constant current to the terminals 19 of the heating element 18 and then measuring the voltage over the terminals 21. The voltage may then be transformed into a temperature using a known conversion technique. During thermal cycling, the power output may be calculated by proportional-integral (PI) controller and a switching power supply operating at 10 kHz may vary the power to the heater by varying the duty-cycle of the output from 0% to 100%. The temperature may be plotted using the program Lab VIEW.
The device 1 may be heated and cooled according to a predetermined temperature cycle 28. The test sample may be heated from a first temperature level 29 of about 50°C to a second temperature level 30 of about 95°C before being cooled to the first temperature level 29 again. The test sample may be heated to a third temperature level 31 of about 72°C before being heated to the second temperature level 30. The temperature may be heated and cooled in up to 50 successive cycles. The reaction chamber 2 may be cooled by natural convection trough at least one of the heat exchanging surfaces located on the first and second layers 4, 5. The illustrated plot shows the measured temperature inside the reaction chamber 2 in an ideal situation where the heating and cooling of the device 1 have been optimised. The x-axis 32 shows the time in seconds and the y-axis 33 shows the temperature in degrees Celsius. In this configuration, a full temperature cycle may be completed after about 30 sec. and the temperature may be increased at a rate of about 10 /s. The tern- perature may be decreased, i.e. cooled, at a rate of about 5 /s. The heating and cooling have been optimised so that the transition between two temperature levels 29, 30, 31 is reduced to a minimum. The dwell time at each temperature level 29, 30, 31 is kept constant during a full cycle 28.

Claims

1. A single-use device for diagnostic testing comprising:
- at least one reaction chamber formed by a laminate of at least three layers, wherein the reaction chamber is configured to receive a biological test sample and a reagent for generating a chemical reaction with a biological test sample, wherein the reaction chamber is connected to at least one inlet via a fluid channel for receiving the test sample,
- wherein the reaction chamber is defined by a first inner surface of a first layer con- nected to a second inner surface of a second layer via one or more sidewalls formed in at least a third layer, wherein the first and second layers both comprise an outer surface facing away from the reaction chamber,
characterised in that
- the at least three layers forming the laminate are made of a polymer material, and - at least one electrical heating element is integrated in the device and arranged on at least one of the outer surfaces, wherein the heating element is configured to be coupled to an external power source for producing and transferring heat to the reaction chamber.
2. A single-use device according to claim 1, characterised in that at least one of the first and second layers has a thickness between 10- 100 μιη, or that the third layer has a thickness of 100-500 μιη.
3. A single-use device according to claim 1 or 2, characterised in that the device com- prises one or more temperature sensor units arranged relative to the reaction chamber, the sensor unit being configured to sense the temperature inside the reaction chamber.
4. A single-use device according to claim 3, characterised in that the sensor unit is arranged on at least one of the outer surfaces of the first and second layers or arranged inside the laminate between the first and second layers.
5. A single-use device according to any one of claims 1 to 4, characterised in that at least one window, such as a transparent window, is arranged in at least one of the outer surfaces.
6. A single-use device according to any one of claims 1 to 5, characterised in that the reaction chamber is an amplification chamber, wherein the reagent is configured to copy a genetic sequence in the test sample into a plurality of copies and for detecting one or more specific gene sequences in the copied genetic sequences.
7. A single-use device according to any one of claims 1 to 6, characterised in that a second heating element or cooling means for cooling the reaction chamber is arranged on the other outer surface and relative to the reaction chamber.
8. A single-use device according to any one of claims 1 to 7, characterised in that the device comprises at least one electronic circuit, e.g. a microprocessor unit, configured to control at least operation of the device, e.g. the temperature control, wherein the electronic circuit is arranged on at least one of the outer surfaces of the first and second layers or on at least a fourth layer of the laminate.
9. A single-use device according to any one of claims 1 to 8, characterised in that the reaction chamber and the reagent are configured to detect nucleic acids or pathogens, such as microorganisms, bacteria, fungus, parasites, vira, spores of bacteria or fungus, or malign tumour cells and/or neoplastic cells.
10. A method for manufacturing a single-use device according to any one of the preceding claims, the device comprising a laminate of at least a first layer, a second layer and a third layer, where the method comprises the steps of:
- forming at least one reaction chamber connected to at least one inlet via a fluid channel in the third layer of the laminate which has a predetermined length and width, - arranging the first layer of the laminate onto the third layer and coupling the first layer to the third layer, e.g. by means of an adhesive,
- arranging the second layer of the laminate onto the third layer and coupling the second layer to the third layer, e.g. by means of an adhesive,
characterised in that - the material of the third layer defining the structures of the reaction chamber, the inlet and the fluid channels are removed in a first unit, and then fed to at least a second unit wherein at least one of the first and second layer is laminated to the third layer.
11. A method according to claim 10, characterised in that the third layer is fed through a laser cutting tool wherein the material of the third layer is cut away by means of at least one laser beam, or that that the third layer is fed through a punch press wherein the material of the third layer is mechanically removed by moving a first cutting tool, such as a punch, through the third layer.
12. A method according to claims 10 or 11, characterised in that the method further comprises the steps of:
- feeding the laminate into a third unit, such as cutting tool, wherein the excess material of the three layers is cut away.
13. A method for controlling a process in a single-use device, where the method comprises the steps of:
- transferring a test sample into a reaction chamber formed inside the single-use device according to any one of claims 1-9,
- applying a reagent to the reaction chamber for generating a chemical process inside the reaction chamber when energy from an energy source is transmitted to the reagent,
- measuring the temperature inside the reaction chamber by means of a temperature sensor arranged relative to the reaction chamber, and
- transferring the measured temperature to a control unit which in turn controls the transfer of energy inside the reaction chamber by means of at least one heating element thermally coupled to the reaction chamber,
characterised in that
- the temperature is thermally cycled between at least a first temperature level and a second temperature level in a predetermined number of cycles, wherein a full cycle is performed within a time period of less than 30 sec.
14. A method according to claim 13, characterised in that a full cycle is performed within a time period of less than 20 sec.
15. A method according to claim 13 or 14, characterised in that the first temperature level is selected between 45 and 55 °C and the second temperature level is selected between 90 and 100°C and a third temperature level is selected between 65 and 80°C.
16. A method according to claim 15, characterised in that the temperature is further cycled to and from a third temperature level located between the first and second temperature level, wherein the third temperature level is selected between 70 and 74 °C.
17. A method according to any one of claims 13 or 16, characterised in that the tem- perature is increased from the first temperature level towards the second temperature level at a rate of at least 10 /s and/or decreased from the second temperature level towards the first or third temperature level at a rate of at least 5 /s.
18. A method according to claim 17, characterised in that the temperature is increased at a rate of 20 /s and/or decreased at a rate of 10 /s.
19. Use of a single-use device according to any one of claims 1-9 to control thermal cycling of a sample located inside a reaction chamber in the single-use device, preferably during a PCR process.
20. Use of a single-use device according to any one of claims 1-9 or the method according to any one of claims 13 to 18 in a diagnostic test for infectious diseases in mammals, in particular humans or animals, caused by staphylococcus and/or pneumo- coccus species, such as S. aureus or methicillin resistant S. aureus (MRS A).
PCT/DK2014/050216 2013-07-10 2014-07-09 Single-use device with a reaction chamber and a method for controlling the temperature in the device and uses thereof WO2015003722A1 (en)

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