WO2010140982A1 - Dispositif pour amplification d'acide nucléique - Google Patents

Dispositif pour amplification d'acide nucléique Download PDF

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Publication number
WO2010140982A1
WO2010140982A1 PCT/SG2010/000207 SG2010000207W WO2010140982A1 WO 2010140982 A1 WO2010140982 A1 WO 2010140982A1 SG 2010000207 W SG2010000207 W SG 2010000207W WO 2010140982 A1 WO2010140982 A1 WO 2010140982A1
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WO
WIPO (PCT)
Prior art keywords
body part
sample
thermal contact
sample mount
contact surface
Prior art date
Application number
PCT/SG2010/000207
Other languages
English (en)
Inventor
Lars Thomsen
Kian Kok Johnson Ng
Original Assignee
Biochip Devises Pte Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biochip Devises Pte Ltd filed Critical Biochip Devises Pte Ltd
Priority to SG2011089547A priority Critical patent/SG176653A1/en
Publication of WO2010140982A1 publication Critical patent/WO2010140982A1/fr

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Classifications

    • 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
    • B01L7/525—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 with physical movement of samples between temperature zones
    • B01L7/5255—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 with physical movement of samples between temperature zones by moving sample containers
    • 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
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • 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
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • 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

Definitions

  • the present invention generally relates to a device for performing nucleic acid amplification reactions.
  • Nucleic acid based diagnostics is by far the most accurate and scientifically validated method for determining the presence of a potential pathogen in a clinical sample.
  • PCR Polymerase chain reaction
  • thermal cycling steps include at least a denaturing step to physically separate the strands of double stranded DNA at very high temperature, typically about 94 to 96 degree C, to produce single stranded DNA, an annealing step to bind the primers to the target region on the single stranded DNA at lower temperature, typically about 50 to 60 degree C, and a replicating step to synthesize and thereby amplify the target DNA at a higher temperature, typically about 70 to 74 degree C.
  • the thermal cycling process as described above has to be repeated a number of times, typically for at least 20 to 35 cycles, to reach the level of amplification necessary to allow detection of the amplified target. Therefore, the main disadvantage in using the PCR technique is the high amount of time consumed in the inefficient thermal cycling process.
  • the thermal cycling process for PCR is most commonly conducted by placing the PCR reaction solution on a thermal block and ramping the temperature of the thermal block up and down to the required temperatures.
  • this is a highly energy demanding task that limits the possibility to make portable devices driven by batteries or solar panels for example.
  • Rapid portable DNA diagnostics is on demand since the world is facing the threat of potential pandemic events such as avian flu. For new and unknown viruses in particular, rapid diagnostics critical to quickly finding and isolating infected individuals. As an example, screening at airports with rapid portable DNA diagnostics can assist in quickly determining if a passenger who reveals symptoms of avian flu is indeed infected with the disease . DNA diagnostics from sputum or nasal samples can reveal this even though the infected person's body has not yet began presenting antibodies against the disease. In order for a device to be successful in this setting, the device needs to be able to operate quickly and automatically. However PCR devices currently available are expensive and bulky in size.
  • a main disadvantage of using a single thermal block for the PCR thermal cycling process is that the rate of heating or cooling the thermal block is slow, typically only about 3 to 4 degree C per second and the rate of cooling is even slower, typically only about 1 to 2 degree C per second. There is therefore a need to provide a device or method of exposing the PCR reaction solution to different temperature zones without having to waste time waiting for the change in the temperature .
  • PCR reaction vessels for example capillaries
  • cassettes which are not in direct thermal contact with the thermal blocks. This results in inefficient heating of the reaction vessel and a decreased rate of heat change. This also results in inefficient energy consumption.
  • Water baths maintained at the various required temperatures for thermal cycling have also been used in the past.
  • using the water baths eliminates the time wasted in waiting for the thermal block to reach the desired temperature and water baths provide a more stable and efficient transfer of thermal energy to the PCR reaction solution submerged therein.
  • a high thermal energy has to be supplied to the water baths to maintain the temperatures therein.
  • devices of this nature are prone to leakages. It is also difficult to maintain a uniform temperature of the water bath due to convectional currents in the heated water.
  • the PCR reaction solution is dipped into a first water bath that is maintained at a first temperature, and then removed therefrom by taking the PCR reaction solution out from the water bath into the atmospheric environment and subsequently dipping into a second water bath that is maintained at a second temperature and so on and so forth. It will be appreciated therefore that considerable time is required to remove the PCR reaction solution from one water bath to the other in both manual and automatic PCR systems. Automation of this process also poses some difficulties, such as the need to use more than one actuator to move the PCR reaction solution in at least two dimensional planes from one water bath to another, thereby increasing the complexity of the automation.
  • the PCR reaction solution Upon removal from one water bath, the PCR reaction solution is exposed to the atmospheric environment. This results in a loss of thermal energy from the PCR reaction solution since the thermal cycling temperatures are typically above the normal atmospheric temperature.
  • a device for selectively maintaining a reactive sample at different temperatures during the course of a reaction comprising: a body having a first part and a second part, at least one of the body parts being moveable relative to the other; at least one sample mount provided in the first body part for mounting the reactive sample therein; temperature control means thermally coupled to the second body part for selectively and independently controlling the temperature of at least two regions of said second body part, said regions being disposed from each other; means for moving at least one of the first and second parts relative to the other for enabling said sample mount to expose the reactive sample to said at least two regions that are at different temperatures during the course of the reaction.
  • the first and second body parts are moveable in one planar direction relative to the other.
  • the complexity of the device is greatly reduced in comparison to any device that requires movements in more than one plane.
  • the temperature control means is coupled to at least two metal blocks.
  • each of the metal blocks can be set to maintain a temperature in the range of about" 0 to about 200 degree C.
  • the device comprises at least four metal blocks.
  • one of the metal blocks is maintained a temperature substantially lower than the required reaction temperature to provide for supercooling, i.e. to allow for rapid cooling of the reactive sample.
  • one of the metal blocks is maintained a temperature substantially higher than the required reaction temperature to provide for superheating, i.e. to allow for rapid heating of the reactive sample.
  • the first and second body parts are configured such that during a stationary mode, said at least two regions (also referred herein as reaction chambers) that are substantially fluidly sealed from each other are formed between said first and second body parts.
  • the first body part may comprise a recess having a wall with an undulating exterior surface extending from the base to the top of the reaction chamber.
  • the undulating surface helps to increase fluid dynamics within the reaction chamber.
  • each of the reaction chambers is maintained at a certain required temperature, so that no time is wasted in waiting for the adjustment of the temperature required to reach the required temperature .
  • thermo-eyeling device having a body comprising a first part and a second part, at least one of the body parts being moveable relative to the other; at least one sample mount provided in the first body part for mounting the reactive sample therein; temperature control means thermally coupled to the second body part for selectively and independently controlling the temperature of at least two regions of said second body part, said regions being disposed from each other; a controller for independently and selectively controlling said temperature of said at least two regions; and means for moving at least one of the first and second body parts to thereby enable said sample mount to expose the reactive sample to said regions at different temperatures during the course of the reaction when in a moving mode .
  • a method for selectively maintaining a reactive sample at different temperatures during the course of a reaction comprising the steps of : providing a thermo-cycling device having a body- having a first part and a second part, at least one of the body parts being moveable relative to the other,- providing at least one sample mount in the first body part for mounting the reactive sample; providing temperature control means thermally coupled to the second body part for selectively and independently controlling the temperature of at least two regions of said second body part, said regions being disposed from each other; and providing means for moving at least one of the first and second body parts relative to the other for enabling said sample mount to expose the reactive sample to said regions at different temperatures during the course of the reaction.
  • a device for selectively maintaining a reactive sample at different temperatures during the course of a reaction comprising: a heating block comprising at least two thermal contact surfaces that are configured to operate at different temperatures from each other; at least one sample mount for mounting the reactive sample therein; biasing means configured to abut said sample mount and said heating block toward each other; means for moving at least one of said sample mount and said heating block relative to each other for enabling said sample mount to subject the reactive sample to thermal contact with said at least two thermal contact surfaces that are at different temperatures during the course of the reaction.
  • the at least two contact surfaces have a profile which is complimentary to at least part of the sample mount such that at least part of the sample mount's surface is in direct thermal contact with the thermal contact surfaces when biased toward the thermal contact surfaces .
  • reactive sample refers to a material that is undergoing or is about to undergo a chemical reaction such as nucleic acid amplification.
  • sample may include a biological sample or a non-biological sample.
  • a “biological sample” may be selected from the group consisting of dermal swabs,. cerebrospinal fluid, blood, sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, and urine.
  • a “non-biological sample” may be a liquid suspension comprising powders, particles from air samples, and particles from earth samples and surface swipes. The biological and non-biological samples may be cultured to facilitate the evaluation of the presence of a microorganism for example, such as B. anthracis .
  • nucleic acid may include, for example, but is not limited to deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) , and artificial nucleic acids such as peptide nucleic acid (PNA) , morpholino and locked nucleic acid
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PNA peptide nucleic acid
  • LNA glycol nucleic acid
  • GAA glycol nucleic acid
  • nucleic acid in the present context, 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-naturalIy occurring nucleobases, sugars and covalent internucleoside
  • nucleic acid analogues or nucleic acid mimics
  • nucleic acid mimetics are peptide nucleic acid (PNA-), Locked Nucleic Acid (LNA-), xylo-LNA- , phosphorothioate- , 2 ' -methoxy- , 2 ' -methoxyethoxy- , morpholino- and phosphoramidate- comprising molecules or functionally similar nucleic acid derivatives .
  • PNA- peptide nucleic acid
  • LNA- Locked Nucleic Acid
  • xylo-LNA- xylo-LNA-
  • phosphorothioate- 2 ' -methoxy-
  • 2 ' -methoxyethoxy- 2 ' -methoxyethoxy-
  • morpholino- and phosphoramidate- comprising molecules or functionally similar nucleic acid derivatives .
  • the term "about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • certain embodiments may ⁇ be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges.
  • a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range.
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3 , from 1 to 4 , from 1 to 5 , from 2 to 4 , from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range .
  • the first body part is disposed within said second body part, wherein the surfaces of the first and second body parts are not in contact with each other.
  • the first body part comprises of at least one protruding arm having a first base end coupled to the first body part and a protruding end configured to support the sample mount.
  • the protruding arm may be made of a flexible material, such as rubber or thin plastic, such that during the moving mode, the sample mount and any container containing the reactive sample will not abrade against the surface of the second body part and/or heating block. This may possibly prevent breakage of the container especially if the container is made of fragile material such as glass.
  • the temperature control means is thermally coupled to the thermal contact surface .
  • the temperature control means is configured to heat said thermal contact surface to enable a heating rate of 5 degrees centigrade per second when said sample mount is in direct thermal engagement with said thermal contact surface.
  • the thermal contact surface is in the form of at least two heat-conducting structures.
  • the heat-conducting structures may be metal blocks.
  • the metal block is formed of a material that exhibits a high thermal conductivity.
  • the at least two heat conducting structures may be made from a resiliently deformable material.
  • the material may have a thermal conductivity in the range of about 10 to about 1000 W/m-K, preferably about 20 to about 800 W/m-K, preferably about 20 to about 600 W/m- K, preferably about 100 to about 500 W/m-K, and more preferably about 200 to about 400 W/m-K.
  • the material may be selected from the group consisting of copper, stainless steel and aluminium.
  • the first and second body parts may be configured such that during a stationary mode said at least two regions (also referred herein as reaction chambers) that are substantially fluidly sealed from each other are formed between said first and second body parts.
  • the first body part is disposed within the second body part and the interface between the first and second body parts are substantially abut each other during the stationary mode to form the reaction chambers therebetween.
  • the sample mount is provided in the first body part for mounting of the reactive sample in at least ' one of the reaction chambers during the stationary mode.
  • the temperature control means is thermally coupled to the second body part for selectively and independently controlling the temperature of the individual chambers during the stationary mode .
  • the first body part may be generally circular in shape and may be configured to rotate about a central axis with respect to the second body part.
  • the first and second body parts are moveable relative to the other in one planar direction, such as by rotation. Accordingly, the size of device is relatively smaller when compared to any device wherein movement in more than one planar direction is required.
  • only the first body part is moveable. In another embodiment, only the second body part is moveable. In yet another embodiment, both the first and second body parts are moveable.
  • the means for moving the first body part comprises a motor coupled to the central axis of the first body part. In one embodiment, at least one of the first and second body parts rotates at a speed of about 10 to about 20 revolutions per second, preferably about 15 to about 18 revolutions per second.
  • the height of the undulating exterior surface is in the range of about 1.3 mm to about 2 mm, preferably about 1.3 mm to 1.5 mm.
  • the length of the undulating exterior surface is in the range of about 3.5 mm to about 4.5 mm, preferably about 3.8 mm to 4.2 mm.
  • the temperature control means that is thermally coupled to the second body part controls and maintains the temperature of a liquid contained within the reaction chambers .
  • the reactive sample is substantially fully submerged within the liquid contained within the reaction chambers.
  • the liquid may be oil.
  • Oil has a higher boiling point than water and is therefore less volatile than water. Accordingly, there will be lesser heat loss when oil is used in comparison to. water.
  • this allows for better and more stable maintenance of the required temperature within each of the reaction chambers.
  • the body of the device further comprises a sealing material between the first and the second body parts. This is to provide enhanced fluid sealing between the first and the second body parts, thereby preventing leakage of any fluid contained within the reaction chambers .
  • the sealing material may also act as a cleaning material that helps to remove any liquid on at least one of the first and second body part surfaces to thereby prevent cross contamination between a first liquid that is contained within a first reaction chamber and a second liquid that is contained within a second reaction chamber.
  • the first body part comprises a tube that is connected to a central axis by a plurality of connecting limbs.
  • the tube is a circular tube .
  • the circular tube comprises at least one recess which corresponds to the respective reaction chambers of the first body part.
  • the limbs are disposed adjacent to the at least one recess.
  • the at least one recess comprises a wall that is shaped such that the majority of a fluid contained within the chamber is substantially retained in the chamber during movement of said first and second body parts .
  • the limbs may comprise a central hollow bore to promote insulation of the reaction chambers, since air is a poor thermal conductor.
  • the above described structure also helps to reduce the bulk weight of the first body part so that less energy is required for facilitating the movement of the first body part.
  • the reactive sample is contained within a capillary.
  • the capillary may be about 35 mm in length with an inner diameter of about 0.75 mm and the outer diameter of about 1 mm.
  • the capillary is a glass capillary.
  • the glass capillary may be made of borosilicate glass or any other material that has excellent thermal properties and can withstand the rapid changes in temperature without breaking.
  • the specific heat capacity of the material of the capillary is lower than that of the reactive solution. This is so that a minimum time is required to transfer heat energy across the wall of the capillary.
  • the capillary is thin-walled and the reactive sample volume is small. Advantageously, this allows for rapid thermo-eyeling.
  • the sample mount is moved such that the capillary containing the reactive sample is exposed to one of the thermally coupled regions when in a stationary- mode. Heat exchange may then occur between the reactive sample and the region it is exposed thereto.
  • the rate of heat exchange between the reactive sample and the region is more than about 5 degree C per second.
  • the capillary does not have to be encapsulated by the thermal contact surface of the heat conducting structures for efficient heat transfer, but is urged into contact with the thermal contact surface of the heat-conducting structures by the biasing means.
  • a pair of sidewalls respectively extends between the edges of the base and top of the wall having the undulating exterior surface.
  • the sample mount may comprise a pair of bores respectively in the pair of sidewalls, and the bores may be dimensioned to receive the respective top and bottom ends of the capillary containing the reactive sample.
  • the first body part is configured to move generally parallel to the thermal contact surface of the second body part .
  • the first body part comprises a biasing means in the form at least one protruding arm having a base end coupled to the first body part and a protruding end configured to support the sample mount.
  • the surface of said second body has a thermal contact surface for at least partially enveloping said sample mount.
  • the thermal contact surface of the second body has a recessed profile that is complementary to the surface of said sample mount.
  • the thermal contact surface of said second body is configured to allow direct thermal engagement with said sample mount .
  • a corresponding surface of said sample mount is configured to lie flush against said thermal contact surface of said second body.
  • the device comprises a biasing means configured to abut said sample mount relative to said thermal contact surface of said second body.
  • the biasing means may be made from a flexible or elastic material, for example rubber, synthetic rubber or Nylon. It will be appreciated that any suitable material known to those of skill in the art may be suitably employed.
  • the biasing means may comprise a spring, for example a compression spring.
  • the thermal contact surface has an undulating profile and said biasing means biases said sample mount toward said undulating profile as said sample mount is moved lateral to said second body.
  • the undulating profile of the thermal contact surface comprises a recess comprising a base disposed between adjacent curved walls and wherein said sample mount is configured to reside within said recess during said reaction.
  • the preferred embodiment is easy to use and more economical to produce.
  • the power requirements of the device can be greatly reduced as there is no need for complex automated temperature control since each thermal contact surface is maintained at the required temperature.
  • the temperature at the thermal contact surfaces can be kept uniform.
  • the reaction is nucleic acid amplification reaction or polymerase chain reaction.
  • the nucleic acid amplification reaction may be preceded by a reverse transcription step that converts RNA into DNA. Therefore, it is to be appreciated that the disclosed device may be used to amplify nucleic acid from various types of RNA such as viral RNA, messenger RNA and transporter RNA.
  • the polymerase chain reaction may occur in less than about 1.5 hours, less than about 1 hour, less than about 30 min, and preferably less than about 15 min to obtain a detectable amount of the amplified nucleic acid.
  • Any material having favorable thermal properties such as low thermal conductivity and low thermal expansion may be used for manufacturing the device. Thermal expansion or contraction in the device may result in leakage of a liquid contained within the reaction chambers or obstruction in the movement of at least one of the first and second body parts .
  • the material has a thermal conductivity of less than about 5 W/(m-K), less than about 3 W/(m-K), less than about 2 W/(m-K), less than about
  • the material used is polyaryletheretherketone (PEEK) or poly(aryl ether ether ketone ketone) (PEEKK) .
  • the device comprises temperature- sensing elements, such as a thermally sensitive metal- based resistor (thermistor) .
  • the thermistor may have a positive temperature coefficient (PTC), that is, the thermistor exhibits increasing electrical resistance with increasing temperature and decreasing electrical resistance with decreasing temperature.
  • the thermistor may be selected from the group of materials comprising copper, nickel, iron, aluminium, platinum, or alloys thereof.
  • the thermistor may have different shapes and/or dimensions.
  • the thermistor may be in the shape of a sheet, a plate, a disc, a wire, or a rod.
  • the thermistor may also be a wire- formed electrode.
  • the device comprises one or more thermal heating elements .
  • the heating elements may have different shapes and/or dimensions and may also be operated based on different physical principles.
  • the heating element may be an electrode that is in the shape of a sheet, a plate, a disc, a wire, or a rod.
  • the heating element may also an infrared light source .
  • the heating element may be made of electrically conductive material, such as that selected from the group consisting of nickel-chrome (NiCr) , iron-chrome-aluminum (FeCrAl) , iron-nickel-chrome (FeNiCr) or other heating element alloys.
  • NiCr nickel-chrome
  • FeCrAl iron-chrome-aluminum
  • FeNiCr iron-nickel-chrome
  • the device further comprises an optical detection unit that is capable of detecting the presence and/or amount of nucleic acid in the reactive sample before, during and/or after the reaction.
  • the disclosed system further comprises a programmable unit to facilitate controlling, monitoring, and/or manipulating of the device prior to operation, when in operation, and/or after operation.
  • the programmable unit preferably comprises at least one computer having one or more computer programs stored within data storage means associated therewith, the computer system being adapted to for controlling the device.
  • the programmable unit may be chosen from the group consisting of a general purpose computer, a personal computer (PC) , a programmable logic control (PLC) unit, a soft programmable logic control (soft-PLC) unit, a hard programmable logic control (hard-PLC) unit, an industrial personal computer, or a dedicated microprocessor, and combinations thereof.
  • the programmable unit for controlling, monitoring, and/or manipulating the device prior to operation, under operation, and/or after operation preferably is preferably adapted for operation under harsh conditions, such as artic climate, tropical climate, and combat environment, in particular combat zones having being subjected to atomic, biological, and/or chemical warfare (ABC-warfare) .
  • the programmable unit complies with the relevant military specifications for such units.
  • the programmable unit provides the function of ensuring that the glass capillary containing the reactive sample therein is properly fitted in the sample mount. This is to prevent misassembling of the glass capillary that may undesirably result in breakage of the glass capillary.
  • the device may comprise an internal power supply.
  • the internal power supply may comprise a battery or a generator that may be portable.
  • the portable power generator may also be utilized as an external power supply.
  • the portable power generator may be recharged using, or simply consist of, a solar module, a battery charger (such as an alternating current or car battery charger) , or a fuel combusting generator.
  • the device may be equipped with a display enabling a visual readout of the experimental results.
  • the display can be in the format of a light emitting source (a LED, a light bulb or similar) , a screen, a digital readout or any combinations thereof .
  • the readout can be communicated in the form of audio signals.
  • the device comprises a component that allows for wireless communication.
  • wireless communication examples include 802.11 Mobile Wireless LAN, cellular, Bluetooth*, GPS, and Ultra Wideband.
  • the communication can comprise transport of data from the system or transport of data to the system, or any combination thereof.
  • Established communication can further be expanded to inter-device communication, i.e., establishment of an ad-hoc network enabling one device to trigger the initiation of sampling of another device thus facilitating the monitoring of, for example, a number of systems equipped with automated means for sampling and sample preparation and used to monitor a building, a farm, a food industry, a pharmaceutical production line etc.
  • the device is a light weight and/or portable device.
  • the device may weigh less than about 10 kg, less than about 8 kg, less than about 6 kg, less than about 4 kg, less than about 3 kg, less than about 2 kg, less than about 1 kg, or preferably less than about 800 g, less than about 600 g, less than about 500 g, less than about 400 g, less than about 300 g, less than about 200 g, less than about 150 g, or less than about 100 g.
  • the device may weigh in the range of about 500 g to 1000 g, about 100 g to about 1000 g, about 100 g to about 500 g, or about 250 g to 500 g.
  • Fig. IA is a schematic diagram of a PCR device in accordance with one embodiment as disclosed herein.
  • Fig. IB is a schematic diagram of a PCR device in accordance with another embodiment as disclosed herein.
  • Figs. 2A-2B are schematic diagrams of a chamber casing of a PCR device in accordance with another embodiment as disclosed herein.
  • Figs. 3A-3D and 4A-4B are schematic diagrams of an inner core of a PCR device in accordance with the embodiment of Figs . 2A and 2B .
  • Fig. 5 is a schematic diagram of the recess on the inner core of a PCR device in accordance with the embodiment of Figs. 2A and 2B.
  • Fig. 6 is a schematic diagram of a PCR device in accordance with an embodiment as disclosed herein.
  • Fig. 7 is a schematic diagram of a system for conducting PCR.
  • like numerals denote like parts.
  • Fig. IA shows the top view of a PCR device 80 in accordance with one embodiment disclosed herein.
  • the PCR device 80 has four regions in the form of chambers (12A, 12B, 12C, 12D) and a cylindrical cavity 16 within a chamber casing 10 for housing an inner core 30 therein.
  • each of the chambers (12A, 12B, 12C, 12D) respectively has an aperture (not shown) to allow for the insertion of a heating element and/or thermal sensor (not shown) for adjusting and maintaining the required temperature of the thermal contact surface (82A, 82B, 82C, 82D) .
  • the inner core 30 has a protruding arm 86. At the end of the protruding arm is a sample mount having a curved surface 88 substantially along the full length of the protruding arm 86 for holding a capillary 60 containing a PCR reaction solution therein.
  • the space within the cylindrical cavity 16 that is not occupied by the inner core 30 helps to increase insulation within the structure since air acts as an insulator in that it is not an efficient conductor of heat.
  • the inner core 30 also has a central axis 48 for allowing the fitting of a rotational motor (not shown) to the inner core 30.
  • the PCR device 80 has an optical detection module 90 that can be used for detecting signals in the reaction sample before, during and/or at the end of the course of the reaction.
  • a rotational motor (not shown) is fitted to the central axis 48 so that the inner core 30 rotates about the longitudinal axis of the shaft when the motor is in operation.
  • Each of the enclosed chambers (12A, 12B, 12C, 12D) contains a thermal contact surface (82A, 82B, 82C, 82D) that is maintained at a prescribed temperature necessary for conducting PCR. Typically, two different temperatures are required for conducting the thermal cycling process employed in PCR techniques.
  • the thermal contact surfaces (82A, 82C) are each maintained at the two different temperatures required for conducting the thermal cycling process.
  • the temperature of thermal contact surface 82A ' is set at 94-95 degree C to provide the denaturation temperature
  • the temperature of thermal contact surface 82C is set at 55-60 degree C to provide the annealing/extension temperature required for PCR.
  • the thermal contact surface 82B is set at 10-15 degree C to provide a "supercooling" temperature
  • the thermal contact surface 82D is set at 110-120 degree C to provide a "superheating" temperature, that facilitates cooling and heating of the reaction solution, thereby- increasing the efficiency and therefore decreases the residence time of PCR reaction.
  • the capillary 60 is brought into contact with thermal contact surface 82A to allow for the denaturation of the nucleic acid in the reaction solution.
  • the inner core 30 rotates to bring the capillary 60 into contact with the thermal contact surface 82B to allow rapid cooling of the reaction solution until it reaches the annealing/extension temperature.
  • the inner core 30 rotates to bring the capillary 60 into contact with the thermal contact surface 82C, wherein the temperature is maintained necessary for annealing/extension.
  • the inner core 30 rotates to bring the capillary 60 into contact with the thermal contact surface 82D to allow rapid heating of the reaction solution until it reaches the denaturation temperature.
  • the inner core 30 then rotates again to bring the capillary 60 into contact with the thermal contact surface 82A again.
  • the procedure repeats until completion of the course of the reaction as required.
  • the thermal contact surface 82B has a wider cross sectional area to facilitate heat dissipation.
  • the thermal contact surface 82B also has a peltier element 84 to provide for the cooling of the thermal contact surface 82B.
  • Fig. IB shows the top view of a PCR device 80 in accordance with another embodiment disclosed herein.
  • the features as shown in Fig. IB are similar to those as shown in Fig. IA, except for the following details as described below.
  • each of the thermal contact surfaces (82A, 82B, 82C, 82D) extends beyond the inner curved surface 92 of the chamber casing 10.
  • the protruding arm 86 is made of a flexible material, such as rubber or thin plastic, which allows the protruding arm 86 to substantially be bent when a slight pressure is applied onto it.
  • the curved surface 88 of the sample mount is designed to hold the capillary 60 such that one side, herein referred to as the right surface 96, of the capillary is exposed.
  • Fig. IB The operation of the PCR device 80 as shown in Fig. IB is similar to that as shown in Fig. IA.
  • the capillary 60 is first brought into contact with thermal contact surface 82A to allow for the denaturation of the nucleic acid in the reaction solution.
  • the right surface 96 of the capillary 60 is in contact with the left surface 94A of the thermal contact surface 82A.
  • the inner core 30 rotates to bring the capillary 60 into contact with the thermal contact surface 82B to allow rapid cooling of the reaction solution until it reaches the annealing/extension temperature.
  • the protruding arm 86 is not flexible, the capillary 60 will collide against the left surface 94A of the thermal contact surface 82A and break.
  • the protruding arm 86 is made of a flexible material, upon rotation of the inner core 30, the pressure applied onto the right surface 96 of the capillary 60 and henceforth on the curved surface 88 of the sample mount causes the protruding arm 86 to bend backwards . This is so that the pressure on the right surface 96 of the capillary 60 is sufficiently reduced and therefore the capillary 60 will not break but slide against the left surface 96A of the thermal contact surface 82A and eventually pass thermal contact surface 82A.
  • the capillary 60 is then brought into contact with the thermal contact surface 82B. Similar to that described above, the right surface 96 of the capillary 60 is brought into contact with the left surface 94B of the thermal contact surface 82B.
  • Fig. 7 shows a system for conducting PCR.
  • the system 200 has a user interface 210, a controller 220, a rotational motor 230 and a PCR device 80 as described above.
  • the user inputs the prescribed temperature and time protocol for the conducting of the PCR reaction at the user interface 210.
  • the controller 220 is then able to independently and selectively control the movement of the rotational motor 230 that is connected to the PCR device 80 and the temperature of the heating blocks 82A, 82B, 82C, 82D.
  • Figs. 2A and 2B respectively show the top and side views of a chamber casing 10 of a PCR device disclosed herein.
  • the chamber casing 10 comprises of four chambers (12A, 12B, 12C, 12D) surrounding a cylindrical cavity 16 for housing an inner core 30 (as shown in Figs. 3A-3D, which will be further described below) .
  • the chamber casing 10 also has a base 18, having an aperture 20 at the centre, to seal the bottom of the chambers (12A, 12B, 12C, 12D) .
  • the outer circumference 32 of the inner core 30 (as shown in Figs. 3A-3D) is in contact with the inner surface (14A, 14B, 14C, 14D) . This is so that when in operation, with the inner core 30 housed in the cavity 16, the chambers (12A, 12B, 12C, 12D) , together with the base 18 and outer circumference 32, each form a concealed cavity that is able to hold liquid therein without leakage.
  • each of the chambers (12A, 12B, 12C, 12D) respectively comprises an aperture (22A, 22B, 22C, 22D) to allow for the insertion of a heating element and/or thermal sensor (not shown) for adjusting and maintaining the required temperature of the liquid contained in the chambers (12A, 12B, 12C, 12D) .
  • Figs. 3A, 3B, 3C and 3D respectively show the oblique (top) , top, oblique (bottom) and bottom views of an inner core 30 of a PCR device in accordance with the embodiments of Figs. 2A and 2B.
  • the inner core 30 has a wave-shaped recess 40 carved into substantially the full length of the outer surface 32. At the respective top and bottom ends of the recess are a bore 42 and a bore 44 for holding a capillary (not shown) .
  • the inner core 30 has air chambers (34A, 34B, 34C, 34D and 36A, 36B, 36C, 36D) to increase insulation as well as to reduce the weight of the inner core 30 to thereby reduce friction when in operation, and to provide support for the structure.
  • the inner core 30 also has side limbs (38A, 38B, 38C, 38D) and a base 46 substantially across the bottom of the inner core 30 to provide support for the inner core 30 structure .
  • the inner core 30 has a central axis 48 (as shown in Figs. 3A and 3B) having a cavity 50 (as shown in Figs. 3C and 3D) therein.
  • the cavity 50 is substantially circular in shape with a segment 52.
  • the shaft of a rotating motor (not- shown) is inserted into the cavity 50 and the shaft of the rotating motor is locked in place by the segment 52 so that the inner core 30 rotates when the rotating motor is in operation.
  • Figs . 4A and 4B respectively show the front and cross-sectional views of the inner core 30.
  • Fig. 4A shows an inner core comprising a capillary 60 containing PCR reaction solution therein.
  • Fig. 4B is the cross-sectional view of Fig. 4A along the axis X-X marked in Fig. 4A.
  • Fig. 5 shows the cross-sectional view of the recess 40 on the inner core 30 and the capillary 60 contained therein, and their dimensions.
  • the outer diameter of the capillary 60 is 1 mm.
  • the height and length of the wave shape of the recess 40 is respectively 1.4 mm and 4.0 mm.
  • the PCR device disclosed herein has a chamber casing 10 and an inner core 30.
  • the inner core 30 is fitted in the cavity 16 of the chamber casing 10, and the dimensions are such that the outer surface 32 of the inner core 30 is in direct contact with the inner surface (14A, 14B, 14C, 14D) of the chamber casing 10, creating enclosed chambers (12A, 12B, 12C, 12D) .
  • a rotational motor (not shown) is fitted to the PCR device.
  • the shaft of the rotational motor penetrates through the aperture 20 of the chamber casing 10 and into the cavity 50.
  • the shaft is locked in place by the segment 52 so that the inner core 30 rotates about the longitudinal axis of the shaft when the motor is in operation.
  • Each of the enclosed chambers (12A, 12B, 12C, 12D) contains oil (not shown) that is maintained at a prescribed temperature necessary for conducting PCR. Typically at least two different temperatures are required for conducting the thermal cycling process employed in PCR techniques. Therefore, the oil contained in each of the chambers (12A, 12B, 12C, 12D) is maintained at the different temperatures required for conducting the thermal cycling process. In a particular embodiment, the temperatures of the oil in chambers 12A, 12B, 12C and 12D are respectively set at temperatures T 1 , T 2 , T 3 and T 4 . Also referring to Figs. 4A and 4B, a capillary 60 containing PCR reaction solution therein is inserted into the bore 42 and held in place by the bore 42 and bore 44. The capillary and the PCR reaction solution contained therein are therefore exposed to the oil contained in the chamber 12C that is maintained at temperature T 3 .
  • the inner core 30 is rotated by the action of the rotational motor (not shown) in the clockwise direction 66 so that the capillary 60 moves to the next chamber 12D.
  • the oil contained within the recess 40 exits the recess 40 in the directions 62, 64 (as shown in Fig. 4B) due to fluid dynamics within the recess 40 structure.
  • the oil contained therein can be substantially removed from the recess 40 into the chamber 12C.
  • the oil of temperature T 3 will substantially remain in chamber 12C and not be brought over to the next chamber.
  • the oil contained in chamber 12D quickly flows into the recess 40 in the directions 62, 64, again due to the fluid dynamics within the recess 40 structure. It is to be appreciated that the oil substantially fills up the whole recess 40 to thereby surround the capillary 60. This ensures that the capillary 60 and the PCR reaction solution contained therein are substantially fully submerged in the oil.
  • the inner core 30 is then rotated by the action of the rotational motor in the clockwise direction 66 so that the capillary 60 moves to the next chamber 12A.
  • the capillary 60 can be moved in one continuous motion across more than one chamber (12A, 12B, 12C, 12D) .
  • the rotational motor can be programmed to rotate the inner core 30 such that the capillary moves from chamber 12A to chamber 12C and then to chamber 12B .
  • the temperature in any one of the chambers 12A, 12B, 12C, 12D can be changed while the capillary 60 is in the other chambers.
  • the temperature at chamber 12C can be changed from temperature T 3 to a next temperature T 5 that may be required in the process later on.
  • Fig. 6 shows a PCR device 100 in accordance with one embodiment as disclosed herein.
  • the PCR device 100 has four thermal contact surfaces (182A, 182B, 182C, 182D) arranged linearly on a horizontal axis, and each thermal contact surface has a heating element (not shown) .
  • Each of the four thermal contact surfaces has a recess (140A, 140B, 140C, 140D) along their longitudinal axis that is of complementary configuration to the shape of a capillary 160.
  • a linear actuator 110 is located on a horizontal axis directly above the thermal contact surfaces .
  • the linear actuator 110 comprises of a motor 107, a leadscrew 109 and a guide rail 108.
  • a movable arm 186 is fitted on the linear actuator 110.
  • a sample mount 104 is attached to a holder 105 at the opposite end of the movable arm 186.
  • the movable arm 186 is provided with a spring 106 such that, in use, the holder 105 and consequently the capillary 160 are urged into contact with any one of thermal contact surfaces 182A, 182B, 182C or 182D.
  • the linear actuator 110, movable arm 186, holder 105 and thermal contact surfaces (182A, 182B, 182C, 182D) are held together by a frame 101.
  • the capillary 160 containing the PCR reaction solution therein is inserted into the sample mount 104.
  • the motor 107 drives the movable arm 186 along the leadscrew 109, and the guide rail 108, so that the capillary 160 is brought into contact with the thermal contact surface 182D.
  • the capillary 160 is urged against the thermal contact surface and is located in the recess 140D of the thermal contact surface.
  • the capillary 160 is in direct contact along its length with the thermal contact surface 182D to permit rapid heat transfer to the reaction solution in the capillary 160.
  • the capillary 160 will collide against the side of the thermal contact surface 182D and break.
  • the pressure exerted on the capillary 160 as it is brought into contact with the side of the thermal contact surface 182D is transferred to the spring 106 as it moves to rest within the recess 140D of the thermal contact surface 182D.
  • the pressure exerted on the capillary 160 is sufficiently reduced and the capillary slides against the side of the thermal contact surface 182D and into the recess 140D of the thermal contact surface 182D. Breakage of the capillary 160 is substantially avoided.
  • the thermal contact surfaces (182A, 182B, 182C, 182D) are maintained at the different temperatures required for conducting the thermal cycling process by the heating element (not shown) .
  • the capillary 160 is brought into contact with the next thermal contact surface and the procedure repeats as described above until completion of the reaction.
  • the disclosed device involves rapid and energy efficient thermal cycling process .
  • the device can also be portable and/or battery driven.
  • a main advantage of the disclosed device is that no time is required to wait for the temperature to change to the temperatures required for conducting the reaction.
  • the reactive sample is contained within a glass capillary and the glass capillary is in thermal contact with a metal block to provide the necessary heating or cooling of the reactive sample during the course of the reaction.
  • the capillary is in direct thermal engagement with the thermal contact surface substantially along its longitudinal length to ensure maximum direct thermal contact area, thereby enabling efficient heat transfer.
  • the first body part comprises a biasing means such that breakage of the capillary is substantially prevented during movement of the body parts .
  • the device comprises of four metal blocks when two temperatures are required during the course of the reaction. Accordingly, two of the metal blocks are each set at the prescribed temperatures required for reactions to occur, and the other two metal blocks are each set at a supercooling and a superheating temperature, to respectively allow for rapid cooling and heating of the reactive sample.
  • this further reduces the time required to change the temperature of the reactive sample during the course of the reaction.
  • the reactive sample is substantially completely submerged in a liquid body at any one point in time to provide the necessary heating or cooling of the reactive sample during the course of the reaction.
  • the speed of the movement of the device in combination with the structure of the first body part, allows for rapid inflow and outflow of the liquid within the reaction chambers. This prevents cross contamination of the liquids that may be contained within the different reaction chambers, which may otherwise affect the temperature maintained within each reaction chamber.
  • the capillary is thin-walled, and the reactive sample volume is small.
  • this allows for rapid thermo-eyeling as the rate of heat exchange with the reactive sample is more than about 5 degree C per second.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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Abstract

La présente invention concerne un dispositif permettant de maintenir sélectivement réaction à différentes températures un échantillon de pendant le déroulement d'une réaction. Ce dispositif comprend: un corps en deux parties dont l'une au moins est mobile par rapport à l'autre; au moins un porte-échantillon équipant la première partie du corps et dans lequel on placera l'échantillon de réaction; des organes de régulation thermique, thermiquement couplés à la seconde partie du corps de façon à réguler sélectivement et indépendamment la température d'au moins deux zones de la seconde partie du corps, ces deux zones étant distinctes l'une de l'autre; et des organes permettant de déplacer l'une au moins des deux parties par rapport à l'autre de façon à permettre au porte-échantillon d'exposer l'échantillon de réaction aux deux zones considérées qui sont à des températures différentes pendant le déroulement de la réaction.
PCT/SG2010/000207 2009-06-02 2010-06-02 Dispositif pour amplification d'acide nucléique WO2010140982A1 (fr)

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SG2011089547A SG176653A1 (en) 2009-06-02 2010-06-02 Device for nucleic acid amplification

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US10641772B2 (en) 2015-02-20 2020-05-05 Takara Bio Usa, Inc. Method for rapid accurate dispensing, visualization and analysis of single cells
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KR102264863B1 (ko) * 2016-06-30 2021-06-14 주식회사 씨젠 핵산 증폭 기기 및 핵산 증폭 방법
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KR102301305B1 (ko) * 2016-06-30 2021-09-13 주식회사 씨젠 핵산 증폭용 튜브 및 핵산 증폭용 반응 용기
EP3479101A4 (fr) * 2016-06-30 2020-05-06 Seegene, Inc. Appareil d'amplification d'acide nucléique et dispositif de détection de fluorescence
US11602752B2 (en) 2016-06-30 2023-03-14 Seegene, Inc. Apparatus for amplificating nucleic acid and fluorescence-detecting device
KR20190007094A (ko) * 2016-06-30 2019-01-21 주식회사 씨젠 핵산 증폭 기기 및 형광 검출 장치
US11460405B2 (en) 2016-07-21 2022-10-04 Takara Bio Usa, Inc. Multi-Z imaging and dispensing with multi-well devices

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