WO2008080106A1 - Commande thermique sans contact de petit volume et appareil associé correspondant - Google Patents

Commande thermique sans contact de petit volume et appareil associé correspondant Download PDF

Info

Publication number
WO2008080106A1
WO2008080106A1 PCT/US2007/088662 US2007088662W WO2008080106A1 WO 2008080106 A1 WO2008080106 A1 WO 2008080106A1 US 2007088662 W US2007088662 W US 2007088662W WO 2008080106 A1 WO2008080106 A1 WO 2008080106A1
Authority
WO
WIPO (PCT)
Prior art keywords
temperature
pyrometer
pcr
heating
sample
Prior art date
Application number
PCT/US2007/088662
Other languages
English (en)
Inventor
James. P. Landers
Michael G. Roper
Christopher J. Easley
Original Assignee
University Of Virginia Patent Foundation
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 University Of Virginia Patent Foundation filed Critical University Of Virginia Patent Foundation
Publication of WO2008080106A1 publication Critical patent/WO2008080106A1/fr

Links

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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J5/00—Radiation pyrometry, e.g. infrared or optical thermometry
    • G01J5/52—Radiation pyrometry, e.g. infrared or optical thermometry using comparison with reference sources, e.g. disappearing-filament pyrometer
    • G01J5/53—Reference sources, e.g. standard lamps; Black bodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J5/00—Radiation pyrometry, e.g. infrared or optical thermometry
    • G01J5/80—Calibration
    • 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
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/148—Specific details about calibrations
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • 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
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium
    • B01L2300/1844—Means for temperature control using fluid heat transfer medium using fans
    • 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
    • B01L2300/1861—Means for temperature control using radiation
    • B01L2300/1872—Infrared light

Definitions

  • the present invention relates to methods and apparatus for rapidly and accurately measuring and controlling the temperature of a small volume sample. More specifically, the present invention relates to methods and apparatus for measuring the temperature of the sample in performing non-contact thermocycling on small, micro to nanoliter, volume samples.
  • thermocycling Numerous analytical methods require that a sample be heated to a particular temperature and then cooled to a particular temperature. Often, sequential heating and cooling steps, known as thermocycling, are required. Various methods involve cycling through two or more stages all with different temperatures, and/or involve maintaining the sample at a particular temperature stage for a given period of time before moving to the next stage. Accordingly, thermocycling of samples can become a time consuming process, hi addition, these methods often require the precise control of temperature at each stage of the cycle; exceeding a desired temperature can lead to inaccurate results.
  • thermocycling Two factors that are typically important, therefore, in the performance of effective thermocycling on a sample are the speed and homogeneity of the apparatus and the methods used. Cycle times are largely defined by how quickly the temperature of the sample can be changed, and relate to the heat source itself and the rate of heat transfer to the sample. Uniformity of sample temperature is important to ensure that reproducible and reliable results are obtained. Typically, increasing cycle speeds makes it harder to maintain homogenous sample temperatures.
  • PCR polymerase chain reaction
  • RNA DNA
  • This procedure requires the repetition of heating and cooling cycles in the presence of an original DNA target molecule, specific DNA primers, deoxynucleotide triphosphates, and DNA polymerase enzymes and cofactors. Heating accounts for a denaturing of the sample while cooling results in annealing of the sample. At a temperature typically between the denaturing and annealing temperatures, extension of the annealed primers using an enzyme occurs to replicate the DNA strand or portion of the strand.
  • PCR based technology has been applied to a variety of analyses, including environmental and industrial contaminant identification, medical and forensic diagnostics, and biological research. There are a number of biochemical reactions that require accurate and rapid thermocycling. Additionally, there are reactions whose specificity can be enhanced when conducted in a rapid and accurate thermocycling environment. The PCR reaction places very high demands on the accuracy of the thermocycling parameters and is, therefore, an ideal assay to test the accuracy of the thermocycling method and apparatus.
  • U.S. Pat. No. 4,683,202 generally describes the PCR concept, in which a stretch of DNA is copied using a polymerase.
  • the procedure involves annealing a piece of primer DNA at a first temperature to any stretch of single- stranded DNA template with a complementary sequence.
  • the DNA polymerase copies the primed piece of DNA at a second given temperature.
  • the newly copied DNA and the primer dissociate from the template DNA, thereby regenerating single-stranded DNA.
  • the temperature of the sample is returned to the first temperature to allow the primer to attach itself to any strand of single-stranded DNA with a complementary sequence, including the DNA strands that were synthesized in the immediately preceding cycle.
  • the template DNA is amplified or reproduced any number of times, depending on how many times the template DNA occurs in the sample, and the number of cycles completed.
  • the procedure can also be performed using RNA.
  • thermocycling In benchtop instrumentation, Most existing methods and techniques of thermocycling in benchtop instrumentation are indirect with respect to the effect of the heating source on the sample. Most thermocycling approaches heat and/or cool a circulating medium, such as water or air, that affects the container which holds the sample and, subsequently, subjects the sample itself to the desired thermocycling process. The rate of the cycling process depends on the effectiveness of the heat transfer between the circulating medium and the sample.
  • a circulating medium such as water or air
  • U.S. Pat. No. 5,504,007 discloses a thermocycle apparatus having a body containing a thermally conductive liquid.
  • the liquid is contained within the body of the apparatus, and the temperature of the liquid alternated between lower and higher temperatures in repeating cycles.
  • a well or container for holding a sample of material is held in contact with the liquid and conducts the cyclic temperature changes of the liquid to the sample.
  • U.S. Pat. No. 5,576,218 discloses a method for the thermocycling of nucleic acid assays using a blended fluid stream produced from constant velocity, constant volume, and constant temperature fluid streams. Using these streams, a variable temperature, constant velocity, constant volume fluid stream is introduced into a sample chamber for heating and cooling the samples contained therein. The temperature of the blended fluid stream is varied by diverting and altering the ratio of the constant temperature fluid streams relative to one another.
  • U.S. Pat. No. 5,508,197 discloses a thermocycling system based on the circulation of temperature controlled water directly to the underside of a thin-walled polycarbonate microtiter plate.
  • the water flow is selected from a manifold fed by pumps from heated reservoirs.
  • U.S. Pat. No. 5,187,084 discloses an apparatus and method for performing thermocycling on a sample using an array of sample containing vessels supported in a reaction chamber, through which air at controlled temperatures is forcibly circulated as a heat-transfer medium in heat exchange relationship with the vessels.
  • the temperature of the air is controlled as a function of time to provide a preselectable sequence defining a temperature profile.
  • the profile is a repetitive cycle that is reproduced to effect replication of and amplification of the desired sequence of the DNA.
  • U.S. Pat. No. 5,460,780 discloses a device for rapidly heating and cooling a reaction vessel through various temperatures in PCR amplification utilizing a device for heating at least one side wall of a reaction vessel, device for cooling the heating device at repeated intervals and device for moving the reaction vessel and/or heating and cooling relative to each other.
  • heated air is used to heat the reaction vessel.
  • U.S. Pat. No. 5,455,175 demonstrates that rapid, non-contact PCR can be accomplished in glass capillaries using air heated by foam lining the chamber in which the capillaries are placed; the foam is heated first by a halogen lamp.
  • thermocycling is through intimate contact between a reaction vessel holding the reaction medium and a heating block that is rapidly heated and cooled (for example, by using a Peltier element that can both heat and cool). That is the basis of most commercially available PCR instrumentation.
  • U.S. Pat. No. 5,525,300 discloses an apparatus for generating a temperature gradient across a heat conducting block.
  • U.S. Pat. No. 5,498,392 discloses chip-like devices for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide polymerization reaction.
  • the devices comprise a substrate microfabricated to defme a sample inlet port and a mesoscale flow system, which extends from the inlet port.
  • a polynucleotide polymerization reaction chamber containing reagents for polymerization and amplification of a polynucleotide is in fluid communication with the inlet port.
  • a heat source and, optionally, a cooling source are used to heat and/or cool the chip.
  • the device contained disposable polypropylene liners to retain the PCR mixture which could be cycled between two temperatures using polysilicon heaters in direct contact with the PCR chamber and cooled either passively or by air drawn along the heater surfaces of the reaction chamber.
  • the device was interfaced with the electrophoretic chip by forcing it into the 1 mm drilled holes in the electrophoretic chip.
  • thermocycling using both a non-contact heating source and a non- contact cooling source.
  • the heating source is provided by optical energy from an IR source.
  • the cooling source is provided by forcing air across the reaction vessel.
  • the temperature sensor in the system is a thermocouple that requires direct contact with the sample fluid.
  • thermocycling on a device in which temperature control is achieved using a temperature sensor that is predetermined by the initial design of the chip are limited, as the location of the temperature sensor is typically part of the chip itself.
  • those microdevices used in thermocycling are spatially constrained; and the devices are not flexible with respect to temperature sensing on different locations within or at the microdevice structure.
  • IR-mediated PCR in microdevices has been shown to be capable of fast thermocycling.
  • the use of a broadband tungsten lamp/convective fan allows for remote heating and cooling, and facilitates building the temperature control hardware into the instrumentation and not into the device, allowing for simple and cost-effective microdevice fabrication.
  • thermocouples for selective heating of microdomains on a fluidic chip, it must be coupled with a comparably simple but sensitive method for temperature sensing. While we have frequently utilized manually-inserted thermocouples for direct sensing of solution temperature, these require surface passivation to avoid inhibition of PCR (as would a thermocouple fabricated into the chamber). A temporary but effective solution to this problem has been the creation of a thermocouple reference chamber adjacent to the PCR chamber. However, a more viable solution involves a method that, like the heating, senses temperature through a non-contact process, again minimizing the cost of microchip fabrication. Additionally, remote (non-contact) temperature sensing is most applicable on microfluidic chips where the channels or chambers are so small (less than about 100 ⁇ m) that a thermocouple will not fit therein.
  • thermocylcing such as that for the polymerase chain reaction (PCR) amplification.
  • Remote temperature sensing is used herein to describe temperature measuring without directly
  • Remote sensing of the temperature of a solution within a small volume chamber can be accomplished by using a pyrometer, which is normally used for remotely measuring the temperature of a surface by measuring the radiation from that surface.
  • a pyrometer which is normally used for remotely measuring the temperature of a surface by measuring the radiation from that surface.
  • the small volume of the present invention is typically a closed reservoir or container
  • the present inventor has developed methods and apparatuses to use the pyrometer to interrogate the temperature inside the closed reservoir or vessel, rather than the temperature of the surface of the material enclosing the reservoir or container.
  • the "closed" vessel does not necessarily imply that the vessel is closed to the outside, but to convey that the fluid in the vessel is not directly exposed to interrogation by the remote temperature sensor.
  • the vessel is contained inside a microfluidic device.
  • a layer of material, that forms the microfluidic device e.g. glass, encloses the vessel.
  • the channel is open to allowing fluids to flow to and from the vessel via micro
  • a pyrometer generally contains an optical system and a detector.
  • the optical system focuses the energy emitted by the surface of an object onto the detector, which is sensitive to the radiation.
  • the output of the detector is proportional to the amount of energy radiated by the target object, and the response of the detector to the specific radiation wavelengths. This output can be used to infer the objects temperature by the Stefan Boltzmann equation:
  • FIG. 1 shows a preferred thermocycling system of the present invention
  • FIG. 2 shows the instrumental layout of pyrometer-controlled PCR of Example 1.
  • the pyrometer was placed off axis from the tungsten lamp to ensure the pyrometer was not heated during experiments.
  • the microdevice was secured to the setup to ensure the same location above the PCR chamber was measured for accurate temperature sensing.
  • the digital control lines from the data acquisition (DAQ) card to the relays are shown as dotted lines.
  • FIG, 3 shows a calibration curve of the pyrometer to a thermocouple. A.
  • thermocouple black line, left Y-axis
  • red line right Y-axis
  • Data values from each hold step were averaged and used in attaining the calibration constants. It is apparent that the time lag between the two temperatures was larger in the initial cycles compared to the later cycles and this effect was likely due to the entire device heating as the cycles progressed (explained in more detail at the end of the text).
  • B. The lag time between the maximum heating rates measured by the thermocouple and pyrometer are plotted for the various transition periods of the cycling shown in Figure 2A.
  • FIG. 4 shows non-contact amplification of ⁇ -phage DNA.
  • A Thirty cycles of a two-temperature heating protocol using the pyrometer calibrated with the trace shown in Figure 2 A.
  • B After thermal cycling was complete, analysis of the product within the PCR chamber by capillary gel electrophoresis demonstrated that an amplified product was present which migrated at the expected size (shown by *). The other peaks present at 3.5 and 4.0 minutes correspond to primer and dimer peaks, respectively.
  • Amplification with no template DNA was subsequently performed and is shown as the bottom electropherogram.
  • FIG. 5 shows calibration of the pyrometer using boiling points.
  • Water black line
  • an azeotrope grey line
  • the derivative of the azeotrope heating trace is shown in the inset with the unbroken arrow indicating the time point that boiling occurred. This time point was then used to determine at which pyrometer output voltage (broken line) corresponded to the boiling temperature.
  • FIG. 6. shows control of PCR using boiling point calibration.
  • the pyrometer was calibrated using the data in FIG. 3 and used to control amplification of a gene fragment from B. anthracis in a 250 nL PCR chamber.
  • the product peak (shown by *) migrated at the expected size relative to a DNA ladder with size of fragments given above ladder peaks in bp.
  • the inset details the sizing information for the 211 bp product (open square).
  • a control amplification showed no detectable amplified product at the expected size (bottom electropherogram).
  • FIG. 7 shows increased heating rates using a Au-coated mirror.
  • a gold- coated mirror was placed 5 cm above the PCR chamber and used to focus stray IR radiation back onto the device. With this mirror, the cycling time, relative to Figure 3A and 5, was reduced 44% to 18.8 minutes.
  • FIG. 8 shows increased overall temperature of the microfluidic device.
  • the average normalized outputs recorded by both the thermocouple and pyrometer at each denature, anneal, and extend steps were plotted for each cycle in a 30 cycle, thermocouple-controlled mock PCR.
  • the thermocouple traces are relatively constant since the temperature is being controlled with this sensor; however, the pyrometer signal increases over the first 15 cycles indicating the temperature of the microdevice surface is increasing during this time.
  • Plots are named by the method used to sense and the hold step, for example, "TC Denature” are values recorded from the thermocouple during the denature holds and "Pyro Anneal” are values recorded from the pyrometer during the anneal holds. One standard deviation is shown for each data point for clarity.
  • FIG. 9 shows thermal cycling in glass devices of 2.2-mm thickness was found to be much slower ( ⁇ 0.5 h) than the polymer counterparts ( ⁇ 10 min).
  • FIG. 10 shows (a) a typical microchamber; (b) a microchamber with contact heating, an indirect heating mechanism; and (c) non-contact heating, a direct heating mechanism requiring less overall energy.
  • FIG. 11 shows that the PID feedback control algorithm, optimized for the 72° C hold, was effective for thermal cycling.
  • FIG. 12 shows that the use of pulse-width modulation (PWM) allowed precise and accurate non-contact temperature control on microchips, even with cycling rates exceeding 20 0 C s '1 .
  • PWM pulse-width modulation
  • FIG. 13 shows a standard method for thermocouple calibration developed using a conventional PCR instrument, (a) An example trace and (b) calibration curve are shown (76.6 mV 0 C 1 ).
  • FIG. 14 shows water boiling with the thermocouple position (a) at the center of the chamber; and (b) at the edge of the chamber.
  • the inaccurate boiling point seen in (a) was indicative of the thermocouple absorbing IR radiation.
  • the present invention is generally directed to an apparatus and method for performing remote, rapid, accurate temperature measurement on small volume samples.
  • Remote temperature measurement in the context of this application, is used to describe temperature measuring without directly contacting the solution of interest.
  • small volume refers to volumes in the picoliters (pL) to microliters ( ⁇ L) range, preferably about 100 pL to about 100 ⁇ L, most preferably about 1 nL to about 10 ⁇ L.
  • the present invention uses a pyrometer, preferably an infrared (IR) pyrometer, to remotely measure temperature of a small volume sample.
  • a pyrometer generally contains an optical system and a detector. The optical system focuses the energy emitted by the surface of an object onto the detector, which is sensitive to the radiation. The output of the detector is proportional to the amount of energy radiated by the target object, and the response of the detector to the specific radiation wavelengths. This output can be used to infer the objects temperature by the Stefan- Boltzmann equation:
  • J SaT 4 (eq. 1) where J is the energy detected by the detector, ⁇ is the emissivity of the object, and ⁇ is the Stefan-Boltzmann constant.
  • Pyrometer are well known in the art and have been used in various capacities to measure surface temperatures. The present invention, however, uses the pyrometer to measure the temperature of the small volume enclosed inside a microfluidic chip, rather than the temperature of the surface of the material enclosing the small volume. In an embodiment of the present invention, several temperature sensors can be used to simultaneously detect temperatures at a plurality of locations on a microfluidic chip.
  • thermocycling method of the present invention are numerous and generally encompass any analytical system in which the temperature of a sample is regulated and/or changed.
  • the present invention is particularly applicable to analytical systems wherein fast or ultrafast transition from one temperature to the next is needed, and in which it is important that exact or nearly exact temperatures be achieved.
  • the present apparatus and methods are suitable for testing and incubation and treatment of biological samples typically analyzed in a molecular biology laboratory or a clinical diagnostic setting.
  • the accuracy of the thermocycling method of the present invention makes it particularly suitable for use in nucleic acid replication by the polymerase chain reaction (PCR). Any reaction that benefits from precise temperature control, rapid heating and cooling, continuous thermal ramping or other temperature parameters or variations can be accomplished using this method discussed herein.
  • thermocycling apparatus and methods, representing only some of the possible applications.
  • a common procedure in the protocols of molecular biology is the deactivation of proteins through heat.
  • One of the most basic procedures in molecular biology is the cleavage of proteins and peptides into discrete fragments by proteases/digestion enzymes, such as trypsin.
  • a thermocycling procedure is typically used to activate the enzyme at an elevated temperature followed by: the incubation of the enzyme during the reaction to sustain the enzymatic catalysis; the heat inactivation of the enzyme; and the final treatment/analysis at ambient temperature.
  • the reaction components are incubated at 4O 0 C for 60 minutes until the reaction is completed, after which the enzyme activity has to be stopped to avoid unspecific cleavage under uncontrolled conditions.
  • enzymes such as trypsin
  • trypsin can be irreversibly inactivated by incubation for 10 minutes at higher temperature, such as 95 0 C.
  • the sample is then cooled back to ambient temperature and ready for downstream analysis.
  • deactivation of enzymes is taught, for example, in Sequencing of proteins and peptides: Laboratory Techniques in Biochemistry and Molecular Biology, ed G. Allen, pages 73-105.
  • the same principle of heat inactivation can be used to inactivate restriction endonucleases that recognize short DNA sequences and cleave double stranded DNA at specific sites within or adjacent to the recognition sequence.
  • the appropriate assay conditions for example, 4O 0 C for 60 min
  • the digestion reaction can be completed in the recommended time.
  • the reaction is stopped by incubation of the sample at 65 0 C for 10 minutes.
  • Some enzymes may be partially or completely resistant to heat inactivation at 65 0 C, but they may be inactivated by incubation for 15 minutes at 75 0 C.
  • Such methods are taught, for example, by Ausubel et al. Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc. (1995) and Molecular Cloning: A Laboratory Manual, J. Sambrook, Eds. E. F. Fritsch, T. Maniatis, 2nd Ed.
  • the sample processing of proteins for electrophoretic analysis often requires the denaturation of the protein/peptide analyte before the separation by electrophoretic means, such as gel electrophoresis and capillary electrophoresis, takes place.
  • electrophoretic means such as gel electrophoresis and capillary electrophoresis
  • a 5 minute heat denaturation (which provides for the destruction of the tertiary and secondary structure of the protein/peptide) at 95 0 C. in an aqueous buffer in the presence or absence of denaturing reagents, such as SDS detergent, allows the size dependent separation of proteins and peptides by electrophoretic means. That is taught, for example, in Gel Electrophoresis of Proteins: A Practical Approach, Eds. B, D. Hames and D. Rickwood, page 47, Oxford University Press (1990).
  • Thermocycling of samples is also used in a number of nonenzymatic processes, such as protein/peptide sequencing by hydrolysis in the presence of acids or bases (for example, 6M HCl at 11O 0 C. for 24 hours) into amino acids.
  • acids or bases for example, 6M HCl at 11O 0 C. for 24 hours
  • binding characteristics such as kinetic association/dissociation constants.
  • thermocycling taught by the present invention will find use, for example, as a diagnostic tool in hospitals and laboratories such as for identifying specific genetic characteristics in a sample from a patient, in biotechnology research such as for the development of new drugs, identification of desirable genetic characteristics, etc., in biotechnology industry-wide applications, and in scientific research and development efforts.
  • samples subjected to the thermocycling methods of the present invention will vary depending on the particular application for which the methods are being used. Samples will typically be biological samples, although accurate heating and cooling of non-biological samples is equally within the scope of this invention.
  • a suitable vessel or reservoir according to the methods of the present invention is one in which extremely low volumes of sample can be effectively tested, including sample volumes in the nanoliter range.
  • the sample vessel must be made of a material that allows the penetration of IR light wavelengths, such as quartz glass, glass, silicon, transparent plastics, and the like.
  • the reaction vessel or container will have a high surface-to-volume ratio.
  • a high surface-to-volume ratio leads to a decrease in the thermal time constant, which can lead to an increase in the efficiency of the thermocycling.
  • a high surface-to-volume ratio, while not as important for the heating step, is related to the effectiveness of the cooling step.
  • suitable reaction vessels can be given, including but not limited to, microchambers, capillary tubes, microchips and microtiter plates.
  • a preferred example of a suitable reaction vessel or reservoir is a microchamber made from thin-walled glass.
  • capillary tube Another preferred embodiment is a glass capillary tube.
  • Such capillaries are typically used in capillary electrophoresis ("CE").
  • Suitable inner diameters of the capillaries having an outer diameter of about 370 ⁇ m typically vary between about 15 ⁇ m and 150 ⁇ m.
  • Thermal gradients that lead to convection are substantially reduced in capillary tubes which are available commercially.
  • the vessel vessel or reservoir may be any component of a microfluidic system where it is desired to measure the temperature of the fluid contained therein.
  • reaction vessel is the channel structure incorporated into a microfabricated device, such as the micro fabricated substrate described by Wilding et al. in Nucleic Acids Res., 24:380-385 (1996), and U.S. Patent Nos. 5,726,026 and 6,184,029, which are encorporated herein by reference.
  • Other reaction vessels with characteristics suitable for rapid thermocycling are shown in U.S. Patent Nos. 6,413,766 and 6,210,882, which are incorporated herein by reference.
  • reaction vessel such as a microtiter plate (96, 384 or 1636 wells)
  • the vessel is made of a material which allows FR radiation to directly heat the sample and has a surface-to-volume ratio sufficient to allow for cooling within the time parameters discussed below.
  • a method for preparing a suitable microfabricated device is discussed in the example section. Further guidance in preparing such microfabricated device is provided, for example, in U.S. Pat. Nos. 5,250,263; 5,296,114; Harrison et al., Science 261 :895-897 (1993); and McCormick et al., Anal. Chem., 69:2626-2630 (1997), which are incorporated herein by reference.
  • FIG. 1 shows the system of the present invention in which microchip 600 contains at least one closed reservoir 602.
  • the temperature of the small volume of fluid inside the reservoir can be measured using a remote temperature sensor (pyrometer) 606.
  • the microchip 600 is preferably placed on a movable stage 608, which may be generally ring-like to leave the underside of the microchip 600 exposed, and which can be motorized or moved manually.
  • a cooling source 614 is directed underneath the reservoir 602. Although only one cooling source 614 is shown, multiple cooling jets may be used to direct air above and/or below the reservoir 602.
  • a heating source 610 may be a lamp having its emitted light whose frequency can be controlled, for example, by a filter.
  • the light to which the reservoir 602 is exposed may be further limited by an aperture and/or a light restricting device to control the amount of heating to the reservoir 602.
  • the microchip 600 may sit on stage that is a frame that supports the microchip 600 on its periphery so that the microchip 600 is exposed to the light from the lamp 610.
  • the stage 608 may be moveable by a motor or manually so that different reservoirs 602 on the same chip may be interrogated by moving the reservoir 602 in proximity of the remote temperature sensor 606.
  • multiple remote temperature sensors 604 can be used to monitor multiple reservoirs 602 on the microchip 600. It is preferable that the remote temperature sensors 606, the cooling source
  • the remote temperature sensor 606 may electrically connected to the microprocessor 622 via a data acquisition card.
  • the cooling source 614 may be a cooling fan or associated with a compressed gas source for cooling the small volume reservoir 602 in the microchip 600.
  • the heating source 610 and the cooling source 614 may be electronically connected directly to and controlled by the microprocessor 622.
  • the microprocessor 622 preferably contains systems for controlling and receiving data from each of the heating source 610, the cooling source 614, and the remote temperature sensor 606.
  • FIG. 1 shows only one remote temperature sensor 604 associated with one reservoir 602, a plurality of temperature sensors/reservoirs are appropriate for the present invention.
  • the number of remote temperature sensors used can equal the number of reservoirs being used on the microchip.
  • a remote heat source, a remote cooling source, and remote temperature sensors are used. That allows for the repeated introduction of any number of reaction vessels in and out of the apparatus.
  • the present invention provides an economic advantage over other thermocycling apparatus, in that it is only a relatively inexpensive microchip, capillary tube, or other reaction vessel that must be changed for every sample.
  • reaction vessel could be completely cleaned to ensure that contamination from one sample to another did not occur, a new chip attached to a new heating, cooling, and/or temperature sensing device would have to be provided for every sample. While for ease of reference, only two sample-containing vessel were shown and/or described in FIG. 1, it is equally within the scope of the present invention to thermocycle more than two samples at the same time.
  • the heating and cooling sources are relatively stationary in the apparatus of the present invention, the reaction vessel can be moved in any direction relative to the heating and/or cooling sources. Heating of the sample is preferrably accomplished through the use of optical energy from a remote heat source.
  • this optical energy is derived from an IR light source which emits light in the wavelengths known to heat water, which is typically in the wavelength range from about 0.775 ⁇ m to 7000 ⁇ m.
  • the infrared activity absorption bands of sea water are 1.6, 2.1 , 3.0, 4.7 and 6.9 ⁇ m with an absolute maximum for the absorption coefficient for water at around 3 ⁇ m.
  • the IR wavelengths are directed to the vessel containing the sample, and because the vessel is made of a clear or translucent material, the IR waves act directly upon the sample to cause heating of the sample. Although some heating of the sample might be the result of the reaction vessel itself absorbing the irradiation of the IR light, heating of the sample is primarily caused by the direct action of the IR wavelengths on the sample itself.
  • the heating source will be an IR source, such as an IR lamp, an IR diode laser or an IR laser.
  • An IR lamp is preferred, as it is inexpensive and easy to use.
  • Preferred IR lamps are halogen lamps and tungsten filament lamps. Halogen and tungsten filament lamps are powerful, and can feed several reactions running in parallel.
  • a tungsten lamp has the advantages of being simple to use and inexpensive, and can almost instantaneously (90% lumen efficiency in 100 msec) reach very high temperatures.
  • a particularly preferred lamp is the CXR, 8 V, 50 W tungsten lamp available from General Electric, That lamp is inexpensive and convenient to use, because it typically has all the optics necessary to focus the IR radiation onto the sample; no expensive lens system/optics will typically be required.
  • the optical energy is focused on the sample by means of IR transmissible lenses so that the sample is homogeneously irradiated. That technique avoids "hotspots" that could otherwise result in the creation of undesirable temperature differences and/or gradients, or the partial boiling of the sample.
  • the homogeneous treatment of the sample vessel with optical energy therefore contributes to a sharper temperature profile.
  • the homogenous sample irradiation can further be enhanced through the use of a mirror placed on the opposite site of the IR source, such that the reaction vessel is placed between the IR source and the mirror. That arrangement reflects the radiation back onto the sample and substantially reduces thermal gradients in the sample.
  • the radiation can be delivered by optical IR-transparent fiberglass, for example, optical fiberglass made from waterfree quartz glass that is positioned around the reaction vessel and that provides optimal irradiation of the sample.
  • various optical instruments such as lenses, mirrors, filters, apertures, etc.
  • various optical instruments may be used to efficiently deliver the energy to the reservoir.
  • mirrors and lens may be used to focus the radiation on the reservoir to improve heating efficiency.
  • Heating can be effected in either one step, or numerous steps, depending on the desired application. For example, a particular methodology might require that the sample be heated to a first temperature, maintained at that temperature for a given dwell time, then heated to a higher temperature, and so on. As many heating steps as necessary can be included.
  • cooling to a desired temperature can be effected in one step, or in stepwise reductions with a suitable dwell time at each temperature step.
  • Positive cooling is preferably effected by use of a non-contact air source that forces air at or across the vessel.
  • a non-contact air source that forces air at or across the vessel.
  • that air source is a compressed air source, although other sources could also be used.
  • positive cooling results in a more rapid cooling than simply allowing the vessel to cool to the desired temperature by heat dissipation. Cooling can be accelerated by contacting the reaction vessel with a heat sink comprising a larger surface than the reaction vessel itself; the heat sink is cooled through the non-contact cooling source. The cooling effect can also be more rapid if the air from the non-contact cooling source is at a lower temperature than ambient temperature.
  • the non-contact cooling source should also be positioned remotely to the sample or reaction vessel, while being close enough to effect the desired level of heat dissipation.
  • Both the heating and cooling sources should be positioned so as to cover the largest possible surface area on the sample vessel.
  • the heating and cooling sources can be alternatively activated to control the temperature of the sample. It will be understood that more than one cooling source can be used. Positive cooling of the reaction vessel dissipates heat more rapidly than the use of ambient air.
  • the cooling means can be used alone or in conjunction with a heat sink.
  • a particularly preferred cooling source is a compressed air source. Compressed air is directed at the reaction vessel when cooling of the sample is desired through use, for example, of a solenoid valve which regulates the flow of compressed air at or across the sample.
  • the pressure of the air leaving the compressed air source can have a pressure of anywhere between 10 and 60 psi, for example. Higher or lower pressures could also be used.
  • the temperature of the air can be adjusted to achieve the optimum performance in the thermocycling process. Although in most cases compressed air at ambient temperature can create enough of a cooling effect, the use of cooled, compressed air to more quickly cool the sample, or to cool the sample below ambient temperature might be desired in some applications.
  • Monitoring and controlling is accomplished by use of a microprocessor or computer programmed to monitor temperature and regulate or change temperature.
  • a microprocessor or computer programmed to monitor temperature and regulate or change temperature.
  • An example of such a program is the Lab VIEW program, available from National Instruments, Austin, TX.
  • Feedback from the temperature sensor is sent to the computer.
  • the temperature sensor provides an electrical input signal to the computer and/or other controller, which signal corresponds to the temperature of the sample.
  • Signals from the computer control and regulate the heating and cooling means, such as through one or more switches and/or valves.
  • the desired temperature profile is programmed into the computer, which is operatively associated with heating and cooling means so as to control heating and cooling of the sample based upon feedback from the temperature sensor and the predetermined temperature profile.
  • the methods of the present invention provide for the use of virtually any temperature profile/dwell time necessary.
  • cleavage of proteins through use of proteases or digestion enzymes might require use of different temperatures, each of which must be precisely maintained for various amounts of time.
  • Activation of restriction endonucleases might similarly require achieving and maintaining two or three different temperatures.
  • Protein or peptide sequencing can require the steady maintenance of a high temperature for an extended period of time.
  • the above apparatus provide for rapid heating and cooling of a sample in a precise and easy to replicate manner. Heating can be effected for example as quickly as 1O 0 C per second when using approximately 15 to 50 ⁇ L volumes of sample in a microchamber and as rapidly as 100°C.
  • Cooling can be effected quickly, typically in the range of between about 5 and 5O 0 C per second.
  • the increased effectiveness of heating and cooling improves the cycling process and sharpens the temperature profile. This means that the desired reaction can be conducted under more optimal thermal conditions than in conventional instruments.
  • Thermal gradients in the reaction medium frequently observed in instrumentation using a contact heat source are detrimental to the specificity of the reaction. Those thermal gradients are substantially reduced in the IR mediated heating, particularly when the heat source is strong enough to penetrate the aqueous mixture and provide sufficient irradiation to the opposite side of the reaction vessel.
  • Non-contact, remote rapid cooling, heating, and temperature sensing such as that provided in the present invention, also contributes to the ability to obtain sharp transition temperatures in minimum time and to achieve fast and accurate temperature profiles.
  • the pyrometer In operation, the pyrometer must be properly calibrated to measure the temperature of the small volume inside the closed reservoir of the microfluidic device rather than just the external wall temperature.
  • the calibration account for the lag time associated with temperature equilibrium between the surface of the microfluidic device and the small volume inside the closed reservoir of the micofluidic device.
  • the method involves the use of the transition temperatures of two different calibration substances.
  • the calibration substances are reference solutions.
  • the calibration method involves the use of the boiling points of at least two different reference solutions.
  • the criteria for choosing the reference solutions included 1) boiling points that were within the range of temperatures normally used for the small volume (in the case of PCR, approximately 60-95 0 C); and 2) the solutions needed to approximate IR absorption in a manner similar to absorption by the solution used inside the small volume (in the case of PCR, at least 10% water).
  • the reference solutions include either water and an azeotrope or two different azeotropes.
  • the azeotrope or azeotropes may be selected from any suitable azetrope known in the art, for example, from those described in the CRC Handbook of Chemistry and Physics.
  • each solution is measured through surface sensing (above the reservoir) with the pyrometer which generated the pyrometer a voltage versus time plot.
  • the temperature remained constant during the phase change, which can be detected with the pyrometer as a change in slope.
  • the pyrometer voltage minima can be identified and used to calibrate the pyrometer against the known boiling point for the reference solution. This method yields at least two reference points with which to calibrate the pyrometer. More reference points may be obtained by using more than two reference solutions.
  • the microfluidic device can be prefabricated with two calibration chambers (reservoirs), each chamber being filled with a different reference solution.
  • the calibration process would be the same where each of the chamber is heated and the boiling point is determined. This prefabricated device saves the user of having to prepare and flow the reference fluids into the chamber for calibration.
  • the calibration substance is a solid.
  • a solid with a flash point at a certain temperature may be used as one or more of the calibration substances, with the flash point acting as a reference temperature.
  • the solids are selected from the alkali metals and alkaline earth metals.
  • the calibration substance is a gas that undergoes a pseudostate change at a specific temperature, with the pseudostate change acting as a reference temperature.
  • Any gas with a detectable pseudostate transition at a suitable temperature may be used as a calibration substance of the present invention.
  • the gas may undergoes a pseudostate transition from clear to cloudy or from cloudy to clear at the reference temperature.
  • a simple, effective, and robust temperature sensing method which, together with IR-mediated heating, enables completely non-contact temperature control for performing PCR in microfluidic devices.
  • Thermal modeling is used to define physical properties of the device and environment needed to achieve this rapid equilibration, and the model verifies an experimentally- observed thermal equilibration of the device during the initial PCR thermal cycles.
  • calibrating the surface temperature relative to the PCR solution temperature is accomplished using the boiling point of water and an azeotrope within the chip.
  • the photomask design consisted of two ellipses, each 0.75 mm wide and 3 mm long. The two ellipses were separated by 1.5 mm (from one center of the ellipse to the other center). Two channels, each impinging on the end of each ellipse, were used for filling the ellipses with PCR solution and insertion of the thermocouple. Etch depths were either 200 ⁇ m deep or 100 ⁇ m deep producing PCR reaction volumes of 550 nL and 230 nL, respectively. Thermocouple and Pyrometer PCR Setups
  • the PCR setup was similar to systems described by Easley et al. ⁇ Lab Chip 2006, 6, 601-610, which is incorporated herein by reference) and Legendre et al. ⁇ Anal Chem. 2006, 78, 1444-1451 , which is incorporated herein by reference). Briefly, the microdevice was mounted on a Plexiglas stage that allowed access to the infrared radiation and convective cooling from the bottom of the stage. Two solid state relays (CMX60D10, Crydom Corp., San Diego, CA, USA) were placed in series with a 12 V power supply to power both the fan and the tungsten lamp (CXR, 8 V, 50 W, General Electric, Cleveland, OH, USA).
  • CMX60D10 Crydom Corp., San Diego, CA, USA
  • the pyrometer (MI-N5, Mikron Infrared, Inc., Oakland, NJ, USA) was oriented 45° from vertical above the microdevice and a red diode laser within the pyrometer was used to align the sensing area of the pyrometer onto the surface of the device above the PCR chamber ( Figure 1).
  • the microdevice was placed on the stage and aligned with both the pyrometer sensing area and the focal spot of the tungsten lamp, the physical location of the microfluidic chip was recorded using double-sided tape.
  • This voltage was recorded using a data acquisition (DAQ) card (PCI- 6014, National Instruments, Austin, TX, USA).
  • DAQ data acquisition
  • PCI- 6014 National Instruments, Austin, TX, USA.
  • the pyrometer voltage output was fed into the same DAQ card using a 1 k ⁇ resistor across the current output of the pyrometer.
  • thermocouple The microdevice and thermocouple were then placed on a conventional PCR thermal cycler (GeneAmp 2400 PCR System, Perkin-Elmer, Wellesley, MA, USA) and the temperature of the conventional cycler was increased in increments of 5 0 C and held for 30 s at each increment.
  • the average thermocouple voltage versus temperature of the conventional cycler was used to calibrate the thermocouple.
  • the pyrometer was calibrated by placing the thermocouple within the microdevice and the IR-lamp used to perform 5 mock PCR cycles with 15 s hold times at 95, 60, and 72 0 C.
  • the average pyrometer output voltage at the average temperature recorded by the thermocouple was used for calibration. When reporting the data for the pyrometer or thermocouple, all values are average + one standard deviation.
  • PCR Procedure Microdevices were rinsed with acetone, dried, and passivated with 10 ⁇ L SigmaCote (Sigma-Aldrich, St. Louis, MO) prior to making PCR mastermix solutions. All PCR reagents were from Sigma-Aldrich and primers were from MWG Biotech, Inc. (High Point, NC, USA).
  • Protocol for amplification of a 211 bp fragment of the virulence B gene on pXOl of the anthrax genome consisted of a 60 s initial denaturation at 95 0 C, followed by 30 cycles of 95 0 C for 5 s, 62 0 C for 5 s, and 72 0 C for 5 s. A final extension at 72 0 C was performed for 30 s. Control amplifications for each of the thermal cycling protocols were performed without template DNA and are shown in the appropriate figures.
  • amplified product was removed from the device, diluted with 24 ⁇ L of water, and 0.8 ⁇ L of a PCR marker (N3234S, New England BioLabs, Inc., Ipswich, MA, USA). This mixture was then separated on a commercial capillary electrophoresis instrument using laser induced fluorescence detection as described by Legendre et al. Sizing results of amplified PCR products are given as average + standard deviation as determined by comparison of the migration time of the amplified product to the migration time of the sizing ladder.
  • thermopile a collection of thermocouples in series.
  • the basis for using a pyrometer as a temperature sensor is that as an object is heated, the blackbody radiation from the object shifts to shorter wavelengths which fall into the range that can be detected by the pyrometer.
  • the pyrometer is composed of a thermopile situated behind a Ge lens which is transparent to wavelengths between 8-14 ⁇ m.
  • the solution in the PCR chamber is heated by the IR radiation from the tungsten lamp, the glass above the chamber is also heated producing blackbody radiation that is detected by the pyrometer and recorded as a change in the output voltage of the thermopile.
  • Output voltages of the thermopile were calibrated to temperatures using either a thermocouple, or the boiling points of reference solutions and used to successfully control PCR amplification in microdevices.
  • the model also defined several parameters that could minimize this time lag - among these were fabrication of a device with a thin cover plate (the thinner the top plate, the more accurately the two temperatures would correlate), a material with a large thermal diffusivity, and performing the experiments in an environment with a small convective heat transfer coefficient (e.g., in a pre-heated chamber).
  • the use of the pyrometer for these cases is currently under investigation.
  • T 5 is the set temperature
  • T is the temperature signal being measured.
  • the control system was then tested for its ability to reach a set temperature of 72 0 C and maintain that temperature. As can be seen in the figure, the set temperature was reached rapidly without overshooting and was maintained without significant ringing at 72.18 ⁇ 0.05 0 C.
  • the system was subsequently tested for its ability to carry out a mock PCR cycling (94 0 C, 15 s; 3 cycles of 60 0 C for 2 s, 72 0 C for 3 s, and 94 0 C for 3 s; 72 0 C). As shown in FIG.

Landscapes

  • Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne des procédés et un appareil pour la commande thermique et/ou de mesure de température de fluides sans contact dans un dispositif microfluidique. L'appareil utilise un capteur de température à distance contenu dans un pyromètre étalonné pour mesurer la température d'un petit volume contenu dans un récipient à l'intérieur du dispositif microfluidique. La présente invention concerne également des procédés et un appareil pour mesurer la température de l'échantillon dans la réalisation de thermocyclage sans contact (à distance) sur des échantillons de petit volume (de l'ordre du microlitre au nanolitre), chaque cycle étant complété dans un intervalle de temps de l'ordre de quelques secondes. La présente invention concerne en outre des procédés d'étalonnage de pyromètres mettant en œuvre certaines substances d'étalonnage.
PCT/US2007/088662 2006-12-21 2007-12-21 Commande thermique sans contact de petit volume et appareil associé correspondant WO2008080106A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US87640106P 2006-12-21 2006-12-21
US60/876,401 2006-12-21

Publications (1)

Publication Number Publication Date
WO2008080106A1 true WO2008080106A1 (fr) 2008-07-03

Family

ID=39562951

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/088662 WO2008080106A1 (fr) 2006-12-21 2007-12-21 Commande thermique sans contact de petit volume et appareil associé correspondant

Country Status (1)

Country Link
WO (1) WO2008080106A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011075568A1 (fr) * 2009-12-18 2011-06-23 Waters Technologies Corporation Appareil de détection thermique de l'écoulement et méthode de chromatographie liquide à haute performance
US20140295441A1 (en) * 2013-03-27 2014-10-02 Zygem Corporation Ltd. Cartridge interface module
WO2014178964A1 (fr) * 2013-04-29 2014-11-06 Waters Technologies Corporation Appareil et procédés de commande de la température d'une colonne de chromatographie
CN108614600A (zh) * 2018-07-13 2018-10-02 中国科学院天津工业生物技术研究所 一种高精度芯片反应系统及方法
CN110487445A (zh) * 2019-03-22 2019-11-22 中国计量科学研究院 一种pcr仪温度校准装置的校正装置及校正方法
US11207677B2 (en) 2018-03-07 2021-12-28 University Of Virginia Patent Foundation Devices, systems, and methods for detecting substances
DE102020209296A1 (de) 2020-07-23 2022-01-27 Wilhelm Bruckbauer Verfahren und Einrichtung zum Kalibrieren eines Temperatursensors

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5576218A (en) * 1994-01-11 1996-11-19 Abbott Laboratories Method for thermal cycling nucleic acid assays
US20030113907A1 (en) * 2001-12-18 2003-06-19 Roberts Peter C. Electro-chemical analysis device with integrated thermal sensor and method for monitoring a sample using the device
US20050287661A1 (en) * 2002-10-08 2005-12-29 James Landers Methods and systems for multiplexing ir-mediated heating on a microchip

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5576218A (en) * 1994-01-11 1996-11-19 Abbott Laboratories Method for thermal cycling nucleic acid assays
US20030113907A1 (en) * 2001-12-18 2003-06-19 Roberts Peter C. Electro-chemical analysis device with integrated thermal sensor and method for monitoring a sample using the device
US20050287661A1 (en) * 2002-10-08 2005-12-29 James Landers Methods and systems for multiplexing ir-mediated heating on a microchip

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011075568A1 (fr) * 2009-12-18 2011-06-23 Waters Technologies Corporation Appareil de détection thermique de l'écoulement et méthode de chromatographie liquide à haute performance
US8943887B2 (en) 2009-12-18 2015-02-03 Waters Technologies Corporation Thermal-based flow sensing apparatuses and methods for high-performance liquid chromatography
US9429548B2 (en) 2009-12-18 2016-08-30 Waters Technologies Corporation Flow sensors and flow sensing methods with extended linear range
US20140295441A1 (en) * 2013-03-27 2014-10-02 Zygem Corporation Ltd. Cartridge interface module
US10563253B2 (en) 2013-03-27 2020-02-18 Lockheed Martin Corporation Cartridge interface module
WO2014178964A1 (fr) * 2013-04-29 2014-11-06 Waters Technologies Corporation Appareil et procédés de commande de la température d'une colonne de chromatographie
US20160038853A1 (en) * 2013-04-29 2016-02-11 Waters Technologies Corporation Apparatus and methods for controlling the temperature of a chromatography column
US11207677B2 (en) 2018-03-07 2021-12-28 University Of Virginia Patent Foundation Devices, systems, and methods for detecting substances
CN108614600A (zh) * 2018-07-13 2018-10-02 中国科学院天津工业生物技术研究所 一种高精度芯片反应系统及方法
CN108614600B (zh) * 2018-07-13 2024-04-30 中国科学院天津工业生物技术研究所 一种高精度芯片反应系统及方法
CN110487445A (zh) * 2019-03-22 2019-11-22 中国计量科学研究院 一种pcr仪温度校准装置的校正装置及校正方法
DE102020209296A1 (de) 2020-07-23 2022-01-27 Wilhelm Bruckbauer Verfahren und Einrichtung zum Kalibrieren eines Temperatursensors

Similar Documents

Publication Publication Date Title
US6210882B1 (en) Rapid thermocycling for sample analysis
US6586233B2 (en) Convectively driven PCR thermal-cycling
Lagally et al. Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system
US8263392B2 (en) Methods and compositions related to continuous flow thermal gradient PCR
Hühmer et al. Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes
Kricka et al. Microchip pcr
Roper et al. Infrared temperature control system for a completely noncontact polymerase chain reaction in microfluidic chips
WO2008080106A1 (fr) Commande thermique sans contact de petit volume et appareil associé correspondant
US20080176289A1 (en) System and method for rapid thermal cycling
US20050287661A1 (en) Methods and systems for multiplexing ir-mediated heating on a microchip
Erill et al. Development of a CMOS-compatible PCR chip: comparison of design and system strategies
Ragsdale et al. A disposable, continuous-flow polymerase chain reaction device: Design, fabrication and evaluation
AU2014357646B2 (en) Non-contact infrared thermocycling
WO1999039120A1 (fr) Commande de fluide par expansion thermique de dispositifs microfluidiques
US20040072334A1 (en) Thermal cycler
Chen et al. Using an IR lamp to perform DNA amplifications on an oscillatory thermocycler
US20040131504A1 (en) Remote temperature sensing of small volume and related apparatus thereof
Lee et al. Miniaturization of polymerase chain reaction
Chien et al. A micro circulating PCR chip using a suction-type membrane for fluidic transport
Crews et al. Thermal gradient PCR in a continuous-flow microchip
US20090209030A1 (en) Thermal Cycler
Ghalekohneh et al. Novel Fabrication Method of a Microfluidic Continuous Flow PCR
Lee et al. A new thermal cycling mechanism for effective polymerase chain reaction in microliter volumes
Zheng et al. Continuous-flow submicroliter-scale PCR chip for DNA amplification
Lagally et al. Monolithic integrated PCR reactor-CE system for DNA amplification and analysis to the single molecule limit

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07865986

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07865986

Country of ref document: EP

Kind code of ref document: A1