EP2798054A1 - Procédés et dispositif pour équilibrer le transfert de rayonnements - Google Patents

Procédés et dispositif pour équilibrer le transfert de rayonnements

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Publication number
EP2798054A1
EP2798054A1 EP12861881.6A EP12861881A EP2798054A1 EP 2798054 A1 EP2798054 A1 EP 2798054A1 EP 12861881 A EP12861881 A EP 12861881A EP 2798054 A1 EP2798054 A1 EP 2798054A1
Authority
EP
European Patent Office
Prior art keywords
substrate
light
temperature
sample
energy
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP12861881.6A
Other languages
German (de)
English (en)
Other versions
EP2798054A4 (fr
Inventor
Juergen Hans PIPPER
Sankar THULASINGA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agency for Science Technology and Research Singapore
Original Assignee
Agency for Science Technology and Research Singapore
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 Agency for Science Technology and Research Singapore filed Critical Agency for Science Technology and Research Singapore
Publication of EP2798054A1 publication Critical patent/EP2798054A1/fr
Publication of EP2798054A4 publication Critical patent/EP2798054A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating 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
    • HELECTRICITY
    • H05ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
    • H05BELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
    • H05B7/00Heating by electric discharge
    • H05B7/18Heating by arc discharge
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1805Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1861Means for temperature control using radiation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1861Means for temperature control using radiation
    • B01L2300/1872Infrared light

Definitions

  • the invention relates to methods and device for heating and cooling biological material for analysis.
  • Multiplex polymerase chain reaction is a modification of PCR in order to rapidly detect deletions or duplications in a large gene.
  • genomic nucleic acid samples are amplified using multiple primers and a temperature- mediated polymerase in a thermal cycler.
  • Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon size, that is, their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
  • kits for PCR are available and used by many forensic laboratories to amplify degraded DNA samples.
  • Commercial kits have a number of advantages over in-house multiplexing methods. Quality control measures are undertaken by the manufacturer of the kit and ensure that reactions are uniform across all kits. This avoids the preparation of PCR master mixes which require pipetting and use of multiple assay tubes, increasing the risk of operator error and contamination. This increased reliability allows profiles obtained from commercial kits to be admitted into court which is pivotal in large criminal trials.
  • the use of specific kits over a number of laboratories also allows for profile results to be compared as long as the same STR markers have been used in each kit. [006].
  • Some of the applications of multiplex PCR include: Pathogen Identification; High Throughput SNP Genotyping; Mutation Analysis; Gene Deletion Analysis; Template Quantification; Linkage Analysis; RNA Detection; Forensic Studies.
  • the current systems available are very big and heavy machines. The machines are not able to do both real time PCR and multiplexing. The size and fragility of cuixent systems means they are lab based and not able to be used in field conditions.
  • Real-time polymerase chain reaction also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain
  • KPCR is a technique based on PCR, which is used to amplify and simultaneously quantify a targeted nucleic acid molecule.
  • Real Time-PCR enables both detection and quantification.
  • the quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes.
  • the procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time.
  • the product of the reaction is detected at its end.
  • Two common methods for detection of products in real-time PCR are: ( 1) non-specific fluorescent intercalating dyes that disrupt any double-stranded DNA, and (2) sequence-specific nucleic acid probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary nucleic acid target.
  • the object of the invention is to alleviate some of the problems of the cuixent devices and methods.
  • a first aspect of the invention provides a method for adjusting the temperature of a sample comprising the step of heating a substrate with a laser diode light; said light projected on to the substrate to absorb the light and convert the light energy to a heat energy thereby raising the temperature of the substrate corresponding to the intensity of the light energy, the substrate configured to transfer the thermal energy substantially homogenously to the sample.
  • Another aspect of the invention provides a device for adjusting the temperature of a sample comprising: a laser diode for projecting a light to a substrate; said light adapted to be projected substantially homogeneously on to the substrate to absorb and convert the light energy to heat energy thereby raising the temperature of the substrate corresponding to theintensity of the light energy, the substrate configured to transfer the thermal energy substantially homogenously to the sample.
  • Another aspect of the invention provides a method for amplifying and/or analyzing a nucleic acid sample comprising the steps of: a. placing the nucleic acid sample in contact with a substrate;
  • the substrate configured to transfer the thermal energy substantially homogenously to the nucleic acid sample;
  • Figure 1A Isotopic view of a partially (only outer surface of ring blackened) black substrate according to the present invention.
  • Figure IB Isotopic view of a partially (black band on outer surface of the ring) black substrate according to the present invention.
  • Figure 1C Isotopic view of a partially (black circles on outer surface of the ring) black substrate according to the present invention.
  • Figure ID Isotopic view of a partially (black slots/holes that accommodate the sample) black substrate according to the present invention.
  • Figure IE Isotopic view of a transparent substrate holding PCR tubes with PCR samples, whereby the PCR samples contain a black substrate according to the present invention. Alternatively, the black substrate might be adsorbed to the PCR sample tubes.
  • Figure IF Reactions vessel as disposable substrate according to the present invention.
  • Figure 2 A Top view of a set-up that utilized a JOLD-4.2-BAXH- 1E laserdiode from Jenoptik with built-in optical components for beam shaping/collimation.
  • a 1 x3 diffractive beamsplitter (2D grating) is used to evenly split the beam into three beamlets.
  • Figure 2B Top view of a set-up that utilized a JOLD-4.2-BAXH- 1 E laserdiode from Jenoptik with built-in optical components for beam shaping/collimation.
  • 4 beamsplitters (50% transmission and reflection each) are used to evenly split the beam into 4 beamlets.
  • Figure 3A Top view of a set-up that utilized a 1 x3 diffractive optical element (DOE) for beamshaping according to the present invention.
  • Figure 3B Temperature uniformity at 60°C for various beamshaping configurations. All setups resulted in temperature uniformities of less than -bQ.5 ( °C. Centrosymmetric arrangements with more than three beamlets achieve temperature uniformities of less than ⁇ 0 C. Fiberoptics, diffractive optical elements (DOEs), and beamsplitters were used for beamshaping.
  • Figure 3C Top view of a set-up that utilized a two beamsplitters and three mirrors to generate three beamlets of equal intensity (1 : 1 : 1) according to the present invention.
  • Figure 3D Top view of a set-up that utilized a combination of beamsplitters and diffractive optical elements to generate 12 beamlets of equal intensity ( 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1) according to the present invention.
  • Figure 4A Isotopic view of a thermocycler device that utilized a 1 x3 fiberoptical beamsplitter for uniformly heating the ring according to the present invention.
  • Figure 4B Isotopic view of a thermocycler device that utilized arrayed beamsplitters for uniformly heating the ring according to the present invention.
  • Figure 4C Heating as well as active and passive cooling rates for the ring according to the present invention.
  • Figure 4D Overall power consumption for the thermal management of the device according to the present invention.
  • Figure 5A Side view of a dual-channel fluorescence detector employed in the device.
  • FIG. 5B Limits of detection (LODs) for the fluorescence detectors used for this device.
  • Figure 6A Standard curve for avian influenza H5.
  • Figure 6B Standard curve for HIV- 1.
  • Figure 6B Standard curve for tuberculosis (TB).
  • Figure 7A Melt curve analysis (MCA) for avian influenza H5 as target.
  • Figure 8A PCR sample temperature measured by a Ptl OOO RTD temperature sensor versus ring temperature monitored by a thermopile.
  • Figure 8B One PCR thermocycle (5s each at 95°C, 60°C, and 72°C, respectively) monitored in closed loop by a thermopile according to the present invention, igure 8C: Based on the same input optical power, a partially blackened ring reaches 96°C (denaturation temperature) faster. 'Blank' means that the inside of the ring is bare polished aluminum metal.
  • Figure 9 Heating using magnetic particles dispersed in the qPCR solution according to one aspect of the present invention.
  • Figure 10A Top view of one device according to the present invention.
  • Figure 10B Bottom view of one device according to the present invention.
  • Figure IOC Side view of one device according to the present invention.
  • Figure 11 Side view of one device according to the present invention. Detailed Description
  • a method for adjusting the temperature of a sample comprising the step of heating a substrate with a laser diode light; said light projected on to the substrate to absorb the light and convert the light energy to a heat energy thereby raising the temperature of the substrate corresponding to the intensity of the light energy, the substrate configured to transfer the thermal energy substantially homogenously to the sample.
  • Heating the substrate substantially homogenously will ensure that the substrate is heated up quickly using a minimum amount of energy.
  • the method is designed to be carried out in field conditions where it is difficult to access a large power supply it is necessary to efficiently heat the substrate and thereafter transfer the heat to the sample.
  • the light should be absorbed on the substrate at least at two locations almost simultaneously to allow efficient heating.
  • the substrate or the laser diode are rotated in which case the one souse of light is absorbed resulting in a uniform distribution of themial energy on the substrate whereby the faster the spinning, the more homogenously the thermal energy distribution is over the substrate surface.
  • two or more laser diode light sources are used to heat the substrate.
  • the method may further comprising the step of optical manipulation of the light by focusing, collimating, splitting, diffracting, switching or reflecting to generate multiple beamlets spatially distributed and directed on to the substrate .
  • One example of optical manipulation is where the light beam from the laser diode is split, diffracted and/or reflected into at least two beamlet paths allowing the light radiation to project on the substrate at two locations so that the radiated energy is absorbed and converted to heat energy thereby raising the temperature of the substrate corresponding to the radiated light intensity and transfer the thermal energy to the sample.
  • optical manipulation light beam from the laser diode light source is optically manipulated to generate plurality of beamlets by splitting, diffracting and reflecting the light beam. These beamlets are spatially directed to a stationary substrate at several locations thereby resulting in a uniform generation of thermal energy on the substrate
  • more than one laser diode light sources can be used and optically manipulated to generate plurality of beamlets by splitting, diffracting and reflecting the light beams.
  • the method may further comprises the step of removing the light energy from the substrate to lower its temperature and maintain the substrate to a new temperature. This allows the substrate to cool via the ambient air temperature.
  • the cooling of the substrate can be further assisted by actively cooling the substrate with a sprayed mist or a fan or by other methods known in the art.
  • the method may further comprise the step of detecting the temperature of the substrate via a sensor not in contact with the substrate.
  • a sensor may include a thermopile.
  • the method may further comprise the step of detecting a signal from the sample via a sensor.
  • a sensor may include a fluorescence detector or a sensor that detects a change in magnetic resonance or any sensor capable of detecting changes in a biological sample.
  • the sample is nucleic acid samples.
  • the method and device described can be used for amplification of nucleic acid samples. This has the advantage that it conforms to the traditional workflow.
  • the method may further comprise the steps of; cooling the substrate to in turn cool the sample; and detecting the fluorescence of the sample as known in the art.
  • a laser diode for projecting a light to a substrate; said light adapted to be projected substantially homogeneously on to the substrate to absorb and convert the light energy to heat energy thereby raising the temperature of the substrate corresponding to the intensity of the light energy, the substrate configured to transfer the thermal energy substantially homogenously to the sample.
  • the device is compact and light weight to allow it to be easily portable and field deployable.
  • the device is designed to use a minimum amount of energy by efficiently heating the substrate. This is achieved by the excitation light being absorbed substantially homogeneously effectively avoiding thermal gradients to conduct a temperature corresponding to the excitation light to the substrate and transferred as heat to the sample. This ensures that the sample is heated up quickly, substantially homogenously and using a minimum amount of energy.
  • the device is designed to be used in field conditions where it is difficult to access a large power supply it is necessary to efficiently heat the substrate and thereby transfer the heat to the sample.
  • the substrate that generates thermal energy is external to the sample holder and transfers the thermal energy to the sample by radiation, conduction and convection. In another embodiment the substrate that generates thermal energy holds the sample. In another category of embodiment the substrate that generates thermal energy is suspended within the sample. [0030]. In one embodiment a portion of the substrate that comes into contact with the light has high emissivity. In this embodiment preferably the portion of the substrate that comes into contact with the light is black. Surface properties of an opaque substrate are important. Surfaces incident to the laserdiode light should be matte-black for maximum absoiption (the emissivity of a black body is 1).
  • surfaces not incident to the laser diode light should not be black in order to reduce radiation losses during the heating processes (especially, heating to 95°C and maintaining 95° during the polymerase hot-start activation and the denaturation step, respectively). Reducing radiation losses at temperatures above ambient temperatures markedly reduces the overall power consumption, as less electric al/optical input power is needed to reach and/or maintain activation, denaturation, and annealing/extension temperatures.
  • Selective (e.g. by masking) black anodization of metals e.g. aluminum
  • Such surfaces might be generated by plating techniques (e.g. 'black gold').
  • One example to selectively generate a non-black surface would be by physical and/or chemical polishing.
  • a portion of the substrate that does not come into contact with the light is non-black.
  • Surface properties of the substrate may be important. Matte-black surfaces will radiate much more efficiently than shiny bare metal in the visible spectrum.
  • a shiny metal surface has low effective emissivity due to its low surface area.
  • the emissivity in the visible spectrum is closely related to color. For most materials, the emissivity in the visible spectrum is similar to the emissivity in the infrared spectrum.
  • the surface of the substrate that comes into contact with the excitation light is black to maximize thermal transfer from the substrate to the sample, however, the portions of the substrate that do not come into contact with the excitation light are preferably white or shiny bare metal to avoid unnecessary heat loss from the device.
  • the substrate is made of a material with low density, low specific heat capacity, high thermal conductivity or high absoiption coefficient.
  • a low (thermal) mass enables fast temperature transition rates/changes.
  • a low thermal mass correlates with a low heat capacity, thus keeping the overall power consumption for heating and cooling at a minimum.
  • high thermal conductivity provides an efficient heat transfer, prevents the buildup of temperature gradients, and thus allows for a uniform temperature distribution within the substrate.
  • the temperature uniformity can be further enhanced by rotating the substrate relative to the beamlets. Also, this rotation allows analyzing multiple qPCR samples by moving them past a miniaturized fluorescence detector. All components in physical contact with the substrate, except the sample and/or sample tubes, should be thermal isolators with low/poor heat capacities.
  • the substrate may be made of the following types of materials: metals, metal alloys, and metal composites (e.g., aluminum, magnesium, and steel), ceramics (e.g.
  • thermally conductive polymers e.g. polypropylene, polycarbonate, polyamides, polyolefin, and liquid ciystal polymers
  • diverse carbon species e.g. diamond, carbon nanotubes, and carbon fibers/particles
  • any combinations thereof e.g. a gold coated thermally conductive polymer
  • the substrate is in the form of a band such as a ring.
  • a band such as a ring.
  • This may take on several forms for example the entire outer surface of the ring may be made matte- black by selective anodic oxidation of a polished aluminum ring using diverse masking techniques (e.g. wax) (see Figure 1A).
  • the height of the ring correlates with the diameter of the laserdiode light beamlets.
  • accommodating the PCR tubes might be blackened or not.
  • the substrate is in the shape of the ring, whereby only a small matte-black band covers the outer surface of the ring (see Figure I B).
  • This set-up has a smaller matte-black surface area and thus reduced radiation losses and a lower overall power consumption.
  • the diameter of the laserdiode light beamlets can be varied by using different types of collimators/focusers (typically 0.5-5mm beam diameters over a free- space traveling distance of 30-200mm of the laserdiode light). Additionally, the holes/slots for accommodating the PCR tubes might be blackened or not.
  • the substrate is in the shape of the ring, whereby only a few matte-black circles cover the outer surface of the ring (see Figure 1 C).
  • this set-up represents one of the most economic solutions and does not require the substrate being rotated.
  • the substrate is in the shape of a transparent ring, whereby only the holes/slots accommodating the PCR tubes are blackened (see Figure I D).
  • the substrate may be formed of a foam.
  • a metallic foam For example a metallic foam
  • the substrate may be in the form of particles within the sample.
  • the particles within the sample might be in the form of a solution, suspension, (colloidal) dispersion, or an emulsion.
  • examples for such substrates might be [but not limited to] quantum dots (e.g. gold particles), magnetic particles, synthetic diamonds, photosensitizers, and any combinations thereof.
  • quantum dots, magnetic particles, and photosensitizers could be employed as probes for additional sensing purposes (e.g. a spectral shift in the emission spectrum of a quantum dot could be used as a temperature sensor).
  • a magnetic particle e.g. silica-coated paramagnetic particles
  • Figure I F shows an illustrative example of a substrate, which is manufactured by co-injection molding of a matte-black thermally conductive polymer (e.g. polypropylene) and a native transparent polypropylene.
  • a matte-black thermally conductive polymer e.g. polypropylene
  • a native transparent polypropylene e.g. polypropylene
  • the transparent bottom or lid will enable optical detection.
  • a disposable substrate also prevents cross-contamination between subsequent qPCR runs -an important aspect in clinical diagnosis of infectious diseases.
  • the substrate may be in the form of a metal mesh or metal foam within the PCR sample solution
  • the device includes a laserdiode light source. In one embodiment the device further includes an additional laserdiode light source. Two or more laserdiodes can be operated at different wavelengths, frequencies, phases, or optical output powers. Laserdiode light is also referred to herein as beam(s) or beamlet(s). For routing the laserdiode light to the substrate, different optical components, such as collimators/ focusers, beamsplitters (e.g. plate beamsplitters), optical switches, diffractive optics (e.g. 2D gratings or diffusers), mirrors, etc. can be used. One or all of these optical elements can already be an integral part of a laserdiode.
  • beamsplitters e.g. plate beamsplitters
  • diffractive optics e.g. 2D gratings or diffusers
  • mirrors etc.
  • One such example would be a laserdiode with an in-built Fresnel lens for beam shaping collimation (see Figure 2A and 2B).
  • a beam shaped in the form of an annular ring e.g. generated by an axicon
  • This set-up would have the advantage, that one optical element alone would be sufficient to provide a substantially homogeneous illumination of the substrate without the build-up of local temperature gradients.
  • the device further comprises one or more optical elements selected from a beam splitter, a diffractive optical element, a mirror, a lens, a light guide or an optical switch is positioned in the path of a light beam between the laser diode and the substrate.
  • optical elements selected from a beam splitter, a diffractive optical element, a mirror, a lens, a light guide or an optical switch is positioned in the path of a light beam between the laser diode and the substrate.
  • permutations of laser diodes and optical elements should be calculated to heat the substrate substantially uniformly.
  • the device comprises a 1 x3 diffractive optical element (DOE) (as an example of a two-dimensional grating to manipulate laserdiode light) in combination with two mirrors (see Figure 3 A).
  • DOE diffractive optical element
  • the beam of a fiber-pigtailed laserdiode is collimated and split into 3 beamlets of equal intensity (1 : 1 : 1), which are equally spaced ( 120°) along the ring.
  • the beamlet of the Ost order impinges directly upon the ring, whereas the beamlets of the 1 st orders are deflected onto the ring by two mirrors.
  • This particular set-up provides a uniform temperature distribution within a stationary (non-rotating) ring.
  • thermopile temperature sensor in a closed loop is employed for contactless temperature measurements.
  • Various diffraction orders might be used.
  • three-dimensional diffractive optical elements might be employed.
  • the split ratio of the beamlets should be equal.
  • any split ratios can be employed.
  • non-equal split ratios might result in a larger temperature gradient within the ring, the temperature gradient can be easily lowered under ⁇ 0.5°C by varying the rotational velocity of the ring.
  • the temperature uniformity can be effectively controlled within ⁇ 0.5°C by adjusting four independent parameters: the relative orientation, number, and optical output power of the beamlets, as well as the rotational speed of the ring.
  • the device comprises an array of 2 plate beamsplitters in combination with three mirrors (see Figure 3C).
  • the beam of a fiber-pigtailed laserdiode is collimated and split into 3 beamlets of equal intensity ( 1 : 1 : 1), which are equally spaced ( 120°) along the ring.
  • This particular set-up provides a uniform temperature distribution within a stationary (non-rotating) ring.
  • a thermopile temperature sensor in a closed loop is employed for contactless temperature measurements.
  • the number of beamsplitters and mirrors might be varied.
  • any combination/permutation involving arrayed beamsplitters should result in beamlets of equal intensity.
  • the device comprises a combination of 4 arrayed beamsplitters, four mirrors, and 4 1 x3 diffractive optical elements (see Figure 3E)
  • the beam of a fiber-pigtailed laserdiode is collimated and split into 12 beamlets of equal intensity ( 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 ), which are equally spaced (30°) along the ring.
  • This particular set-up provides a uniform temperature distribution within a stationaiy (non-rotating) ring.
  • a thermopile temperature sensor in a closed loop is employed for contactless temperature measurements.
  • a diffuser might be used instead of 1 x3 diffractive optical element.
  • the number of beamsplitters, mirrors, and diffractive optical elements might be varied.
  • any combination/permutation involving arrayed beamsplitters should result in beamlets of equal intensity.
  • non-equal split ratios might result in a larger temperature gradient within the ring, the temperature gradient can be easily adjusted by varying the rotational velocity of the ring.
  • the device may further comprise a fan or a blower to remove thermal energy from the substrate
  • a fan or a blower may actively vary or control the temperature of the substrate (including PCR tubes and PCR samples). Alternatively, a vortex tube or compressed air might be used.
  • the device may further comprise a signal processor for detecting a signal of the sample being analyzed.
  • the device sensor may be a fluorescent signal processor connected to a fluorescence detector for processing a fluorescent signal (emerging from the PCR sample in the PCR rube). Alternatively it could be for the detection of electro
  • the device comprises a portable battery.
  • the laser diode light source used to heat the substrate requires electrical energy for operation.
  • the device is designed to consume less than l OWh of electrical energy to generate the thermal cycles for denaturation, annealing and extension.
  • the device will be compact and light weight which allows it to be easily portable and field deployable.
  • the device can be used in remote settings and powered by Lithium ion batteries comparable to those used in laptops and notebook computers.
  • the device preferably comprises a small form-factor battery (lithium-ion or lithium polymer-based batteries).
  • the substrate may comprise two or more substrates.
  • This possibility is to use one collimated laserdiode light beam (without placing any other optical elements except at least one mirror between the collimator and the substrate) to project the laserdiode light at multiple substrates (see Figure l OA-C).
  • This is an advantage, because only a limited number of optical elements would be needed to affect heating.
  • a further advantage of this set-up is, that the thermal mass of the samples is more or less identical with the thermal mass of the substrates, which would enable faster heating and cooling (compared to one large substrate with a rather large thermal mass).
  • the substrates would act as disposable reaction containers. E.g.
  • FIG. 10A 3 rotating disposable black substrates, which are slotted into a substrate holder, are heated simultaneously by one laserdiode light beam.
  • FIG. 10B 6 rotating disposable black substrates, which are slotted into a substrate holder, are heated simultaneously by one laserdiode light beam.
  • Figure 1 1 shows an example of a printed circuit board (PCB)-based motor, which uses friction force to rotate the substrate holder.
  • PCB printed circuit board
  • the device may further comprise a temperature sensor not in contact with the substrate.
  • thermosensor might be Pt RTD temperature sensors, thermocouples, and non-contact thermal and optical sensors, such as thermopiles, bolometers, or pyrometers.
  • a method for amplifying and/or analyzing a nucleic acid sample comprising the steps of: a. placing the nucleic acid sample in contact with a substrate; b. heating the substrate to a first temperature and maintaining the first temperature with a laser diode light source; said light projected on the substrate to absorb the light and convert the light energy to heat energy thereby raising the temperature of the substrate corresponding to the intensity of light energy, the substrate configured to transfer the thermal energy substantially homogenously to the nucleic acid sample; c. lowering the temperature of the substrate to a second temperature and
  • nucleic acid sample is amplified.
  • the thermal cycle comprises real time polymerase chain reaction to amplify the nucleic acid sample.
  • a detailed method may include the following steps.
  • the method is suitable for amplifying more than one nucleic acid sample.
  • the method comprises real time polymerase chain reaction to amplify the nucleic acid sample.
  • the method relies on photonic principles, whereby an excitation light converts the light energy to thermal energy and transfers the thermal energy to a sample.
  • the method is for amplification of more than one nucleic acid sample.
  • the method uses real time polymerase chain reaction to amplify the nucleic acid sample. It would be understood that the method described herein may be applied to any nucleic acid amplification methods known in the art.
  • the system is able to capture real-time qPCR data using a multiplex-capable fluorescence detector.
  • VIS visible range of the spectrum
  • the system is not limited to 4 optical channels and can be upgraded accordingly, e.g. by introducing additional channels in the ultraviolet (LTV), VIS, and near infrared range (IR).
  • LTV ultraviolet
  • VIS near infrared range
  • 1 -36 samples can be processed at a time.
  • the working volumes are in the range of 1 to l OuL. If needed, larger volumes can be processed by changing the system accordingly.
  • the systems' mode of operation completely relies on photonic principles: light at different wavelengths, frequencies, and phases is used for heating/cooling, temperature sensing, positional sensing, and optical detection.
  • Figure 4A depicts an example of a set-up.
  • An 8W 808nm-laserdiode light beam is equally split into 3 beamlets using a 1 > 3 fiberoptic beamsplitter and projected onto a stepper motor-driven matte-black ring holding multiple PCR sample tubes.
  • a thermopile temperature sensor working above 5 ⁇ in a feedback loop controls the ring temperature by regulating the laserdiode drive current.
  • a fan allows for active cooling using ambient air.
  • An optical interrupter/flag operating at 980nm moves the black ring -and thus PCR sample tube 1 to its home position above an optical detection system for initiating the real-time fluorescence data acquisition, which is perfonned by means of incremental step counting in either a stop-and-go or on-the-fly mode.
  • Two-color optical detection ( Figure 5A) is monitored between 475 and 655nm.
  • A3 aspheric lens of the fluorescence detection system De two-color optical detection system, Fa cooling fan for active cooling with ambient air, Fil -3 3 fiberoptic ends of a 1 x3 fiberoptic beamsplitter, G PCR glass capillary, I optical interrupter together with optical flag for indexing/mapping of the sample positions, LI and L2 LEDs used as excitation lightsources in the fluorescence detector, R black ring for absorbing the laserdiode light guided by the fiberoptics, St stepper motor to rotate the ring, T thermopile temperature sensor for controlling the ring temperature, W wheel for coupling the ring to the motor shaft.
  • the bar size is 10mm.
  • the interrupter might be replaced by an absolute or incremental encoder.
  • Figure 4B depicts yet another set-up, in which a collimated light beam of a 10W 980nm-laserdiode is collimated, equally ( 1 : 1 : 1 : 1) split into 4 beamlets using an array of plate beamsplitters and mirrors and projected onto a stepper motor-driven matte-black ring holding multiple PCR sample tubes.
  • the split ratio of all three beamsplitters is 50:50
  • a matte-black black anodized aluminum ring is used as the substrate holder to absorb infrared radiation from the beamlets (see Figures 1A-E). The absorbed heat is transferred by conduction and/or radiation to the PCR sample tubes held at locations around the substrate holder.
  • the combination of a black anodized aluminum ring in combination with polypropylene (PP)-overmolded glass or plastic PCR sample tube, and polyether ether ketone (PEEK) wheel yields temperature transition rates of more than ⁇ 10°C/s (see Figure 4C) and an average power consumption of around 4W (see Figure 4D).
  • thermally conductive materials such as metals, metal alloys, and metal composites, ceramics, thermally conductive polymers, diverse carbon species, and any combinations thereof can be used. Due to its low specific weight matte-black anodized magnesium is preferable.
  • the entire ring is matte-black.
  • a matte-black surface can be generated by various techniques, such as anodization methods, electrochemical plating, electroless deposition, spray painting, carbonization, incorporation of carbon fibers, incorporation of photosensitizers, etc.
  • a matte-black surface is preferred because it acts like a black body, thereby absorbing all incident electromagnetic radiation over a wide wavelength range.
  • the ring surface has an emissivity value of larger than 0.9.
  • the ring coating has an emissivity value substantially equals to 1.
  • the other portions are not blackened or coated in order to minimize radiation losses, which might contribute as much as 30%.
  • those non-blackened portions should have an emissivity of less than 0.9 (see Figures 1A-E).
  • other wavelengths of the electromagnetic spectrum such as ultraviolet (UV) or visible (VIS) can be utilized.
  • the two-channel fluorescence detector is based on a miniature optical bench populated with common (electro)optical components (see Figure 5A). Utilizing low-cost LEDs, antireflective (AR)-coated aspheric lenses, a hermetically-sealed silicon (Si) photodiode, as well as hardcoated filters does not require any maintenance or calibration during the entire lifecycle of the device. Unlike to an off-axis orientation, the in-axis configuration of the exciation and emission beam pathways adds more flexibility with respect to the PCR sample tube position and geometry. Moreover, each LED is individually modulated, which makes it possible to simultaneously monitor both optical channels under ambient light.
  • Figure 5 A shows a side view of two-color optical detection system.
  • a l and A2 aspheric lenses for collimating LED excitation light, A3 aspheric lens for focusing excitation light into the PCR sample tubes and collimating fluorescence light from the PCR sample tubes onto the photodetector (see Figure 4A and B), Di dualband dichroic mirror, Em dualband emission filter, Ex land Ex2 excitation filters, Fa fan for active cooling with ambient air, Fi l -3 1 x3 fiberoptic beamsplitter adapters, G glass PCR tube, Li lighttrap for minimizing stray light being backscattered from the dicroic mirror, Lo longpass filter, P Si photodiode, R matte-black ring, Sh stepper motor shaft, St stepper motor, T thermopile temperature sensor, W wheel for coupling the ring to the stepper motor shaft.
  • Bar size is 10mm.
  • a bolometer or pyrometer might be used for temperature control.
  • LODs limits of detection
  • Laserdiodes used so far ranged from under 400nm (UV) to more than 2300nm.
  • Figure 7A depics a melt curve analysis for the H5 target (black) and a no-template control (red).
  • the device/system contains additional components, such as a display, global positioning system (GPS), battery/recharging circuit, wireless local area network (WLAN), interlock system(s) to protect the user from radiation hazards during operation, a beam shutter, absolute or incremental position encoders, MCU/embedded PC, controllers), and software for each of those; and a power source for powering said device.
  • additional components such as a display, global positioning system (GPS), battery/recharging circuit, wireless local area network (WLAN), interlock system(s) to protect the user from radiation hazards during operation, a beam shutter, absolute or incremental position encoders, MCU/embedded PC, controllers), and software for each of those; and a power source for powering said device.
  • Figure 8A shows the correlation of the qPCR sample temperature measured by a Ptl OOO resistive temperature device (RTD) sensor and a thermopile, which was calibrated for temperatures between room temperature and 100°C.
  • RTD Ptl OOO resistive temperature device
  • Figure 8B depicts one full thermocycle (5s at 95°C, 5s at 60°C, and 5s at 72°C), whereby the temperature was monitored using a thermopile. Black indicates the object temperature and blue the reference temperature within the thermopile.
  • the invention is a thermal system for amplification of a nucleic acid sample comprising:
  • an optics manipulation system that splits the light beam from the laser diode and generates spatially distributed multiple light beamlets using optic fibres
  • collimators/focusers plate beam splitters, diffraction gratings and mirrors
  • a substrate positioned in a path of above said light beamlets and configured to absorb the radiated light energy and generate heat energy ;
  • a holding device to hold said nucleic acid sample, said holding device in physical contact with the substrate, said holding device configured to transmit the heat from the substrate to the nucleic acid sample;
  • nucleic acid sample containing at least one nucleic acid which undergoes
  • thermopile sensors in response to the thermal cycle a cooling system to cool said substrate and samples to a different temperature setting Platinum RTD sensors, themiocouples and non-contact thermopile sensors to accurately measure the temperature of the substrate and samples,
  • a feedback PID control system for the above said thermal management and to produce thermal cycles required for nucleic acid processing such as 95°C for denaturation and 60°C for annealing/extension for example.
  • nucleic acid sample containing at least one nucleic acid which undergoes
  • nucleic acid sample of Embodiment 1 and the addition of nucleic acid samples placed in physical contact with the substrate and spaced apart and thermally communicating with one another via the said substrate;
  • the substrate configured to rotate so that separate nucleic acid samples are positioned in a path of radiated light from each electromagnetic source at a time and each thermal cycle experienced by each nucleic acid sample is identical.
  • thermocycler device comprising:
  • a fluorescent signal processor connected to said detection system for processing a fluorescent signal detected by said detection system
  • thermo system as defined in Embodiment Example 1 or Embodiment Example2 above; optional components such as display, GPS, battery/recharging circuit, WiFi, optical position sensor for detection system, MCU/ embedded PC, controller(s) and software for each of those; and
  • a power source for powering said device.
  • the device is designed to be used with a Software that: a) interfaces, qPCR data manipulation at the end of the run, log service (GPS), real-time data streaming (WiFi GSM in combination with dedicated server), healthcare services, GMP compliance, minimum requirement for scientific intendons, remote diagnosis function.
  • GPS log service
  • WiFi GSM real-time data streaming
  • GMP compliance minimum requirement for scientific doctrineons, remote diagnosis function.
  • the invention described herein may include one or more range of values (eg size, concentration etc).
  • a range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

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Abstract

L'invention concerne un procédé et un dispositif pour ajuster la température d'un échantillon par chauffage d'un substrat avec une lumière de diode laser, ladite lumière étant projetée sur le substrat pour qu'il absorbe la lumière et convertisse l'énergie lumineuse en énergie thermique en augmentant ainsi la température du substrat en fonction de l'intensité de l'énergie lumineuse, le substrat étant conçu pour transférer l'énergie thermique à l'échantillon de manière sensiblement homogène. Le dispositif ou le procédé conviennent à l'amplification d'un échantillon d'acide nucléique.
EP12861881.6A 2011-12-28 2012-12-28 Procédés et dispositif pour équilibrer le transfert de rayonnements Withdrawn EP2798054A4 (fr)

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Publication number Priority date Publication date Assignee Title
CN107921399A (zh) * 2015-07-30 2018-04-17 加利福尼亚大学董事会 光腔pcr

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WO2015144783A1 (fr) * 2014-03-28 2015-10-01 Curiosity Diagnostics Sp. Z O.O. Dispositif d'application de cycles thermiques simultanés et uniformes d'échantillons et ses utilisations
DE102014108144B4 (de) * 2014-06-10 2015-12-31 Kist Europe-Korea Institute of Science and Technologie Europe Forschungsgesellschaft mbh Verfahren zum Betreiben eines Echtzeit-Polymerase-Kettenreaktionssystems (PCR) sowie eine Vorrichtung zum Betreiben des Verfahrens.
DE102015205003B4 (de) 2015-03-19 2016-12-08 Glp German Light Products Gmbh Beleuchtungsvorrichtung
SG10202003076TA (en) * 2020-04-02 2021-11-29 Delta Electronics Int’L Singapore Pte Ltd Thermal cycling system

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WO1996041864A1 (fr) * 1995-06-13 1996-12-27 The Regents Of The University Of California Chambre de microreaction chauffee par laser a diode dotee d'un moyen de detection d'echantillons
US6210882B1 (en) * 1998-01-29 2001-04-03 Mayo Foundation For Medical Education And Reseach Rapid thermocycling for sample analysis
AU2003287029A1 (en) * 2002-10-08 2004-05-04 University Of Virginia Patent Foundation Methods and systems for multiplexing ir-mediated heating on a microchip
US7279721B2 (en) * 2005-04-13 2007-10-09 Applied Materials, Inc. Dual wavelength thermal flux laser anneal
BRPI0819691A2 (pt) * 2007-11-30 2021-03-16 Cobertt Research Pty Ltd. Aparelho e método para controlar a temperatura de uma mistura de reação mantida dentro de um recipiente de reação

Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN107921399A (zh) * 2015-07-30 2018-04-17 加利福尼亚大学董事会 光腔pcr

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