Variable temperature reactor, heater and control circuit for variable temperature reactor

Abstract

A variable temperature reactor for receiving a predetermined reaction therein is described. The reactor includes a reaction cell, a heater, and a heat sink. The reaction cell has a thickness H V and width W V where W V >4H V and one of the faces of the larger area of ​​the reaction volume has thickness H W The reactor volume is defined by a surface bounded by an outer wall having a heater in contact with the outer wall. The heater comprises a heat-generating heating element disposed on a surface closer to the reaction volume and a heater support on an opposite surface. The heater support is in contact with a heat sink, and the heater support provides a thermal resistance R between the heating element and the heat sink. T The reactor has a thermal diffusion coefficient D V When the material is filled with a reagent having a diffusion time t V and t V =H V 2 / D V t V is the reaction time constant t R The outer wall has a thermal diffusion coefficient of D W and the thermal diffusion time is t W =H W 2 / D W <t V is.

Description

[Technical Field]

[0001] The present invention relates to a reactor for a reaction requiring rapid temperature adjustment. The present invention also relates to a heater for the reactor, as well as drive and sensing circuits, and a corresponding method for operating the reactor.

[0002] One example of a process requiring such a reactor is DNA amplification by polymerase chain reaction (PCR), where the reactor is suitable for rapid thermal cycling to shorten the time to complete the PCR. Another example is DNA sequencing by synthesis, where the addition of bases can be optimized by adjusting the temperature at each step of the multi-step reaction.

[0003] The reactor of the present invention is also suitable for monitoring the progress of PCR by calorimetry and detection of oligonucleotides in solution. The reactor is also suitable for digital PCR or multiplex PCR, which involves independent analysis of subsamples at multiple locations within a continuously connected liquid sample.

[0004] The present invention also relates to a heater that rapidly adjusts the temperature of a reaction volume and features high temperature uniformity and precise temperature control.

[0005] The present invention also relates to an apparatus for differential scanning calorimetry (DSC) or differential thermal analysis (DTA) that uses a sample heater and a reference heater configured within a Wheatstone bridge circuit. [Background technology]

[0006] PCR requires repeated temperature cycles between approximately 60°C and 95°C. Traditionally, heating and cooling have been performed using a Peltier element; when the temperature needs to be raised, heat is transferred from the heatsink to the sample, and when the temperature needs to be lowered, heat is transferred from the sample to the heatsink. The heatsink is often cooled by a fan.

[0007] This approach has many drawbacks, including the large size and high power consumption of the required equipment. The heat capacity of the parts of the equipment that change the temperature during thermal cycling is significantly larger than that of the sample, increasing energy consumption and slowing down the thermal cycling process. The rate of temperature increase is limited, and the thermal cycling time is extended by the long thermal diffusion time through the Peltier element, the components used to thermally contact and contain the sample, and the sample itself. These factors result in slow and energy-inefficient thermal cycling in PCR.

[0008] Traditionally, thermocyclers use Peltier elements and temperature sensors located away from the sample for control. Inevitably, there is a delay in the thermal response of the temperature sensors compared to the Peltier elements, which complicates the thermal control of the system. While tuned PID algorithms are often used to maximize the rate of temperature rise and minimize overshoot, these can be complex to develop and implement, and may not achieve the intended time and temperature profiles within the sample.

[0009] A further drawback is that the lifespan of the Peltier element is limited due to the mechanical stress resulting from repeated thermal cycling.

[0010] These drawbacks also apply to other reactions that require precise temperature control and rapid temperature changes.

[0011] An alternative conventional approach to thermal cycling involves moving the sample between water baths or heaters at a fixed temperature. Three examples are shown in Figures 1-3. In Figure 1, the sample is transferred between water baths, and in Figure 2, the sample is transferred between heater blocks. These solutions involve significant mechanical complexity and require a large amount of space due to the moving parts. In Figure 3, the sample is passed through a microfluidic chip containing a meandering path over two heater blocks, but this has the disadvantage of exposing the sample to a large surface area of ​​the microfluidic channel. This has two disadvantages: the sample and reagents may adhere to the channel walls, reducing sensitivity; the sample volume requirements increase; reagent costs increase; and the size and cost of the microfluidic chip also increase.

[0012] The large size and surface area of ​​the long, meandering channels shown in Figure 3 are due to the need for the channels to pass between two different temperature zones that are spatially separated by a distance of typically more than 1 mm. Furthermore, since the sample volume is smaller than the internal volume of the meandering channels, the ratio of the channel's surface area to the sample volume becomes large.

[0013] It is desirable to perform thermal cycling at a sufficiently fast rate so that the time required for temperature change does not account for the majority of the total thermal cycling time. The total thermal cycling time is the sum of the time for temperature change and the reaction time, and the longest part of the PCR reaction is the extension phase, which requires at least 1 second for a typical sequence length of 100 base pairs. Therefore, the target time for temperature rise should be less than 1 second. Since the target temperature for PCR is usually between 60°C and 95°C, achieving a temperature rise rate of 70°C / s or higher for heating and cooling is necessary to reduce the time for temperature change to 1 second. The total time required is governed by the reaction time, not the time required for temperature change, so the speed advantage is limited at much higher temperature rise rates (above 200°C / s). [Overview of the project]

[0014] In this invention, the inventors disclose a reactor for performing high-speed thermal cycling at approximately 100°C / second, which does not have the drawbacks of a mechanical transfer system or a long meandering channel. Compared to the case of a meandering channel, it is possible to reduce the surface area of ​​the sidewalls by containing the sample in a wide channel. When using a wide channel, it may be necessary to introduce internal support structures such as ribs or pillars to avoid large unsupported spans and to increase the mechanical strength of the fluid channel.

[0015] In addition, the present invention discloses a reactor including a serpentine channel. While some serpentine channels have drawbacks as described above, another approach allows for the inclusion of the sample in a shorter serpentine channel placed on a single heater whose temperature can be varied over time. In this case, the sample does not flow between different temperature zones during amplification, but rather the heater temperature varies over time. This makes it possible to shorten the length of the serpentine channel, make the internal volume of the channel equal to the volume of the sample, and reduce the ratio of the channel's surface area to the sample's volume. A further advantage is that this configuration is convenient for quantitative PCR experiments, in which the concentration of amplified DNA can be monitored over time at a single location using, for example, a fluorescent probe or insertion dye.

[0016] A reactor comprises three main elements: a reaction cell containing a volume to be temperature-controlled, a heat sink that absorbs heat, and a heater that is in thermal contact with the reaction cell and heat sink. The heater has an exothermic heating element on the side in contact with the reaction cell and a heater support on the side in contact with the heat sink. The thermal resistance of the heater support is selected to provide a low thermal cycle time for a given set of target reaction temperature, heat sink temperature, and power requirements.

[0017] In one embodiment, the present invention provides a variable temperature reactor for accepting a predetermined reaction, the reactor comprising a reaction cell, a heater, and a heat sink, The reaction cell has a thickness of H V and width WV has a reaction volume, where W V > 4H V is such that one of the faces of the reaction volume with a larger area is defined by a face bounded by an outer wall having a thickness H W and is bounded by a face bounded by an outer wall having a thickness H The heater is in contact with the outer wall In this case, the heater includes a heating heater element disposed on a face closer to the reaction volume and a heater support on the opposite face. Since the heater support is in contact with the heat sink, the heater support provides a thermal resistance R between the heater element and the heat sink T to provide In this case, when the reactor is filled with a reagent having a thermal diffusivity D V it has a diffusion time t in the thickness direction V having, t V = H V 2 / D V where t V is the reaction time constant t R and is smaller than The outer wall has a thermal diffusivity D W and has a thermal diffusion time t W = H W 2 / D W ​​​​​​​​​​​​​​​​​​​​​​​​​​​V and length L V Width W is formed by a meandering channel located within a region having V and length L V It includes a reaction volume having the following characteristics.

[0022] Preferably, the reaction volume is the thickness H W The large surface area is bordered on both sides by an outer wall having a certain feature, the heater is in contact with both of the outer walls, and the heat sink is in contact with both heaters.

[0023] Preferably, the heater element is a resistive heating element.

[0024] Preferably, the heater element is made from a conductive material, and the heater support is made from an electrically insulating material.

[0025] Preferably, the heater is separable from the heatsink.

[0026] Preferably, the reactor has a thermal resistance R between the heater element and the heat sink. T The following relationship R T >(T HIGH -T Sink ) / p Heat and 0.5R T,Opt <R T <2R T,Opt Here, R T,Opt =( T HIGH +T LOW -2T Sink ) / p Heat Selected to satisfy the following conditions, The reactor has an output of p Heat A heater and temperature control with Sink Using a heatsink, lower temperature T LOW and higher temperature T HIGH It is configured to repeatedly cycle between and

[0027] Preferably, the reactor has a heater support with a thermal resistance RT The sum of the heat capacity of the filled reaction volume and the heat capacity of the thin outer wall portion located between the reaction volume and the heater element is C. V R T C V <t R The relationship satisfies, where t R It is configured to be the time constant of the reaction.

[0028] Preferably, the reactor has a heat capacity of the filled reaction volume and a heat capacity between the reaction volume and the heater element. The total heat capacity of the thin outer wall section located at this location C V And the heat capacity C of the heatsink S However, relationship C S / C V It is configured to satisfy >100.

[0029] Preferably, the thermal conductivity of the heat sink material is more than 10 times that of the heater support material.

[0030] Preferably, the heat capacity of the thin outer wall portion located between the reaction volume and the heat sink, and the heat capacity of the heater, are lower than the heat capacity of the liquid in the reaction volume.

[0031] Preferably, the thermal permeability of the heat sink material is more than 10 times that of the heater support material, where the thermal permeability e is the thermal conductivity k, density ρ, and specific heat capacity c of the material. p It is a function of and e = sqrt(kρc p ) is defined as.

[0032] Preferably, the heater element extends across the entire region of the reaction volume.

[0033] Preferably, the heater element is resistant and has a rectangular or square shape.

[0034] Preferably, the heater element is manufactured from a conductive material having an absolute value of a temperature coefficient of resistance greater than 500 ppm / K, preferably greater than 2,500 ppm / K, and more preferably greater than 10,000 ppm / K, over the operating temperature range of the heater.

[0035] Preferably, the heater includes a Kelvin contact for measuring electrical resistance.

[0036] Preferably, the heater element is configured such that the heat output per unit area is higher near its periphery than near its center.

[0037] Preferably, the heater element comprises a main heater surrounded by one or more guard heaters.

[0038] Preferably, the main heater is elongated in the direction of current flow, and in this case, the guard heater is positioned adjacent to the long side of the main heater.

[0039] Preferably, the sheet resistance of the heater element increases locally in the end zone located at the edge of the heater element perpendicular to the direction of current flow.

[0040] Preferably, the reaction volume is configured to contain reagents used for polymerase chain reaction (PCR) amplification of nucleic acid sequences during use.

[0041] Preferably, the reactor is configured to perform DNA amplification by polymerase chain reaction (PCR) thermal cycling. [Brief explanation of the drawing]

[0042] Next, an example of the present invention will be described with reference to the attached drawings.

[0043] [Figure 1] This figure shows a system and method of the prior art. [Figure 2] This figure shows a system and method of the prior art. [Figure 3]This figure shows a system and method of the prior art. [Figure 4] This figure shows a system and method of the prior art. [Figure 5] This figure shows a system and method of the prior art. [Figure 6] This figure shows a system and method of the prior art. [Figure 7] This figure shows a system and method of the prior art. [Figure 8A] This figure shows an exemplary reactor according to the present invention. [Figure 8B] This figure shows an exemplary reactor according to the present invention. [Figure 9A] This figure shows an exemplary reactor according to the present invention. [Figure 9B] This figure shows an exemplary reactor according to the present invention. [Figure 10A] This figure shows an exemplary reactor according to the present invention. [Figure 10B] This figure shows an exemplary reactor according to the present invention. [Figure 11] This figure shows an exemplary reactor according to the present invention. [Figure 12] This is a cross-sectional view of the geometric shape of the alternative reactor according to the present invention. [Figure 13A] This figure shows an example of a reaction cell cover that can be used in the present invention. [Figure 13B] This figure shows an example of a reaction cell cover that can be used in the present invention. [Figure 14A] This figure shows an example of a reaction cell cover that can be used in the present invention. [Figure 14B] This figure shows an example of a reaction cell cover that can be used in the present invention. [Figure 15] This is an exploded assembly diagram of the reactor according to the present invention. [Figure 16] This figure shows two types of reaction cells used in the present invention. [Figure 17]This figure shows a simulation of simultaneous DNA amplification and diffusion up to 60 seconds during the operation of the present invention, and the subsequent DNA diffusion after 60 seconds have elapsed. [Figure 18] This figure shows the DNA concentration 100 seconds after the start of PCR amplification for two exemplary cells used in the present invention. [Figure 19] This figure shows the concentration fluctuation in the method using the present invention, with the position at 100 seconds after the start of PCR amplification. [Figure 20] This figure shows a fluid cell array that can be used in the present invention. [Figure 21] This figure shows a fluid cell array that can be used in the present invention. [Figure 22A] This figure shows a fluid cell array that can be used in the present invention. [Figure 22B] This figure shows a fluid cell array that can be used in the present invention. [Figure 23A] This figure shows the reaction volume according to the present invention. [Figure 23B] This figure shows the reaction volume according to the present invention. [Figure 23C] This figure shows the reaction volume according to the present invention. [Figure 24A] This figure shows the reaction volume according to the present invention. [Figure 24B] This figure shows the reaction volume according to the present invention. [Figure 25] This figure shows an example of a heater according to the present invention. [Figure 26] This figure shows an example of a heater according to the present invention. [Figure 27] This figure shows an example of a heater according to the present invention. [Figure 28] This figure shows an example of a heater according to the present invention. [Figure 29A] This figure shows an example of a heater according to the present invention. [Figure 29B] This figure shows an example of a heater according to the present invention. [Figure 29C] This figure shows an example of a heater according to the present invention. [Figure 29D] This figure shows an example of a heater according to the present invention. [Figure 30] This figure shows an example of a heater according to the present invention. [Figure 31] This figure shows a heat sink that can be used in the present invention. [Figure 32] This graph shows the operating characteristics and output characteristics of the present invention during the reaction. [Figure 33] This graph shows the operating characteristics and output characteristics of the present invention during the reaction. [Figure 34] This graph shows the operating characteristics and output characteristics of the present invention during the reaction. [Figure 35] This graph shows the operating characteristics and output characteristics of the present invention during the reaction. [Figure 36] This is another graph illustrating the operating characteristics of the present invention. [Figure 37] This is another graph illustrating the operating characteristics of the present invention. [Figure 38] This is another graph illustrating the operating characteristics of the present invention. [Figure 39] This is a diagram showing the circuit according to the present invention. [Figure 40] This is a diagram showing the circuit according to the present invention. [Figure 41] This is a diagram showing the circuit according to the present invention. [Modes for carrying out the invention]

[0044] Figure 1 (prior art) shows thermal cycling by moving a sample between water baths maintained at different temperatures. The apparatus is shown in schematic form in (A). The temperature and position of the sample are plotted against time in (B). Reference: Farrar, JSand Wittwer, CT (2015) Extreme PCR: Efficient and Specific DNA Amplification in 15-60Seconds, Clinical Chemistry, 61:1 145-153.

[0045] Figure 2 (prior art) shows thermal cycling by moving a sample between heater blocks held at different temperatures. See International Publication No. 2012161566.

[0046] Figure 3 (conventional technique) shows thermal cycling by passing the sample through a meandering path that alternately passes through high-temperature and low-temperature zones. Reference: Trauba, J.Mand Wittwer, CT (2017) Microfluidic Extreme PCR: <1 Minute DNA Amplification in a Thin Film Disposable, J.Biomedical Science and Engineering, 10, 219-231.

[0047] Figure 4 (conventional technique) shows digital PCR by creating droplets of the sample contained in an immiscible liquid, usually a fluorinated oil. Reference: Hindson, BJ (2011) High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number, Anal. Chem, 83, 8604-8610.

[0048] Figure 5 (prior art) shows a top and side view of a slip-chip device. The slip-chip uses the relative motion of two parts to create separated subsamples for digital PCR or multiplex PCR. Reference: U.S. Patent No. 9,415,392B2.

[0049] Figure 6 (Conventional Technology) illustrates the digitization of digital PCR by first filling with a sample liquid, and then filling with a gas that can pass through a gas permeable barrier and divide the sample into detached portions. Reference: International Publication No. 2018094091.

[0050] Figure 7 (prior art) shows two repeated DSC scans of a 0.0244 mM PNA(TG) / DNA bilayer, a 0.0303 mM DNA(TG) / DNA bilayer, and a buffer solution. The temperature scanning rate was 60 K / h, and the cell volume was 0.511 ml. Reference: Chakrabarti, MC and Schwarz, FP (1999) Thermal stability of PNA / DNA and DNA / DNA Duplexes by differential scanning calorimetry, Nucleic Acids Research, 24, 4801-4806.

[0051] In the type of reaction to which the present invention is employed, thermal cycling requires shortening the thermal diffusion time between the heater and the sample compared to the target cycle time. The thermal diffusion time t is given by the following equation: t=R 2 / D T , Here, R is a characteristic length scale, and D T is the thermal diffusivity coefficient of the material. Table 1 below shows an example of the reactor design described below, where the reaction volume and thin outer layer are used. The thermal diffusion time at the wall is shorter in both cases than the PCR reaction time, which is considered to be about 1 second for a 100-base-pair sequence.

[0052] To heat efficiently, it is desirable to minimize the ratio of the heat capacity of the reaction cell material to the heat capacity of the heater relative to the heat capacity of the sample. The heat capacity of the reaction volume must be greater than the majority of the sum of the heat capacity of the reaction cell and the heat capacity of the heater, preferably 10%, and more preferably 50%.

[0053] To heat the reaction volume, the heater element is switched on, and the heat generated by the heater element flows into the reaction volume and the heat sink. To cool the reaction volume, the heater element is switched off, and heat flows from the reaction volume through the heater to the heat sink. The heat capacity of the heat sink is selected to be much larger than the combined heat capacity of the reaction cell and heater in order to limit the temperature rise of the heat sink over the series of thermal cycles required to complete the reaction. In the example provided below, the heat capacity of the heat sink is more than 100 times the combined heat capacity of the heater and reaction cell. To further limit the temperature rise of the heat sink, the heat sink can be continuously cooled, for example, by natural or forced convection, liquid circulation, spray cooling, or by connection to a heat pipe or Peltier device.

[0054] Given temperature profile and heatsink temperature T Sink and heater element output p Heat To minimize the thermal cycling time related to the heater support R T The thermal resistance can be optimized. LOW and T HIGH The time required for thermal cycling between heating and cooling is minimized when the heating time is equal to the cooling time, and this condition is R T =R T,Opt In this case, the following conditions are met: R T,Opt =( T HIGH +T LOW -2T Sink ) / p Heat

[0055] The axial direction is defined as being perpendicular to the contact surface between the heater and the reaction cell, and the lateral direction as being within the plane of the contact surface between the heater and the reaction cell. Since lateral heat flow implies a temperature gradient and temperature non-uniformity within the reaction cell, it is desirable to limit lateral heat flow to reduce the accuracy of temperature control.

[0056] To limit lateral heat flow, the reaction volume is set such that the area of ​​the side is smaller than the area of ​​the surface closest to the heater element, with height HV (Axis direction) and width W V The ratio is selected to have a (horizontal) ratio. This condition is met for square or circular reaction volumes as follows: W V >4H V

[0057] To further restrict lateral heat flow, heater elements may be designed to have higher thermal output near their edges and to extend beyond the reaction volume. The higher thermal output of the heater elements near their edges can compensate for lateral heat flow and provide more uniform temperature conditions throughout the reaction volume.

[0058] An electric heater element may have a heating region in the shape of a square or rectangle, with electrical terminals on two opposite sides of the heating region, and a direction in which current flows from one terminal to the other. One approach to improving the temperature uniformity of such a heater element is to increase the sheet resistance of the heater element near the sides of the electrical terminals so that the heat output per unit area increases locally near these sides compared to the heat output near the center of the heater element. To further improve temperature uniformity, it is also desirable to increase the heat output of the heater element along the sides parallel to the direction of current flow. This increases the current density near these sides, and thus increases the heat output per unit area compared to the heat output near the center of the heater element. This can be achieved by locally reducing the sheet resistance near these sides.

[0059] The resistance of a heater element can be locally increased by reducing the thickness of the material, or by patterning the material with holes, slots, or material variations that have the effect of increasing the length of the electrical conduction path and decreasing the cross-sectional area of ​​the electrical conduction path. These hole or slot features must be small compared to the thickness of the reaction volume so as not to interfere with temperature uniformity within the reaction volume.

[0060] By using guard heaters, lateral heat flow at the edges of the heater can be further restricted. A guard heater is an additional heater positioned near the edges of the heater element and driven to maintain a temperature close to the target temperature of the main heater element. The heat output per unit area of ​​the guard heater is higher than that of the main heater element to compensate for lateral heat losses. The guard heater may operate in closed-loop control with the same temperature setpoint as the main heater element, or it may operate with the same controller or on / off timing as the main heater element but with a different drive voltage that can be adjusted to optimize temperature uniformity with a specific temperature setpoint.

[0061] The reactor can be configured as a single-sided cell or a double-sided cell. A single-sided configuration has a reaction cell with one thin outer wall in contact with the heater, and the heater is in contact with a heat sink. A double-sided configuration has a reaction cell with two thin outer walls in contact with the heater, and the heater is in contact with a heat sink.

[0062] The reaction cell, heater, and heat sink can take on a planar or curved shape. A planar shape may be preferred for ease of construction and for optical monitoring of the reaction. However, other shapes such as partially spherical or cylindrical are also possible, and these may have the advantage of allowing the tensile, flexible reaction cell and heater layers to have good thermal contact with each other and with the heat sink, which is usually a rigid metal component. [Table 1]

[0063] Thermal control of a heater element using spatially isolated temperature sensors presents challenges due to the time lag between temperature changes in the heater element and temperature changes in the temperature sensor. This time lag can cause problems such as overshoot and temperature fluctuations in the heater element. To avoid these problems, the heater element may be configured as a temperature sensor that determines its temperature using the resistance of the heater element. The heater element may have a positive temperature coefficient of resistance (e.g., metal) or a negative temperature coefficient of resistance (e.g., metal oxide or other dielectric). In either case, it is desirable that the temperature coefficient of resistance (TCR) of the heater element be large, preferably exceeding 500 ppm / K, more preferably exceeding 2,500 ppm / K, and most preferably exceeding 10,000 ppm / K.

[0064] The heater element may be equipped with a four-terminal Kelvin contact to enable more accurate measurement of the heater element's resistance and more accurate temperature measurement.

[0065] If the material of the heater element has a lower sheet resistance than the desired resistance, it may be patterned with slots or gaps perpendicular to the direction of current flow to increase the sheet resistance.

[0066] If the heater element has a higher sheet resistance than the desired resistance, then, in this case, interlocking contact terminals may be provided to shorten the path length through the heater element and increase the cross-sectional area of ​​the current flow.

[0067] It is desirable to monitor the reaction state during thermal cycling and at the end of some thermal cycles. In particular, PCR amplification of DNA can be detected using fluorescently labeled dyes or electrochemically active labels. Each of these methods has its drawbacks, such as the costly labeling requirements, the requirements for complex optical instruments and optical interfaces with the sample, and the requirements for electrochemical interfaces with the sample.

[0068] This invention enables calorimetry and detection of amplified DNA by detecting the heat of melting during the heating phase of a thermal cycle, which results in an increase in heat capacity at the melting temperature. This allows for continuous monitoring of PCR amplification in a label-free manner with minimal additional instrumentation hardware.

[0069] The reactor configuration of the present invention is highly suitable for scanning calorimetry detection because its design allows for a rapid temperature rise, and the heat capacity of the sample contained in the reaction volume accounts for a significant portion of the total heat capacity of the heater, reaction cell, and sample. These factors increase the intensity of the calorimetry signal and improve the sensitivity of the calorimetry detection.

[0070] Differential thermal analysis (DTA) can be used to detect the presence of DNA by the increase in heat capacity at the DNA melting temperature. In this method, the temperature difference between the sample cell and the reference cell is measured as a function of temperature or time while the temperature rises past the melting point with equal heat input to both the sample and the reference. By monitoring the progress of the differential temperature scan over the course of the reaction, changes in the concentration of the reaction product can be detected. Another method uses differential scanning calorimetry (DSC), in which the difference in heat flux input to the sample cell and the reference cell is measured as a function of temperature or time while the temperatures of both the sample cell and the reference cell rise and are kept equal.

[0071] By combining AC calorimetry with the present invention using pulsed or oscillating heater drivers, the robustness and sensitivity of calorimetry can be enhanced. One example approach involves driving a resistive heater with a sinusoidal drive voltage at a drive frequency, generating a thermal output that oscillates at twice the drive frequency. The temperature of the heater element also oscillates at twice the drive frequency, and the amplitude of this oscillation depends on the heat capacity of the sample. The amplitude of the temperature oscillation can be detected by measuring the oscillation of the electrical resistance of the heater element, if the heater is also configured as a temperature sensor with a non-zero temperature coefficient of resistance. The advantage of this approach is that it is not affected by slow DC drift in the heater resistance, and external noise sources can be eliminated by using synchronous detection of the temperature oscillation.

[0072] The optimal frequency range for heat input in AC calorimetry can be estimated by considering the degree of intrusion L of temperature oscillations generated by an oscillatory heat source (see: Marin). ,E.(2010)Characteristic dimensions for heat transfer Lat.Am.J.Phys.Educ.,1,56-60).

number

[0073] D T ω is the thermal diffusivity coefficient of the material through which heat is diffused, and ω is the angular frequency of the vibration of the heat source. In the case of a reactor with a heater on only one side, the height H of the reaction volume is... V It is preferable to select conditions that give the following penetration degree L. The advantage of using higher frequency vibrations and smaller penetration degrees is that the calorimetry is H V This makes it less susceptible to fluctuations, thereby reducing sensitivity to differences in the geometric shape of the sample and the reference reaction volume.

[0074] Condition L≦H V The frequency is 2f DRIVE To generate a thermal output that oscillates, a frequency f is applied to the resistive heater element.DRIVE occurs in the sinusoidal electric drive device, where f DRIVE satisfies the following conditions.

Number

[0075] In the case of a reactor equipped with a heater on only one side, and in the case of a typical reaction volume according to the present invention, the height H V = 100 μm, the thermal diffusivity D of water V = 1.43×10 -7 m 2 / s, and f DRIVE ≧ 2.3 Hz.

[0076] In the case of a reactor equipped with heaters on both sides, the penetration degree L is at most half of the height of the reaction volume, and in the case of a typical reaction volume according to the present invention, the height H V = 100 μm, the thermal diffusivity D of water V = 1.43×10 -7 m 2 / s, and f DRIVE ≧ 9.1 Hz is preferred. Generally, in the case of a reactor equipped with heaters on both sides, f DRIVE satisfies the following conditions.

Number

[0077] Also, the penetration degree L is desirably not less than the thickness H of the thin outer wall in contact with the reaction volume. This gives condition W above.

Number

[0078] In the case of a typical reaction volume according to the present invention, the wall thickness H W = 24 μm, the thermal diffusivity D of the polypropylene wall W = 8.26×10 -8 m 2 / s, and f DRIVEThe frequency is ≤23Hz. Preferably, as referred to herein, “volume” may also mean “container” or “cell” that defines the volume, and vice versa.

[0079] When using the AC calorimetry method, it is advantageous to measure the heater resistance fluctuation at a frequency twice the frequency of the AC electric drive. By supplying a sinusoidal electric drive waveform of frequency ω to the heater element, temperature fluctuations occur at frequency 2ω, and consequently, resistance fluctuations occur at 2ω. This further leads to voltage fluctuations at 3ω. By using the "3-ω" method to measure the voltage fluctuations at a frequency three times the frequency of the AC electric drive, narrowband detection techniques can be used, providing an improved signal-to-noise ratio.

[0080] When using differential calorimetry, it is advantageous to configure electrical resistance heater elements for the sample reaction and reference reaction within a Wheatstone bridge circuit. This approach allows for the direct measurement of the difference between the sample temperature and the reference temperature, and avoids the errors associated with individually detecting the sample temperature and reference temperature and then calculating the small difference between the two relatively large quantities.

[0081] In another configuration of the Wheatstone bridge, the sample and reference are each heated by two heater elements, and the four heater elements are configured in a full bridge configuration, so the voltage output of the bridge circuit is doubled with respect to a given temperature difference.

[0082] The difference in resistance between the sample heater element and the reference heater element can prevent the bridge circuit output from becoming zero, potentially leading to uneven heating of the sample and reference. It is desirable to balance the bridge circuit so that its output is zero when the sample and reference are at the same temperature, and also so that the sample and reference receive the same power input when voltage is applied across the bridge circuit. This combination of voltage and power balancing can be achieved with four trim resistors in series or parallel configuration.

[0083] Digital PCR is a technique in which a sample containing PCR reagents is first divided into subsamples within several separate partitions (e.g., droplets or areas separated by walls), and then all subsamples are subjected to temperature cycling to allow the PCR reaction to proceed.

[0084] The objective is to determine the concentration of one or more target oligonucleotides in the original sample more precisely and accurately than is possible with real-time PCR. Real-time PCR is defined as the number of amplification cycles required for the PCR reaction measurement to exceed a threshold value. T The DNA concentration is quantified by determining the starting concentration of DNA. T It is related to C T The value of becomes smaller.

[0085] Statistical analysis of the number of subsamples in which PCR proceeded successfully allows for a more accurate and precise estimation of DNA concentration in a sample. Subsamples in which PCR proceeds must contain one or more target oligonucleotides, while those in which PCR did not proceed are considered to be without them. Statistical methods require a large number of subsamples to accurately measure concentrations; typically, more than 1,000 subsamples are used, and more than 10,000 subsamples are used in applications requiring higher sensitivity.

[0086] A major drawback of existing digital PCR techniques is the need to first split the sample into subsamples. This requirement significantly increases the complexity and hardware costs associated with the process. Droplet digital PCR requires the generation of microfluidic droplets using more complex reaction cells, precisely controlled flow of aqueous samples and immiscible oils, and additional time for droplet generation before PCR amplification. Alternatively, gas can be used to split the reaction volume instead of droplet digital PCR. This requires controlled addition of sample, followed by controlled addition of gas, and the use of a gas-permeable barrier to allow the gas to escape.

[0087] The present invention provides a wall-free, droplet-free digital PCR system and method in which the sample is not divided into separate subsamples. Instead, PCR proceeds (or does not proceed) within several reaction zones within the sample, and these reaction zones are defined only by the temperature range they experience. There are no physical barriers between the reaction zones, and the movement of oligonucleotides from one reaction zone to another is inhibited only by the relatively slow diffusion of oligonucleotides in solution. The sample is contained as a continuous series of liquids with no solid, immiscible liquid, or gas barriers between the subsamples. This is made possible by allowing PCR to be performed with short cycle times, resulting in 30–50 cycles of PCR amplification being completed in less than 100–200 seconds, limiting the time for diffusion and the distance over which diffusion may occur.

[0088] In variations of this approach, partial barriers may be introduced to define the positions of subsamples to facilitate the detection and analysis of subsequent reactions, and to reduce the diffusion rate between adjacent subsample positions, thereby allowing for more precise separation of subsamples, and increasing the number of subsamples relative to the reactor size and total amount of sample.

[0089] The diffusion coefficient D of a double-stranded DNA molecule in water having a length [bp size] DNA This is expressed by the following formula (see: Lukacs, Glet al(2000) Size-dependent DNA mobility in cytoplasm and nucleus.J This can be calculated using Biol Chem. 275(3):1625-9). D DNA =[bp size] -0.72 ×4.9×10 -6 cm 2 / s

[0090] We examine the diffusion of a 100-base-pair DNA sequence, which represents a typical target for PCR amplification. We analyze the diffusion length R on a 100-second timescale. DSince the height is 84 μm, the isolated clusters of amplified DNA will spread approximately this distance over 100 seconds. V Equivalent volume V of sample partition in digital PCR with respect to reaction volume P This can be calculated as follows: V p =πR n 2 H v

[0091] This allows for a volume of 2.2 nl for a reaction volume height of 100 μm, making it possible to divide a typical 5 μl sample into more than 1000 partitions, as is usually required in digital PCR. When quantifying DNA concentration using digital PCR, it is desirable to divide the sample into many partitions, as a larger number of partitions increases the accuracy of the measurement and allows for the measurement of a wider range of concentrations. Therefore, by combining a small reaction volume cell height of about 100 μm with high-speed amplification of about 100 seconds, it becomes possible to analyze a typical amount of PCR sample (usually up to 20 μl) using digital PCR.

[0092] In addition to enabling small partition volumes, the flat reaction volume shape of the reactor described in the present invention is also convenient for fluorescence detection of amplified DNA for digital PCR using array detectors such as digital cameras.

[0093] Multiplex PCR is a technique for analyzing a sample to detect the presence of multiple different oligonucleotide sequences. One conventional approach is to multiplex the detection of different species using fluorescent labels with different fluorescence wavelengths, but this approach is usually limited to detecting up to about six species due to overlapping fluorescence wavelengths. An alternative conventional approach is to subdivide the sample into different reaction volumes separated by barriers that can be solid, immiscible liquid, or gas. This approach involves complexity, as in the case of digital PCR described above. This has the disadvantage of increasing the complexity and lengthening the analysis time.

[0094] Figure 8 shows a single-sided reactor A (left) and a double-sided reactor B (right) according to the present invention. Each reactor comprises a reaction cell with one or more heaters and heat sinks. The heater comprises a heating element on a heater support in contact with the heat sink. The heater support may include a softer, more flexible heat pad layer to achieve good thermal contact between a harder, more rigid heater support layer and a harder, more rigid heat sink. Embodiment A, a reactor with a single-sided heater, comprises a single heater and heat sink and a reactor with side walls, thin outer walls and a cover. In Embodiment A, the reaction volume is surrounded by the cover, side walls and thin outer walls. Embodiment B, a reactor with a double-sided heater, comprises a reaction cell with two thin outer walls surrounding the side walls. In Embodiment B, the reaction volume is surrounded by the side walls and thin outer walls.

[0095] Figure 9 shows side views of the single-sided reactor A (left) and the double-sided reactor B (right) according to the present invention.

[0096] Figure 10 shows side views of a single-sided reactor A (left) and a double-sided reactor B (right) without a heat pad according to the present invention.

[0097] Figure 11 shows the dimensions of the reactor according to the present invention, with the height H of the reaction cell. V , thickness of thin wall H W , heater support height H H , height of the heating pad H P , Heatsink height H S and the width W of the reaction cell V This indicates that.

[0098] Figure 12 shows cross-sectional views of the geometric shapes of various reactors. All include a reaction cell in contact with a heater, which is in contact with a heat sink. Plane geometry is shown in (A), curved geometry in (B), and cylindrical geometry in (C). In all cases, the reaction volume is bounded by two substantially parallel walls of a larger area and a smaller side wall. At least one of the larger walls is a thin outer wall in contact with the heater.

[0099] As described above, the reaction cell can have a planar or curved shape. However, within the reaction cell, one or more reaction volumes to which thermal cycling is applied may be constructed in several ways.

[0100] Figure 13 shows a cover for a reaction cell cover used in an embodiment of the present invention, which comprises a substantially flat portion having through holes forming fluid ports, recessed features defining the reaction volume, and a fluid channel connecting the reaction volume to the fluid ports. The cover is shown in its complete form (A) and in cross-section (B).

[0101] Figure 14 shows a plan view of a reaction cell cover used in an embodiment of the present invention, comprising a reaction volume connected to a fluid port by a fluid channel. The reaction volume may have an elongated or rectangular shape (A) or a substantially square shape (B).

[0102] Figure 15 shows an exploded assembly diagram of a reactor according to the present invention, including an assembly of a reaction cell, heater, and heat sink. The reaction cell contains two reaction volumes, each of which is situated on a heater element that extends laterally beyond the edge of the reaction volume. The heater is in thermal contact with the heat sink (optionally, via a thermal pad not shown in this figure). By using an adiabatic air gap positioned near the periphery of the heater element area, the velocity of heat flow from the heater to the heat sink can be locally reduced, thereby suppressing the tendency for the edges of the heater element area to be cooler than the center and improving thermal uniformity within the reaction volume.

[0103] Figure 16 shows two forms of reaction cells used in embodiments of the present invention: (A) a reaction cell for multiple samples (eight sample forms are shown) and (B) a reaction cell for a single sample having multiple detection sites. The detection sites may be used to perform digital PCR, where the number of sites where amplified DNA is detected is used to calculate the concentration of the target sequence in the sample. The detection sites may also be used to perform multiplex PCR to detect multiple different target sequences, for example by providing different sequence-specific PCR primers at different detection sites. Each reaction cell in Figure 16(A) and Figure 16(B) may have a single reaction volume associated with a single heater element.

[0104] In one embodiment, a wallless multiplex PCR system and method are provided in which the sample is not divided into discontinuous subsamples. Instead, the reactor is pre-loaded with arrays of various reagents for amplification or detection of different oligonucleotide sequences, with the various reagents located at different positions. The sample is then loaded into the reactor and analyzed as a continuous series of liquids. The spacing of target-specific reagents is selected such that the distance from one position to an adjacent position is greater than the diffusion length of PCR, typically 30–50 cycles in the case of oligonucleotide detection, relative to the time required for the detection reaction.

[0105] For PCR amplification of different target sequences, in this example, different primer pairs can be pre-loaded at different locations within the reaction volume by, for example, depositing a row of primer pairs in a row on one of the larger walls of the reaction cell and then drying them. Primer-specific amplification can occur at the location of each primer pair, and the results of this amplification can be determined independently of the results of PCR amplification at adjacent locations, provided that the PCR amplification is performed in a shorter time than the time required for the reaction product or primer to diffuse to the adjacent location.

[0106] Typical primers are oligonucleotide sequences of approximately 20 bases, shorter than the target DNA sequence and consequently having a larger diffusion coefficient. Applying the same equation as DNA diffusion, a 20-base primer can diffuse 151 μm in less than 100 seconds. For independent detection, adjacent locations for multiplexed detection must be at least twice this distance, preferably at least 4-6 times this distance. As an example, a height of 100 μm and an area of ​​50 mm². 2 A reaction volume of 5 μl can be considered, with a spacing of approximately 1 mm between adjacent positions. Each detection position is 1 mm 2 It occupies the area and provides 50 independent detection positions for multiplexing.

[0107] Diffusion-restriction channels can be used to limit the diffusion of reagents and reaction products between adjacent reaction zones for the duration of the reaction. Figure 17 shows a simulation of simultaneous DNA amplification and diffusion up to 60 seconds, and DNA diffusion after 60 seconds, in a reactor according to the present invention. At the start of the simulation, the central cell contains one DNA molecule, which is amplified by PCR with a cycle time of 2 seconds. Reaction cells with diffusion-restriction channels are shown by solid lines, and reaction cells without diffusion-restriction channels are shown by dashed lines. Fast amplification is required for complete amplification in one reaction zone before large diffusion and amplification can occur in adjacent zones. In this example, the DNA concentration in the adjacent cell with a diffusion-restriction channel is approximately 5% of the DNA concentration in the main cell at 60 seconds, while the DNA concentration in the adjacent cell without a diffusion-restriction channel is approximately 35%.

[0108] Figure 18 shows the simulation results of DNA concentration in the method using the present invention at a time of 100 seconds for two cases, where A is a reaction cell without diffusion-restricting channels and B has separate reaction zones (detection locations) separated by diffusion-restricting channels. This is a reaction cell. DNA diffusion to adjacent cells is inhibited by diffusion restriction channels. In both cases, PCR amplification is simulated for 60 seconds (30 cycles of PCR amplification at 2 seconds per cycle), followed by diffusion for another 40 seconds. Each reaction cell in Figure 18(A) and Figure 18(B) may have a single reaction volume associated with a single heater element.

[0109] Figure 19 shows the concentration fluctuations in the method using the present invention, along with the position at 100 seconds after the start of PCR amplification. The diffusion barrier helps maintain high concentrations of DNA near the amplified DNA site and low concentrations at adjacent reaction sites. This allows digital PCR, multiplex PCR, or other reactions to be performed in a reactor with a continuous fluid path for the flow of reagents and samples, eliminating the need for droplets or other means to separate reaction volumes.

[0110] Figure 20 shows two different designs of a fluid cell having a row of reaction zones connected by smaller channels that restrict diffusion between adjacent reaction zones according to the present invention. The reaction zones may contain different reagents, such as PCR primers, to perform multiple amplification and detection of different DNA sequences at different locations. Alternatively, the reaction zones may all contain the same reagent to perform digital PCR. The diffusion-restricting channels allow the row of reaction zones to be filled without the need to generate water droplets in the oil to provide separated reaction zones, thereby simplifying the reaction and reducing the cost of performing the reaction. Each of the fluid cells in Figure 20(A) and Figure 20(B) may have a single reaction volume associated with a single heater element.

[0111] Figure 21 shows details of a fluid cell having a reaction zone 330 separated by a diffusion limiter 340. The fluid pathway from the reaction zone recombines to allow for the collection of reaction products and their transfer out of the reaction cell.

[0112] Figure 22 shows details of the reaction cell outlet where the fluid paths have been recombined. To avoid bubble formation, a series of capillary flow limits are placed in the fluid path, with the capillary burst pressure increasing in the flow direction. In Figure 22(A), the larger radial capillary flow limit 351 has a lower burst pressure than the smaller radial capillary flow limit 352. When the flows from the two channels merge, one channel is fixed at the capillary flow limit until the adjacent channel is filled, at which point the leading meniscuses of the two advancing liquids come into contact, and the flows recombine without bubble formation. In Figure 22(B), the liquid flow is stopped at the smaller capillary radial flow limit point 352, while the liquid flows through the larger capillary radial flow limit point 351. The liquid flows recombine when the advancing meniscuses reach each limit point 352.

[0113] Figure 23 shows that the reaction volume is 220 and the width is W. V Length L V and height H V Another embodiment of a reaction cell having is shown. In this example, the cell structure is more robust by avoiding wide fluid channels with large, unsupported spans. The reaction volume 220 is positioned in proper alignment with the heater element 500 and configured to be heated by the heater element 500, and the reaction volume region W V ×L V Distribute the sample throughout at height H V It is constructed from a meandering fluid channel 270 having the following dimensions, for example, W V =2.5mm, L V =16mm, and H V= 0.1 mm. A reaction cell containing a reaction volume having such a configuration offers several advantages, such as the fluid pathway being easily controlled, bubbles being able to be washed away, and the components being able to be manufactured using embossing and lamination processes to attach the thin outer wall 230 to the fluid cell. To perform PCR amplification, the sample is introduced into the reaction volume, the movement of the liquid is stopped, positive pressure is applied, and then the reaction volume is thermally cycled. PCR primers may be provided as a linear array along a meandering channel. —These may be selected considering their solubility, and as a result, the primers remain substantially fixed along the channel.

[0114] Figure 24 shows another embodiment with a meandering fluid channel. In this embodiment, four reaction volumes 220 are filled from a single sample via a meandering fluid channel 270 and are configured to be thermally cycled by a heater element 500. The width of each reaction volume is W V The length is L V In this embodiment, the dimensions are, for example, W V =2.0mm, L V =3.6mm, and H V It can be 0.1 mm.

[0115] Figure 25 shows a conductive heater element used in an embodiment of the present invention, which has zones of medium sheet resistance, low sheet resistance, and high sheet resistance to increase heating near the edges of the heater element in order to improve temperature uniformity throughout the element. The heater element is formed from a thin film conductor supported on an electrically insulating support. The sheet resistance of the heater element is locally increased by forming holes in the conductive thin film, with a larger percentage of the hole area resulting in a greater increase in sheet resistance. As current flows between the electrical terminals, the increased sheet resistance in the upper and lower high-resistance zones results in a higher heating power density, and the increased current density in the lower left and right low-resistance zones results in a higher heating power density. The heater is also made partially transparent by forming holes in the conductive layer, which is normally opaque. Partially transparent heater elements are useful for enabling optical monitoring of reactions, particularly fluorescence detection of amplified DNA.

[0116] Figure 26 illustrates an alternative approach to edge effect compensation in a heater element used in embodiments of the present invention. A guard heater is provided on one or more edges of the main heater, and the guard heater is electrically driven to the same temperature setpoint as the guard heater. The main heater also has an edge heater zone with higher electrical resistance to provide increased heating near the edge, thereby suppressing the edge effect. The main heater is equipped with a four-terminal Kelvin connection, so its resistance can be accurately measured and used to calculate the temperature of the heater element. The resulting temperature measurements are used to control the heater's driving force and thermal cycling operation.

[0117] Figure 27 shows a heater element used in an embodiment of the present invention, which includes a main heater with guard heaters positioned near two edges. In this case, the main heater is elongated, and the guard heaters are positioned near the longer side. The main heater has a central zone of low resistance and an end zone of high resistance, and the current flowing along the length of the heater increases the thermal output of the high-resistance end zone, and this increased thermal output compensates for the edge effect and improves the temperature uniformity of the heater. The advantage of the elongated heater element is that it is convenient to manufacture and matches the shape of an elongated reaction volume, which is convenient for filling without trapping air bubbles. The perforations in the main heater make the heater partially transparent.

[0118] Figure 28 shows the heater design for two reaction volumes used in embodiments of the present invention. The heater includes two elongated main heater elements, each with three guard heater elements adjacent to the long side of the main heater element. The end zones of the main heater have a higher sheet resistance, created by increasing the area fraction of holes in the thin film conductor (B), while the central zones of the main heater have a lower sheet resistance, created by decreasing the area fraction of holes in the thin film conductor (A). The main heater is equipped with Kelvin contacts, allowing for accurate measurement of the heater resistance. The change in heater resistance with temperature is used to monitor and control the heater temperature. The guard heaters are provided with individual electrical contacts, enabling independent electrical drive and control. The outer guard heaters may be designed to have lower resistance than the inner guard heaters, resulting in the outer guard heaters generating more heat when all guard heaters are driven at the same voltage, which is, Higher heat output compensates for greater heat loss at the heater's edges, thus improving temperature uniformity.

[0119] Figure 29 shows a heater used in an embodiment of the present invention, which has patterned electrodes to vary resistance. Heater (A) has two main heaters, which have a higher resistance region near their short ends and a lower resistance region in the center. The resistance can be adjusted by the geometry of a gap region, which provides a variable conductor geometry. (B) shows an example of a higher resistance region and a lower resistance region. The elongated gap is oriented perpendicular to the direction of current to increase the sheet resistance of the heater element. A gap with a larger aspect ratio provides a higher resistance end zone, and a gap with a smaller aspect ratio provides a lower resistance center zone. The design of the main heater is robust against single-path failure by providing parallel electrical conduction paths. Guard heaters are positioned near the long sides of the main heaters. To provide greater thermal output when driven at the same drive voltage, the outer guard heater (C) has lower resistance than the inner guard heater (D).

[0120] The heater element is the thickness H of the reaction volume. V If any hole with a smaller diameter than or substantially continuous with the heating element, or if there is an elongated gap within the heating element, the gap width is the thickness of the reaction volume H V It will be understood that it will be smaller than that.

[0121] Figure 30 shows a heater element used in an embodiment of the present invention, which uses a combination of low-resistance interlocking electrodes and a high-resistance region for heating and temperature sensing. The high-resistance region can be manufactured using a metal oxide material such as vanadium oxide, which has a large temperature coefficient of resistance (TCR) over the target temperature range, and the low-resistance interlocking electrodes can be manufactured using a thin film of a metal such as gold, aluminum, or copper, which has a lower sheet resistance than the high-resistance heating and sensing region.

[0122] Figure 31 shows a heat sink used in an embodiment of the present invention, perforated to allow optical monitoring of the reaction when used in combination with a partially transparent heater element. The diameter and pitch of the holes are selected to be small enough not to disrupt the temperature uniformity of the reaction cell. In a typical example, a 2 mm thick aluminum heat sink block is used with 1 mm diameter holes at a 2 mm pitch. This configuration allows for optical monitoring of the reaction even when the heater and heat sink are on both sides of the reactor.

[0123] Figure 32 shows the temperature fluctuation over time of a heater connected to a heatsink via a thermal resistor according to the present invention (A). The heater is driven with a fixed power of 10W until it reaches the upper limit set temperature (time = 0.26 seconds), and the heater power is reduced to zero until it reaches the lower limit set temperature (time = 0.59 seconds). Given a given heater power and heater shape, there exists a thermal resistance that minimizes the thermal cycling time, as shown in (B). For a 10W heater, the optimal thermal resistance is approximately 10K / W. The higher the heater output, the lower the thermal resistance required to minimize the thermal cycling time, and the lower the heater output, the higher the thermal resistance required to minimize the thermal cycling time. For example, a 5W heater requires a thermal resistance of 20K / W, and a 20W heater requires 5K / W.

[0124] Figure 33 shows the variation in heater power and thermal resistance for thermal cycling times of 0.4 seconds, 0.8 seconds, and 1.6 seconds in an exemplary reactor according to the present invention. For a given thermal cycling time, there exists an optimal thermal resistance that minimizes the required heater power.

[0125] Figure 34 shows the temperature fluctuation over time of a heater used in an embodiment of the present invention, driven between 90°C and 60°C. The thermal cycling time is 2 seconds. The solid line represents the temperature setpoint, and the dotted line represents the measured temperature.

[0126] Figure 35 shows DNA melting curve data obtained using the reactor of an embodiment of the present invention. The presence of double-stranded DNA is indicated using the insertion dye SYBR-GREEN, which shows a rapid decrease in fluorescence when the temperature exceeds the melting temperature of DNA, in this case around 83°C. The variation in fluorescence with temperature is shown in A, and the gradient -dF / dT is shown in B.

[0127] Figure 36 shows DNA amplification curve data for PCR amplification after thermal cycling using the reactor of an embodiment of the present invention. The upper graph shows the fluorescence of the insertion dye against time, showing growth in DNA concentration above background levels at approximately 120 seconds. The lower graph shows temperature against time. An initial hot start is used to activate the polymerase enzyme, followed by thermal cycling times of 5 seconds at high temperature of 95°C and low temperature of 60°C.

[0128] Figure 37 shows the temperature difference between a sample cell and a reference cell used to perform calorimetry detection of DNA melting in one embodiment of the present invention. The sample cell contains a DNA concentration of 1 μM, and the reference cell does not contain DNA. The increase in heat capacity due to DNA melting causes a temperature decrease when the sample cell and reference cell are driven with equal power. DC driving is shown in A, and AC driving is shown in B. AC driving provides a temperature oscillation of twice the driving frequency when the DNA is undergoing the melting transition. Electrical detection of the signal generated by AC driving may be easier than that of DC driving. The frequency of AC driving is selected to give a heating period approximately equal to the time of heat diffusion through the height of the reactor. The heating period is equal to 1 / 4 of the cycle time of sinusoidal driving.

[0129] Figure 38 shows the temperature profile of the PCR amplification scheme in the method using the present invention, which consists of 30 repetitions of a 20-second hot start period followed by a 5-second thermal cycle. Differential scanning calorimetry is used to detect the amplified DNA, as follows: i.e., a baseline differential temperature scan (recording the temperature difference between the sample cell and the reference cell relative to the temperature) is measured during the temperature rise of the hot start period. An endpoint temperature scan is performed during the temperature rise of the last PCR cycle. The difference between the baseline scan and the endpoint scan is proportional to the heat of DNA melting.

[0130] Figure 39 shows a bridge circuit according to the present invention for calorimetry and detection of DNA melting. The sample cell is heated by resistors R1 and R4, and the reference cell is heated by resistors R2 and R3. In the case of DNA melting, the sample cell will have a lower temperature, the resistance values ​​of R1 and R4 will decrease, a higher voltage will be generated at the positive (non-inverting) input of amplifier X1, and a lower voltage will be generated at the negative (inverting) input of amplifier X1. This will result in a positive output from X1. In this case, a DC drive V1 is shown, but AC drive can also be used. In the case of sinusoidal AC drive, a frequency-selective detection method can be used to detect only signals at twice the drive frequency. In the case of square wave drive (pulse +V or 0V), frequency-selective detection can be used to detect signals at the same frequency as the drive frequency. To generate a measurable signal voltage from a small temperature difference, it is desirable that the heater resistors have resistances with a large temperature coefficient. In another approach, a thermocouple connected between the sample cell and the reference cell generates a signal voltage proportional to the temperature difference between the cells.

[0131] Figure 40 shows a balanced circuit according to the present invention, which uses additional series resistors R5, R6, R7, and R8 to minimize the differential voltage output of the bridge circuit and the difference between the power output of the sample heater with R1 and R4 and the reference heater with R2 and R3. The values ​​of R5, R6, R7, and R8 can be set at manufacturing or adjusted before detection. To enable high amplification gain and highly sensitive detection without saturating X1, it is desirable to minimize voltage imbalance in the bridge circuit. To ensure that both cells reach the DNA melting temperature simultaneously and provide a signal proportional to the difference in DNA concentration between the two cells, it is desirable that the power input to the sample cell and the reference cell be equal.

[0132] Figure 41 shows additional parallel resistors R5, R6, R7, and R8 that may be used in the present invention to minimize the differential voltage output of the bridge circuit and the difference in power output between the sample heater and the reference heater.

[0133] As will be understood by those skilled in the art, several exemplary reactors, heaters, and circuit structures according to the present invention are described, which can be used in combination with any of the other examples described to achieve the present invention.

[0134] The subject matter of the application further includes the following numbered clauses:

[0135] 1. A variable temperature reactor for accepting a predetermined reaction, Equipped with a reaction cell, heater, and heat sink, The reaction cell has a thickness of H V and width W V The reaction volume has the following characteristics, where W V >4H V And one of the surfaces with a larger area of ​​reaction volume has a thickness of H W It is defined by a surface bounded by an outer wall having, The heater is in contact with the outer wall. The heater comprises a heat-generating heater element positioned on the side closer to the reaction volume and a heater support on the opposite side, the heater support being in contact with the heat sink, and therefore the heater support has a thermal resistance R between the heater element and the heat sink. T Provided, The reactor has a thermal diffusivity D V When filled with a reagent having the following properties, the diffusion time t in the thickness direction VIt has, V =H V 2 / D V and t V The reaction time constant is t. R Smaller than, The exterior wall has a thermal diffusivity coefficient of D W It has a thermal diffusion time t W =H W 2 / D W <t V A variable temperature reactor having the following features.

[0136] 2. The heater element functions as both a heater and a temperature sensor in the reactor according to clause 1.

[0137] 3. A controller is also provided, and the heater is connected to the controller and controlled by the controller to raise the reactor temperature to a higher temperature T High and lower temperature T Low The temperature changes between these two points, and both are the temperature of the heatsink T. Sink A reactor that exceeds the limits of clause 1 or clause 2.

[0138] 4. Thickness H of the reaction volume V A reactor of any one of clauses 1 to 3, wherein the diameter is less than 250 microns.

[0139] 5. The reaction cell has a width W. V and length L V Width W is formed by a meandering channel located within a region having V and length L V A reactor according to any one of the clauses 1 to 4, comprising a reaction volume having a reaction volume.

[0140] 6. The reaction volume is defined as the thickness H on both large surfaces when the heaters are in contact with both of the outer walls and the heat sink is in contact with both heaters. W A reactor according to any one of clauses 1 to 5, bounded by an outer wall having the following features.

[0141] 7. The heater element is a resistive heating element, in the reactor according to any one of clauses 1 to 6.

[0142] 8. The heater element is manufactured from a conductive material, and the heater support is manufactured from an electrically insulating material. a reactor according to any one of the clauses 1 to 7.

[0143] 9. The reactor according to any one of the clauses 1 to 8, wherein the heater support is formed from a softer, more flexible layer in contact with a harder, more rigid heat sink layer.

[0144] 10. The heater support is further formed from a harder, more rigid layer, which is in contact with a softer, more flexible layer, in this case the softer, more flexible layer is in contact with the heat sink layer, according to clause 9 of the reactor.

[0145] 11. A reactor according to any one of the clauses 1 to 10, wherein the reaction cell is separable from the heater.

[0146] 12. A reactor according to any one of the clauses 1 to 11, wherein the heater is separable from the heat sink.

[0147] 13. A reactor according to any one of clauses 1 to 12, wherein the heat sink is cooled by forced air cooling, circulating liquid cooling, spray cooling, heat pipes, or active cooling by a Peltier element or heat pump.

[0148] 14. A reaction cell is a reactor according to any one of the clauses 1 to 13, having an inlet channel and an outlet channel that connect the inlet port and outlet port of the fluid to the reaction volume.

[0149] 15. A reactor according to any one of the clauses 1 to 14, wherein the reaction cell comprises a liquid contact material which is an inert polymer.

[0150] 16. Thermal resistance R between the heater element and the heatsink T The following relationships are selected: RT >(T HIGH -T Sink ) / p Heat and 0.5R T,Opt <R T <2R T,Opt Here, R T,Opt =( T HIGH +T LOW -2T Sink ) / p Heat , The reactor has an output of p Heat A heater and temperature control with Sink Using a heatsink at a lower temperature T LOW and higher temperature T HIGH A reactor according to any one of clauses 1 to 15, configured to repeatedly circulate between and .

[0151] 17. Thermal resistance R of the heater support T , and the sum of the heat capacity of the filled reaction volume and the heat capacity of the thin outer wall portion located between the reaction volume and the heater element C V However, R T C V <t R The relationship satisfies, where t R A reactor according to any one of clauses 1 to 16, configured such that is the reaction time constant.

[0152] 18. The sum of the heat capacity of the filled reaction volume and the heat capacity of the thin outer wall portion located between the reaction volume and the heater element C V And the heat capacity C of the heatsink S However, C S / C V A reactor according to any one of clauses 1 through 17, configured to satisfy the relationship >100.

[0153] 19. A reactor according to any one of the clauses 1 to 18, wherein the thermal conductivity of the heat sink material is greater than 10 times that of the heater support material.

[0154] 20. A reactor according to any one of the clauses 1 to 19, wherein the heat capacity of the thin outer wall portion located between the reaction volume and the heat sink and the heater is lower than the heat capacity of the liquid in the reaction volume.

[0155] 21. The thermal permeability of the heat sink material is more than 10 times that of the heater support material. Here, the thermal osmotic coefficient e is given by the thermal conductivity k, density ρ, and specific heat capacity c of the material. p It is a function of and e = sqrt(kρc p A reactor as defined in any one of clauses 1 to 20.

[0156] 22. A reactor according to any one of the clauses 1 to 21, wherein the heater element extends over the entire region of the reaction volume.

[0157] 23. A reactor according to any one of clauses 1 to 22, wherein the heater element is resistive and has a rectangular or square shape.

[0158] 24. A reactor according to any one of Clauses 1 to 23, wherein the heater element is made from a conductive material having an absolute value of a temperature coefficient of resistance greater than 500 ppm / K over the operating temperature range of the heater.

[0159] 25. A reactor according to any one of Clauses 1 to 24, wherein the heater element is manufactured from a conductive material having an absolute value of a temperature coefficient of resistance greater than 2,500 ppm / K over the operating temperature range of the heater.

[0160] 26. A reactor according to any one of Clauses 1 to 25, wherein the heater element is manufactured from a conductive material having an absolute value of a temperature coefficient of resistance greater than 10,000 ppm / K over the operating temperature range of the heater.

[0161] 27. A heater element manufactured by evaporating, sputtering, printing, laminating, lithographing, or laser patterning a conductive material, as specified in any one of Clauses 1 to 26.

[0162] 28. The reactor of any one of clauses 1 to 27, wherein the heater element is made of a conductive material having a positive TCR and may contain one of Pt, Ti, Al, Mo, Ni, Cu, Au.

[0163] 29. The reactor of any one of clauses 1 to 28, wherein the resistive heating element is made of a material having a negative TCR, such as a metal oxide that transitions from electrical insulation to conductivity as the temperature rises, such as vanadium oxide.

[0164] 30. The reactor of any one of clauses 1 to 29, wherein electrical contacts are provided at opposite corners of the heater element, and the current is distributed along the opposite edges of the heater element by an electrically conductive track having a tapered width that narrows from a wider width at the corners of the electrical contacts to a narrower width at the distal side of the electrical contact edges.

[0165] 31. The reactor of any one of clauses 1 to 30, wherein the heater includes Kelvin contacts for electrical resistance measurement.

[0166] 32. The reactor of any one of clauses 1 to 31, wherein the heater element is configured such that the heat output per unit area is higher near the periphery than near the center.

[0167] 33. The reactor of clause 32, wherein the heater element has a square or rectangular shape with electrical contacts along two opposite edges, and has a higher sheet resistance region at the edges of the electrical contacts and a lower sheet resistance region at the other two edges.

[0168] 34. The reactor of any one of clauses 1 to 33, wherein the heater element includes a main heater surrounded by one or more guard heaters.

[0169] 35. The reactor of clause 34, comprising means for controlling the temperature of the guard heater to the same temperature set value as the main heater.

[0170] 36. A reactor according to clause 34 or 35, wherein the guard heater has a lower sheet resistance than the main heater.

[0171] 37. A reactor according to any one of clauses 34 to 36, wherein the main heater is elongated in the direction of the current flow and the guard heater is arranged adjacent to the long side of the main heater.

[0172] 38. A reactor according to any one of clauses 1 to 37, wherein the sheet resistance of the heater element locally increases in an end zone located at the edge of the heater element perpendicular to the direction of the current flow.

[0173] 39. A reactor according to any one of clauses 1 to 38, wherein the sheet resistance of the heater element locally decreases in a side zone located at the edge of the heater element parallel to the direction of the current flow.

[0174] 40. In the reactor according to clause 38, the increased sheet resistance within the heater element is provided by a partial coating of the conductive material, for example, by forming a layer of conductive material perforated by an array of holes or slots.

[0175] 41. A reactor according to any one of clauses 1 to 40, wherein the heater support or heat sink comprises a heat insulation layer located near the periphery of the heater element and between the heater element and the heat sink.

[0176] 42. A reactor according to any one of clauses 1 to 41, wherein the reaction volume is configured to contain reagents used for polymerase chain reaction (PCR) amplification of nucleic acid sequences during use.

[0177] 43. A reactor according to any one of clauses 1 to 41, configured to perform DNA amplification by polymerase chain reaction (PCR) thermal cycling.

[0178] 44. A reactor according to any one of clauses 1 to 43, comprising a controller configured to control the temperature at different temperature set values for different reaction stages in a multi-stage reaction.

[0179] 45. The reactor of Clause 44 is configured to carry out a multi-step reaction for DNA sequencing.

[0180] 46. ​​A heat sink provided for any one of clauses 1 to 45, which has holes to allow optical inspection of the reaction volume.

[0181] 47. A reactor according to any one of the clauses 1 to 46, configured to detect the result of a reaction in the reaction volume using fluorescence or colorimetric or UV absorption or electrochemistry or calorimetry or electrophoresis or oligonucleotide sensing.

[0182] 48. A reactor according to any one of clauses 1 to 47, configured such that the reaction output is detected by monitoring the progress of the reaction measurement across multiple thermal cycles.

[0183] 49. A reactor according to any one of the clauses 1 to 48, wherein the reaction sensor is located inside or in contact with the outer wall of the reaction volume.

[0184] 50. A reactor according to any one of clauses 1 to 49, configured for calorimetry and detection of the results of a reaction, by comprising measuring components for measuring changes in the heat capacity of a sample in the reactor.

[0185] 51. A reactor configured for DTA or DSC, by being configured to use a sample reaction and a reference reaction, according to any one of the clauses 1 to 50.

[0186] 52. A reactor according to any one of the clauses 1 to 50, configured for DTA or DSC with baseline correction by providing means for subtracting a first temperature scan from a second temperature scan measured at different times.

[0187] 53. A reactor according to any one of clauses 1 to 52, configured to detect the result of a reaction without labeling.

[0188] 54. The reactor of clause 51, configured such that measurements are made while heating the sample reaction volume and the reference reaction volume.

[0189] 55. The reactor of any one of clauses 1 to 54, configured to handle a reaction product that is DNA or RNA, and configured such that melting of the reaction product is detected by calorimetry.

[0190] 56. The first DTA or DSC measurement is performed while raising the temperature of the sample and the reference for the hot start phase preceding the thermal cycling for PCR amplification of DNA, and the second DTA or DSC measurement is configured to be performed following the thermal cycling for PCR amplification of DNA. The reactor of clause 51 or 54.

[0191] 57. A plurality of reactors of any one of clauses 1 to 56, wherein at least one reaction volume contains a sample material and at least one reaction volume contains a reference material in use.

[0192] 58. The reactor of any one of clauses 1 to 57, wherein the heater element has a drive device configured to provide a pulsed or oscillatory heat output to the heater.

[0193] 59. A reaction volume having a height H V and a content having a thermal diffusivity D V and a thin reaction chamber wall having a thickness H W and a thermal diffusivity D W are configured such that only one side is heated by a heater, where the frequency of the heater drive device satisfies the relational expression

Number

[0194] 60. A reaction volume having a height H V and a thermal diffusivity DV Contents having a thickness H W and thermal diffusion coefficient D W A thin reaction chamber wall having a [specific feature] is configured to be heated on both sides by heaters, and the frequency of the heater drive is given by the relational expression

number

[0195] 61. A reactor according to any one of the clauses 1 to 60, further comprising a resistive heater element driven in a sinusoidal waveform at a drive frequency, and comprising means for measuring the temperature of the heater element by measuring the variation in the heater element resistance at twice the drive frequency, or by measuring the variation in the heater voltage or current at three times the drive frequency.

[0196] 62. A reactor according to any one of the clauses 1 to 61, wherein the temperature difference between the sample and the reference is sensed using a sample resistance heater, a reference resistance heater, and two fixed resistors arranged in a bridge circuit.

[0197] 63. A reactor according to any one of the clauses 1 to 61, wherein the temperature difference between the sample and the reference is sensed using two sample resistance heaters and two reference resistance heaters located within a bridge circuit, and each side of the bridge circuit includes one resistance heater from the sample container and one from the reference container, respectively.

[0198] 64. A reactor according to clause 62 or 63, using trim resistors to simultaneously balance the output voltage of the bridge circuit with the heater output applied to the sample vessel and the reference vessel.

[0199] 65. A reactor according to any one of clauses 1 to 64, the heater element having holes for enabling optical inspection of the reaction volume.

[0200] 66. A reactor according to any one of the clauses 1 to 65, wherein the heater element is formed by holes, slots, gaps, or meandering paths that allow light transmission and optical monitoring of the reaction.

[0201] 67. The reactor according to any one of the clauses 1 to 66, wherein the heater element is formed from a transparent conductive material such as ITO, graphene, or nanowire material.

[0202] 68. The reaction volume includes multiple spatially separated zones, with different reagents placed in different zones of the reaction volume, and the zone spacing is the reaction time constant t. R A reactor according to any one of clauses 1 to 67, wherein the mass diffusion length of the reaction product and the mass diffusion length of the reagent on the timescale are greater than the larger of the two, and different reactions are monitored independently within each reaction zone.

[0203] 69. Cycle time t C The reaction volume is further provided with means for thermal cycling N times, and the reagent pitch is t C A reactor according to clause 68, wherein the mass diffusion length of the reaction product and reagent at a diffusion time equal to the product of the two factors is greater than the mass diffusion length of the reaction product and reagent.

[0204] 70. A reactor according to clause 68 or 69, wherein, if the same reagent is placed in each zone of the reaction volume in use, means are provided for independently monitoring each zone to detect the presence or absence of reaction products in each zone.

[0205] 71. A reactor according to any one of the clauses 68 to 70, further comprising a processor for processing statistics of the number of zones containing reaction products and zones not containing reaction products, in order to calculate the concentration of the analyte in use.

[0206] 72. A reactor according to any one of the clauses 68 to 71, wherein the reaction volume contains at least 100 reaction zones, or at least 1,000 reaction zones, or at least 10,000 reaction zones.

[0207] 73. A reactor according to any one of clauses 68 to 72, wherein the reaction volume is divided into reaction zones connected by diffusion-restricting channels having a cross-sectional area less than 0.25 times the cross-sectional area of ​​the reaction zone, wherein the diffusion-restricting channels have a length greater than 0.25 times the width of the reaction zone.

[0208] 74. A heater for a variable temperature reactor, equipped with a heating element that functions as both a heater and a temperature sensor.

[0209] 75. The heater element of Clause 74 is resistant and has a rectangular or square shape.

[0210] 76. A heater according to Clause 74 or 75, the heater element being manufactured from a conductive material having an absolute value of a temperature coefficient of resistance greater than 500 ppm / K over the operating temperature range of the heater.

[0211] 77. A heater according to any one of clauses 74 to 76, wherein the heater element is manufactured from a conductive material having an absolute value of a temperature coefficient of resistance greater than 2,500 ppm / K over the operating temperature range of the heater.

[0212] 78. A heater according to any one of clauses 74 to 77, wherein the heater element is manufactured from a conductive material having an absolute value of a temperature coefficient of resistance greater than 10,000 ppm / K over the operating temperature range of the heater.

[0213] 79. Heater elements of any one of clauses 74 to 78, manufactured by evaporating, sputtering, printing, laminating, lithographing, or laser patterning a conductive material.

[0214] 80. A heater according to any one of clauses 74 to 79, wherein the heater element is made from a conductive material having a positive TCR and may include one of Pt, Ti, Al, Mo, Ni, Cu, and Au.

[0215] 81. A resistive heating element is a heater according to any one of the clauses 74 to 80, manufactured from a material having a negative TCR, such as a metal oxide, such as vanadium oxide, which transitions from electrically insulating to conductive as the temperature rises.

[0216] 82. A heater according to any one of clauses 74 to 81, wherein electrical contacts are provided at opposite corners of the heater element, and the current is distributed along the opposite edges of the heater element by electrically conductive tracks having a tapered width that narrows from a wider width at the corners of the electrical contacts to a narrower width distal to the edges of the electrical contacts.

[0217] 83. A heater, including a Kelvin contact for measuring electrical resistance, as specified in any one of clauses 74 to 82.

[0218] 84. A heater according to any one of clauses 74 to 83, wherein the heater element is configured such that the heat output per unit area is higher near its periphery than near its center.

[0219] 85. The heater element of Clause 84, having a square or rectangular shape with electrical contacts along two opposing edges, with higher sheet resistance regions at the edges of the electrical contacts and lower sheet resistance regions at the other two edges.

[0220] 86. A heater element comprising a main heater surrounded by one or more guard heaters, as per any one of the clauses 74 to 85.

[0221] 87. The heater according to clause 86, comprising means for controlling the temperature of the guard heater to the same temperature setpoint as the main heater.

[0222] 88. Guard heaters have lower seat resistance than main heaters, as specified in clause 86 or 87.

[0223] 89. A heater according to any one of the clauses 86 to 88, wherein the main heater is elongated in the direction of current flow, and the guard heater is positioned adjacent to the long side of the main heater.

[0224] 90. A heater according to any one of the clauses 74 to 89, wherein the sheet resistance of the heater element increases locally in the end zone located at the edge of the heater element perpendicular to the direction of current flow.

[0225] 91. A heater according to any one of clauses 74 to 90, wherein the sheet resistance of the heater element is locally reduced in the lateral zone located at the edge of the heater element parallel to the direction of current flow.

[0226] 92. The heater of Clause 91, wherein the increased sheet resistance within the heater element is provided by partial covering of a conductive material, for example by forming a layer of conductive material perforated by an array of holes or slots.

[0227] 93. A drive and sensing circuit for a variable temperature reactor, comprising means for supplying power to a sample resistance heater and a reference resistance heater, and means for sensing the temperature difference between a sample and a reference by monitoring a sample resistance heater, a reference resistance heater, and two fixed resistors arranged in a bridge circuit.

[0228] 94. A drive and sensing circuit for a variable temperature reactor, comprising means for supplying power to two sample resistance heaters and two reference resistance heaters arranged within a bridge circuit, and means for measuring the temperature difference between a sample and a reference by monitoring the two sample resistance heaters and two reference resistance heaters, wherein each side of the bridge circuit is configured to include one resistance heater from the sample container and one from the reference container, respectively, when in use.

[0229] 95. The circuit of clause 93 or 94, further comprising a trim resistor for simultaneously balancing the output voltage of the bridge circuit with the heater output applied to the sample container and the reference container.

[0230] 96. A method for operating a reactor, heater, and circuit of any one of the clauses 1 to 95 in order to provide measurements from a variable temperature reaction.

Claims

1. Among them is a variable temperature reactor for accepting a predetermined reaction, Equipped with a reaction cell, heater, and heat sink, The reaction cell has a thickness of H Ｖ (mm) and width W Ｖ The reaction volume has (mm), where W Ｖ > 4H Ｖ And one of the surfaces with a larger area of ​​the reaction volume has a thickness H Ｗ It is defined by a surface bounded by an outer wall having (mm), The heater is in contact with the outer wall. The heater comprises a heat-generating heater element positioned on the side closer to the reaction volume and a heater support on the opposite side, the heater support being in contact with the heat sink, and therefore the heater support has a thermal resistance R between the heater element and the heat sink. Ｔ (K / W) provided, The heater temperature is the output p Ｈｅａｔ The heater having (W) and the heat sink at temperature T Ｓｉｎ Using the heat sink at k, a lower temperature T ＬＯＷ (°C), and the lower temperature T ＬＯＷ A higher temperature T 35 (°C) higher than (°C) is repeatedly circulated between ＨＩＧＨ (°C), and the heat diffusion coefficient D Ｖ (m ２ / s), when filled with a reagent having, the reactor has a diffusion time t in the thickness direction Ｖ (s), and t Ｖ = H Ｖ ２ / D Ｖ and t Ｖ (s) represents the minimum time for the PCR reaction, and is smaller than the reaction time constant t of 1 second Ｒ The temperature change time is the time required to heat the heater from the lower temperature T ＬＯＷ (°C) to the higher temperature T ＨＩＧＨ (°C), and the time required to cool the heater from the higher temperature T ＨＩＧＨ (°C) to the lower temperature T ＬＯＷ (°C), and the lower temperature T ＬＯＷ (°C) to the higher temperature T ＨＩＧＨ (°C), the time required to heat the heater is 0.26 seconds or less, and the higher temperature T ＨＩＧＨ (°C) to the lower temperature T ＬＯＷ (°C), the time required to cool the heater is 0.33 seconds or less, and the thermal cycle time is the sum of the temperature change time and the reaction time The aforementioned outer wall has a thermal diffusion coefficient D Ｗ (m ２ It has a thermal diffusion time t ( / s), Ｗ = H Ｗ ２ / D Ｗ <t Ｖ It has the thermal diffusion time t Ｗ The unit is s, The reactor is a variable-temperature reactor configured to perform DNA amplification by a polymerase chain reaction (PCR) thermal cycle.

2. The heater element functions as both a heater and a temperature sensor, as described in claim 1. A device.

3. The reactor is further equipped with a controller, the heater being connected to the controller and controlled by the controller to raise the temperature of the reactor to a higher temperature T. Ｈｉｇｈ (°C) and a lower temperature T than the above. Ｌｏｗ The temperature is varied between (°C) and both temperatures are the temperature T of the heat sink. Ｓｉｎ The reactor according to claim 1 or claim 2, wherein k exceeds k.

4. The thickness H of the reaction volume Ｖ The reactor according to any one of claims 1 to 3, wherein the diameter is less than 250 microns.

5. The reaction cell has a width W Ｖ (mm) and length L Ｖ Width W is formed by a meandering channel located within a region having (mm). Ｖ (mm) and length L Ｖ A reactor according to any one of claims 1 to 4, comprising a reaction volume having (mm).

6. The reactor according to any one of claims 1 to 5, wherein the heater element is a resistive heating element.

7. The reactor according to any one of claims 1 to 6, wherein the heater element is manufactured from a conductive material and the heater support is manufactured from an electrically insulating material.

8. The reactor according to any one of claims 1 to 7, wherein the heater is separable from the heat sink.

9. The thermal resistance R between the heater element and the heat sink Ｔ (K / W) is selected such that the following relationship is satisfied, R Ｔ > (T ＨＩＧＨ -T Ｓｉｎｋ ) / p Ｈｅａｔ and 0.5R Ｔ，Ｏｐｔ <R Ｔ <2R Ｔ，Ｏｐｔ Here, R Ｔ，Ｏｐｔ = (T ＨＩＧＨ +T ＬＯＷ -2T Ｓｉｎｋ ) / p Ｈｅａｔ And R Ｔ，Ｏｐｔ The reactor according to any one of claims 1 to 8, wherein the unit is K / W.

10. Thermal resistance R of the heater support Ｔ The sum of the (K / W) heat capacity, the heat capacity of the reaction volume filled with the reagent, and the heat capacity of the outer wall portion located between the reaction volume and the heater element, C Ｖ (J / K) and R Ｔ C Ｖ <t Ｒ The relationship satisfies, where t Ｒ The reactor according to any one of claims 1 to 9, wherein (s) is configured to be the reaction time constant.

11. The sum of the heat capacity of the reaction volume filled with the reagent and the heat capacity of the outer wall portion located between the reaction volume and the heater element, C Ｖ (J / K) and the heat capacity C of the heat sink. Ｓ (J / K) and C Ｓ / C Ｖ A reactor according to any one of claims 1 to 10, configured to satisfy the relationship >100.

12. The reactor according to any one of claims 1 to 11, wherein the thermal conductivity of the heat sink material is more than 10 times that of the heater support material.

13. The reactor according to any one of claims 1 to 12, wherein the heat capacity of the outer wall portion and the heater located between the reaction volume and the heat sink is lower than the heat capacity of the liquid in the reaction volume.

14. The thermal permeability of the heat sink material is more than 10 times that of the heater support material, where the thermal permeability e(W·s) １／２ / m ２ The values ​​k (W / m / K) represent the thermal conductivity of the heat sink material, and ρ (kg / m³) represents its density. ３ ), and specific heat capacity c ｐ Function of (J / K / kg) Therefore, e = sqrt(kρc ｐ A reactor according to any one of claims 1 to 13, as defined as:

15. The reactor according to any one of claims 1 to 14, wherein the heater element extends over the entire region of the reaction volume.

16. The reactor according to any one of claims 1 to 15, wherein the heater element is resistant and has a rectangular or square shape.

17. The reactor according to any one of claims 1 to 16, wherein the heater element is manufactured from a conductive material having an absolute value of a temperature coefficient of resistance greater than 500 ppm / K over the operating temperature range of the heater.

18. The reactor according to any one of claims 1 to 17, wherein the heater includes a Kelvin contact for measuring electrical resistance.

19. The reactor according to any one of claims 1 to 18, wherein the heater element is configured such that the heat output per unit area is higher near its periphery than near its center.

20. The reactor according to any one of claims 1 to 19, wherein the heater element comprises a main heater surrounded by one or more guard heaters.

21. The reactor according to claim 20, wherein the main heater is elongated in the direction of current flow, and the guard heater is arranged adjacent to the long side of the main heater.

22. The reactor according to any one of claims 1 to 21, wherein the sheet resistance of the heater element increases locally in the end zone located at the edge of the heater element perpendicular to the direction of current flow.

23. The reactor according to any one of claims 1 to 22, wherein the reaction volume is configured to contain reagents used for polymerase chain reaction (PCR) amplification of nucleic acid sequences during use.

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