The present invention relates to a heater for providing a variable temperature on a reaction surface of the heater.
One example process where such a heater is required is DNA amplification by the polymerase chain reaction (PCR), where the heater provides fast thermocycling to reduce the time for completion of PCR.
Prior art heaters are fabricated from electrically conductive tracks supported by an electrically insulating substrate. Prior art heaters are controlled using a separate temperature sensor to sense the heater temperature and a control algorithm and electronic drive circuit to modulate the electric drive to the heater.
In order to provide fast thermal response, the heater must have low heat capacity and the heater must be in close thermal contact with the reaction surface. In particular the thermal diffusion time from the heater element to the reaction surface must be less than the required temperature change response time, so the heater and reaction surface can only be separated by a thin layer.
Conventional heaters and temperature control systems have several disadvantages when trying to achieve fast response, precise temperature control and uniform temperature distribution.
For example, use of a temperature sensor separated from the heater introduces a delay in the heater control loop which can reduce the response speed or introduce temperature overshoot.
Additionally, temperature non-uniformity results from heat generation within spatially separated resistive heating tracks located close to the reaction surface, resulting in hotter regions directly above heater tracks and colder regions above the gaps between heater tracks. Temperature non-uniformity on the reaction surface is undesirable as it may reduce the efficiency and specificity of the PCR amplification. Therefore an objective of the invention is to increase the temperature uniformity and references to improved temperature uniformity and increased temperature uniformity are equivalent.
Temperature non-uniformity at the reaction surface can be reduced by using narrower tracks and gaps, but this complicates fabrication using standard printed circuit board techniques. Temperature non-uniformity at the reaction surface can also be reduced by increasing the distance between the heater tracks and the reaction surface, but this increases the thermal diffusion time from heater to reaction surface and slows the heater response.
Temperature non-uniformity also results from edge effects, where the temperature of the heater is reduced at the edges due to lateral heat flow. In prior art, edge effects are reduced by design of heater track patterns with increased heat output near the edge of the heater, for example by reducing the track and gap widths of the heater element in these areas. However this approach needs to be carefully designed for a particular operating temperature and reaction surface geometry and thermal load, and may be difficult to achieve if the heater track and gap widths are already close to the minimum values practical for standard fabrication processes. It is also desirable to minimise the heater track and gap widths in the central area of the heater to minimise temperature non-uniformity and therefore it is difficult to further reduce the heater track and gap widths near the edges of the heater.
In order to allow rapid cooling when the heater power is reduced, a heater may be connected to a heat sink via a controlled thermal resistance. However the heater temperature uniformity will depend on the uniformity of the thermal contact between the heater and heat sink. In particular, any air gaps between the heater and heat sink can introduce substantial thermal resistance and temperature non-uniformity.
In view of the above problems and objectives, the present invention provides a heater for thermocycling to carry out PCR amplification. The heater comprises: a thermal diffusion layer having a reaction surface for transferring heat to a reaction cell; a heater track support layer having a back surface for cooling; an electrically conductive main heater track supported between the heater track support layer and the thermal diffusion layer; and four-terminal electrical contacts to the main heater track adapted to provide electrical connection for driving the main heater track and simultaneously sensing a resistance of the main heater track. The lateral dimensions of the reaction surface are greater than a thickness H of the heater, such that reaction surface area A>H2.
Preferably, the main heater track comprises a central region comprising a plurality of substantially parallel track sections having widths Wtrack and separated by gaps of width Wgap, wherein the thickness HD of the thermal diffusion layer is less than a minimum width of the track sections Wtrack or less than a minimum gap width Wgap, where Wtrack or Wgap are evaluated in the central region of the main heater track. This means that the main heater track can be manufactured using PCB manufacturing techniques. This also means that the heater is thin enough for many applications requiring rapid temperature change.
Preferably, the gap width Wgap and/or the width of the track sections Wtrack is lower for a track section near an edge of the main heater track than for a track section in the central region of the main heater track. This increases temperature uniformity in the central region of the main heater track.
Preferably, the heater further comprises: a guard heater track between the heater track support layer and the thermal diffusion layer, the guard heater track substantially surrounding the main heater track; and two further electrical contacts to the guard heater track independent from the four-terminal electrical contacts to the main heater track. This inhibits lateral heat flow and increases temperature uniformity in the plane of the main heater track.
Preferably, the heater track support layer has a thermal resistance×area product in the range 1×10−4 to 1×10−2 K·m2/W and more preferably in the range 3×10−4 to 3×10−3 K·m2/W.
Preferably, the heater further comprises a reaction surface heat spreader layer located in contact with or within one of the thermal diffusion layer or the heater track support layer. This improves temperature uniformity at the reaction surface.
Preferably, the reaction surface heat spreader layer is more thermally conductive, has a greater lateral thermal conductivity and has a lower heat capacity than the one of the thermal diffusion layer or the heater track support layer.
Preferably, the reaction surface heat spreader layer is located within the heater track support layer at a distance Ls from the main heater track, wherein Ls is less than 20% of the minimum of the heater track width Wrack and heater gap width Wgap evaluated in the central region. This further improves temperature uniformity at the reaction surface.
Preferably, a back surface heat spreader layer is located on the back surface. In addition to improving temperature uniformity at the reaction surface, this improves thermal contact with any heat sink adjacent to the back surface.
Preferably, the heater further comprises a heat sink in contact with the back surface. This has the effect of reducing the temperature of the heater when the heater is not being driven.
In another aspect, the present invention provides a single use consumable comprising a heater and a reaction cell arranged in contact with the reaction surface.
In another aspect, the present invention provides a method of operating a heater or a variable temperature reactor, comprising driving the main heater track, simultaneously sensing a resistance of the main heater track, and calculating a temperature of the main heater track based on the sensed resistance.
Preferably, the method comprises performing feedback-based driving of the main heater track according to a sequence of temperature set points for the main heater track to cycle the temperature of the reaction surface to carry out PCR amplification.
Preferably, the method further comprises driving the guard heater track to provide a higher heat output per unit area than the main heater track.
Preferably, the heater or single use consumable as previously described further comprises a control circuit configured to perform a method as previously described.
Examples of the present invention will now be described with reference to the accompanying drawings, in which:
We describe below an example heater suitable for carrying out thermal cycling for PCR amplification. It is desirable to carry out thermal cycling at a speed fast enough that the time required for temperature changes does not account for the majority of the total time for thermocycling. The total time for thermocycling is the sum of the time for temperature changes and the time for reactions, and the slowest part of the PCR reaction is the extension phase, which requires approximately 1 s or more for a typical sequence length of 100 base pairs. Therefore we target a time of <1 s for temperature ramping. The target temperatures for PCR are typically between 60° C. and 95° C. so we need temperature ramp rates of 70° C./s or more for heating and cooling to reduce the temperature change time to 1 s. Much higher temperature ramp rates (200° C./s or higher) offer limited speed advantage as the total time required will be dominated by the reaction time and not the time required for temperature changes.
In one embodiment described below, a heater for carrying out fast thermal cycling has a temperature ramp rate of approximately 100° C./s without the disadvantages of conventional heaters and temperature control systems.
The heater may, for example, be arranged together with a reaction cell in a single use consumable. The single use consumable may be supplied with the necessary reagents and power to perform a single reaction test and then disposed of.
The heater contains the following elements: main heater tracks configured to enable simultaneous heating and temperature sensing via the heater track's temperature-dependent resistance; guard heater tracks substantially surrounding the main heater tracks; a thermal diffusion layer located between the heater tracks and the reaction surface; and a heater support layer located between the heater tracks and the back surface of the heater. The heater may also be provided with a heat sink in thermal contact with the back surface to allow rapid cooling of the heater when the heater drive power is reduced.
The heater 100 has a reaction surface 110 on one face and a back surface 120 on the opposite face. The reaction surface 110 is heated by the heater to provide a time-variable and substantially spatially-uniform temperature. The back surface 120 is in thermal contact with the heat sink 200 to allow cooling when the heater 100 is not driven.
In the following description, we define an axial direction to be perpendicular to the reaction surface and a lateral direction to be in the plane of the reaction surface.
The heater comprises a main heater track 130 for resistive heating of the reaction surface. However, it is desirable to limit lateral heat flow associated with temperature gradients and temperature non-uniformity across the reaction surface, which reduce the precision of temperature control.
In order to limit lateral heat flow within the area of the main heater track, the main heater track 130 is substantially surrounded by a guard heater track 140.
A guard heater track is an additional heater track located near the edge of a main heater track and driven to maintain a temperature close to or greater than a target temperature of the main heater track. The heat output per unit area of the guard heater track is higher than that of the main heater track to compensate for lateral heat loss. The guard heater track may be driven independently from the main heater. The main heater track 130 and the guard heater track 140 may, for example, be formed from a metal such as copper.
The main heater track 130 and the guard heater track 140 are located between a heater track support layer 150 and a thermal diffusion layer 160. The heater track support layer 150 may, for example, comprise a printed circuit constructed from FR4 or polyimide or another electrically insulating support material.
A reaction surface heat spreader layer 170, 180 is within or in contact with each of the heater track support layer 150 and thermal diffusion layer 160. The reaction surface heat spreader layer 170, 180 is a layer of a material with higher thermal conductivity than the thermal diffusion layer or heater track support layer. The function of the reaction surface heat spreader layers is to increase the temperature uniformity on a reaction surface 110. Each of the reaction surface heat spreader layers has a thickness Hs, thermal conductivity ks, density ρs, and specific heat capacity Cs, while the heater track support layer 150 and the thermal diffusion layer 160 each have respective thicknesses HB, HD thermal conductivities kB, kD, densities ρB, ρD and specific heat capacities CB, CD. In order to increase temperature uniformity while maintaining fast temperature response, the reaction surface heat spreader layer must have a greater lateral thermal conductivity and/or a lower heat capacity than the heater track support layer 150/thermal diffusion layer 160. For the heat spreader layer to have greater lateral thermal conductivity than the thermal diffusion layer, Hsks>HDkD. For the heat spreader layer to have a lower heat capacity than the thermal diffusion layer, HsρsCs<HDρDCD. For the heater support layer, these conditions are replaced by Hsks>HBkB and HsρsCs<HBρBCB respectively.
Each of the reaction surface heat spreader layers 170, 180 is located near to the main heater track 130. In this particular example, the reaction surface heat spreader layer 170 in the thermal diffusion layer 160 is provided at a distance of 10 μm from the upper surface of the main heater track and the reaction surface heat spreader layer 180 in the heater track support layer 150 is provided at a distance of 5 μm from the lower surface of the main heater track.
The back surface 120 is also provided with a back surface heat spreader 190 to increase temperature uniformity on the reaction surface 110.
The heat sink 200 may take any form, including the solid block shown in
To achieve a good thermal contact with the main heater track, a thermal resistance×area product between the main heater track and the back surface heat spreader or the heat sink should preferably be in the range 1×10−4 to 1×10−2 K·m2/W and more preferably in the range 3×10−4 to 3×10−3 K·m2/W.
The heater and heat sink (if used) can have planar or curved forms. A planar form may be preferred for ease of construction and optical monitoring of a reaction for which the heater is to be used. However other forms such as part-spherical or cylindrical are possible and these may have benefits in allowing tensioned flexible reaction cell and heater layers to make good thermal contact with each other and with the heat sink which is typically a rigid metal part.
As illustrated in
A spatially separated temperature sensor could cause a time lag between temperature changes at the main heater track and temperature changes at the temperature sensor. This time lag could cause problems such as overshoot or oscillation of the heater element temperature. To avoid these problems, the main heater track is configured as a temperature sensor where the resistance of the heater element is used to determine its temperature. A metallic heater element will usually have a positive temperature coefficient of resistance while a metal oxide or semiconductor heater element will have a negative temperature coefficient. It is desirable that the magnitude of the temperature coefficient of resistance (TCR) of the heater element is large, preferably greater than 500 ppm/K, and more preferably greater than 2,500 ppm/K.
The main heater track 130 has 4-wire connections comprising electrical drive positive and negative connections 132 and 134, and voltage sense Vsense positive and negative connections 136 and 138. Measurements of Vsense can be used to accurately monitor of the track resistance using a circuit such as shown in
The guard heater track 140 has positive and negative connections 142 and 144 to be driven independently from the main heater track 130.
The current flowing through the heater track 130 is measured using a current sense resistor 320 with known resistance Risense and a voltage measuring circuit 330 for measuring the voltage Visense across the current sense resistor. The current through the heater is calculated as: Iheater=Visense/Risense. The resistance of the heater track 130 is then calculated as Rheater=Vsense/Iheater. Feedback-based driving of the main heater track may then be performed according to a sequence of temperature set points.
Temperature control is implemented by determining setpoint values of Rheater corresponding to desired temperature setpoints and controlling the heater drive to meet the heater resistance setpoint values. Alternatively, temperature control may be performed continuously across a temperature range based on the known temperature coefficient of resistance, TCR. A switch 340, which may be a transistor, is turned on to measure the heater resistance and is then turned off or left on for a predetermined time interval depending on whether Rheater is above or below a currently required setpoint resistance. Alternatively, the switch 340 may driven with a pulse width modulated waveform with duty cycle selected to drive the heater with the required power. In both approaches the switch 340 is used to modulate the electrical drive to the main heater track to cycle the temperature of the reaction surface to carry out PCR amplification.
The guard heater track may be operated with closed loop control with a temperature setpoint equal to or greater than the temperature setpoint of the main heater track or the guard heater track may be operated with the same controller or on/off timing as the main heater element but with a different drive voltage which can be adjusted to optimise the temperature uniformity at a specific temperature setpoint.
Referring back to
Turning to
In
As an example, a heater according to the invention may be used for providing heat to a reaction. In such a usage, the reaction surface of the heater is located in contact with a reaction cell having a reaction volume containing a sample. In order to heat the reaction surface, the heater element is switched on, and heat generated by the heater element flows through the reaction surface into the reaction volume. If rapid cooling is required, the heater can contact a heat sink on its back surface so that when the heater element is switched off, heat flows from the reaction surface, through the heater and into the heat sink.
When the heater is applied for thermal cycling, such as for driving PCR reactions, it is advantageous for the thermal diffusion time between the heater and the sample to be small compared with the target cycle time. In general, thermal diffusion time t for a material sample is given by:
t=L
2
/D,
where L is a characteristic length scale of the material sample and D is the thermal diffusivity of the material. Table 1 below shows an example choice of materials for a heater according to the invention, in which the thermal diffusion time of the thermal diffusion layer is less than the reaction time for PCR, which we take as approximately 1 s for amplification of a 100 base pair DNA sequence.
Additionally, the thermal resistance of the heater track support layer RT can be optimised to minimise the thermal cycling time for a given temperature profile and heatsink temperature TSink and heater power pHeat. The time required for thermal cycling between a TLOW and THIGH is minimised when the heating time is equal to the cooling time and this condition is satisfied when RT=RT,Opt as follows:
R
T,Opt=(THIGH+TLOW−2TSink)/pHeat.
Table 2 (below) shows example values for heater power, optimal thermal resistance and thermal cycle time. These are shown for the case of a reaction surface with area 50 mm2 and with heat capacity 0.04 J/K, cycling between 60° C. and 95° C. with a heat sink temperature of 30° C.
Table 3 (below) shows example values for heater power, optimal thermal resistance and thermal cycle time for a case where the thermal cycle includes a hold step of 1 s duration at 72° C. These are shown for the case of a reaction surface with area 50 mm2 and with heat capacity 0.04 J/K, cycling between 60° C. and 95° C. with a heat sink temperature of 30° C.
The embodiment of
As shown in
The graph in
In the above-described embodiments, the heater is provided in an assembly with a heat sink. However, the invention is also applicable in cases where uniform heating is required, but a heat sink is not required. For example, the heat sink may be omitted for applications where a cooling time is less important.
In the above-described embodiments, the heater is provided with a guard heater track 140. However, in addition to or instead of providing the guard heater track 140, the main heater track 130 may be designed to have higher heat output near its edges and to extend beyond the reaction volume. This higher heat output effect may be achieved by increasing the density of the heater track by reducing the gap width between two or more heater track portions near the edge of the main heater track as compared to heater track portions at the centre of the main heater track. Additionally or alternatively, this effect may be achieved by increasing the resistance of the main heater track by reducing the width or height of one or more heater track portions near the edge of the main heater track as compared to heater track portions at the centre of the main heater track. The higher heat output of the heater element near its edges can compensate for lateral heat flow and provide more uniform temperature conditions across the reaction volume. Furthermore, where a heater has a reaction surface that extends significantly beyond the required reaction volume, it is possible to omit both of the guard heater track and the modifications near the edge of the main heater track.
Additionally, in the above description, comparisons are evaluated between including and omitting each of the heat spreaders. Accordingly, the reader will understand that, while preferably included, each of the heat spreaders 170, 180 and 190 may be omitted in embodiments of the invention.
Number | Date | Country | Kind |
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19188688.6 | Jul 2019 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2020/071059 | 7/24/2020 | WO |