WO2003006161A2

WO2003006161A2 - Microchannel thermal reactor

Info

Publication number: WO2003006161A2
Application number: PCT/CA2002/001057
Authority: WO
Inventors: Dongqing Li; David Erickson
Original assignee: Dongqing Li; David Erickson
Priority date: 2001-07-11
Filing date: 2002-07-11
Publication date: 2003-01-23
Also published as: WO2003006161A3; AU2002319041A1

Abstract

A low power microchannel thermal reactor for a biological or biochemical reaction. During the reaction, the biochemical or biological sample is heated by the microchannel with embedded in-channel heating elements and cooled by conduction and passive convection.

Description

MICROCHANNEL THERMAL REACTOR

FIELD OF INVENTION

The present invention relates to a low power microchannel thermal reactor.

More specifically, this invention concerns a microchannel thermal reactor for a biochemical or biological reaction with heating using in-channel heating elements and cooling by conduction and convection.

BACKGROUND OF THE INVENTION

Biochemical reactions may be facilitated or precipitated by the addition of thermal energy to the reaction solution. Such reactions would include polymerase chain reactions ("PCR"), for reproducing or amplifying nucleic acid sequences from a given sequence of DNA or RNA in a biological sample. The process repeatedly exposes the sample to heating and cooling cycles at specific temperatures to accomplish the desired effect. Typically a PCR cycle consists of 3 heating and cooling stages, each ideally within specific temperature.

A PCR reactor chip of a few square centimeters can produce more than 10 million copies of a target DNA sequence from only a few molecules. The PCR reaction is induced by processing the sample through a specified thermal cycle, each cycle roughly doubling the amount of DNA.

A microchannel PCR reactor has a number of significant advantages over traditional commercially available macro-devices such as that from Cepheid™. Firstly, the amount of the available DNA sample for PCR is typically very small, a chip sized "micro-device" makes the sample handling and processing more efficient. Additionally the high surface area to volume ratio inherent in a microchannel has the potential to allow for very high speed and efficient thermal cycling, minimizing the time required to perform the PCR. Most importantly, however, are the potential cost savings that microchannel PCR reactor chip devices offer (largely because smaller quantities of expensive reagents are required), which could result in more widespread applications of the technology and greater potential revenue from these devices.

One of the significant drawbacks of current on-chip devices is the large amount of electrical power required to generate a significant amount of heat and high temperatures in PCR thermal cycles. This prevents the existing PCR chips from being operated by using low cost batteries. Consequently, while a few studies have performed on-chip PCR, the devices are limited to either "proof-of-the-concept" demonstrations or those which are not designed to be truly stand alone as they require some forms of external support to perform the thermal cycling and/or to transport the sample. A typical example is ref. [3] where the PCR was conducted on a chip but the thermal cycling was performed by placing the entire chip in a commercial oven. Another device is the silicon microchambers of ref. [2], where rapid cycling is achieved in a small chamber with a large oil drop used for insulation and to prevent evaporation. A claim that this device could be adapted for battery operation is unrealistic in view of the fact that it requires 2.0 W per chamber to reach the highest temperature. Additionally it is not a "flow through" device and thus requires significant external pre- and post- handling of the sample. To date the only PCR reactor that does not require external support is a briefcase sized device detailed in ref. [4] and - available from Cepheid™ under the product name "Smart Cycler"™ XC (Extreme Condition) System (The reactor details are given in ref. [5]. This is not a microchannel reactor but a smaller version of a large reactor). This device requires relatively high voltages to perform the cycling and has been made "portable" only by incorporating a large, heavy, expensive, high voltage, rechargeable power source.

Applications of performing temperature controlled reactions in microfluidic systems have been discussed in the prior art. US patent Ser. No. 5,736,314 uses a long capillary tube to run a fluid through individual temperature controlled regions. US patent Ser. No. 6,203,683 discloses the use of electrodes attached to a direct current course to heat fluid to specific temperatures in a microchip. US patent Ser. No. 6,174,675 teaches the principle of resistive electrical heating to control the liquid temperature within a microchannel. This heating method relies on applying an electric field across a microchannel; the electrical current passing through an electrical resistive liquid will generate heat in the liquid. In all of the above cases, a large voltage is required to heat the fluids to those temperatures that are required for biochemical reactions, more specifically PCR. As a result, expensive, external power sources are needed to generate the needed large voltages.

From the above, it is clear that a device and method that allows control and maintenance of the temperature in a safe, inexpensive and efficient manner is highly desirable, preferably one that uses lower voltage.

SUMMARY OF THE INVENTION The present invention relates to a low power microchannel thermal reactor. More particularly, the present invention provides a technique, including methods and devices, for generating a biochemical or biological reaction with heating using in- channel heating elements and cooling by conduction and convection. These methods and devices are useful in a broad range of analytical and synthetic operations. One variation of the invention concerns polymerase chain reactions (PCR), amongst other fields of application.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing two complete ideal thermal cycles for a PCR reaction.

Figure 2 is a diagram showing an overall view of the configuration of a preferred embodiment of an on-chip microchannel reactor with peripheral components forming a reactor chip. Figure 3 is a diagram of a cross-sectional top view parallel to microchannels of a typical reactor, showing 3 microchannels and 3x2 internal heating element configuration.

Figure 4a is a cross-sectional top view showing x-z plane along horizontal symmetry axis of a typical microchannel thermal cycling reactor, 3x2 heating elements per channel configuration (not to scale).

Figure 4b is a cross-sectional side view showing the x-y plane of a single microchannel at an arbitrary location along the z-axis of a typical microchannel thermal cycling reactor. Dashed line represents computational domain.

Figure 5 is a graph of the electro-osmotic velocity profile in computational domain for a 200x200 micron microchannel and an applied voltage of 4V. Ionic concentration of fluid is 10^"3 M.

Figure 6 is a graph showing the influence of total applied electroosmotic driving voltage on per microchannel volume flow rate for microchannels of different cross- sectional area. Results show are for a fluid of concentration 10^"3 M in a 40 mm long glass microchannel (ζ = -56mV).

Figure 7a is a diagram showing temperature contours within a lOmW/channel microchannel reactor during a heating phase for a x-z cross-section showing entire microchannel along the horizontal symmetry axis. Lighter colors represent warmer temperatures and contour labels show temperature in degrees Celsius. Figure 7b is a diagram showing temperature contours within a 10mW/channel microchannel reactor during a heating phase for a x-z cross-section in the x-y plane at z » 20 mm.

Figure 8 is a diagram showing the influence of total heating element power cycle time. Shown is the centerline temperature in a 200x200 micron microchannel and a 4 V total electroosmotic driving force.

Figure 9 is a diagram showing the influence of reactor power on initial heating time. Electroosmotic driving voltage for this case is 4 V.

Figure 10 is a cross-sectional side view of a reactor on a chip. Figure 11 is a diagram of a computer-controlled system incorporating a reactor on a chip.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a low power microchannel thermal reactor and a method for facilitating biological or biochemical reactions using such a device. ^■ This invention also include a microchannel reactor chip which incorporates the microchannel thermal reactor for such reactions, and a computer controlled system which further incorporates the microchannel reactor chip. Such a reactor of possibly a few square centimetres can be used to produce, for example, more than 10 million copies of a target DNA sequence from only a few molecules in a PCR reaction. The reaction is induced by processing the sample through at least one thermal cycle, for example, in the case of DNA replication, typically doubling the amount of DNA during each cycle. Applications for this device and method include PCR reactions, but are not excluded to such.

A reactor chip incorporating the microchannel thermal reactor can accomplish rapid thermal cycling and sample transport using only an inexpensive, lightweight, "off the shelf battery as the only power source. It uses direct in-channel heating and cooling to perform the thermal cycling and a preferred embodiment utilizes low voltage electro-osmotic pump for sample transport. Though it is recognized that the heating and control aspects of the present invention are readily adaptable to systems utilizing other material transport systems.

Essentially all solid surfaces have an electrical double layer when they are in contact with aqueous solutions. The strength and other properties of the electrical double layer field depend on the material of the solid surface (e.g., glass or silicon or plastic) and on the liquid properties. For example, if the wall is negatively charged, it would attract many positively charged ions to the electrical double layer region in the liquid near the wall. So there is a net charge in the double layer region. The reaction may occur while the sample is stationary in a microchannel or while the sample is in transit through the microchannel.

In this document, a thermal reactor 40 (or reactor 40) is defined as a sample reservoir 10 (adapted to receive a single sample fluid), a microchannel network 120 with all its microchannels 110 fluidically connected to the sample reservoir 10, and all product reservoirs 30 receiving reaction product flowing from the said microchannels 110. A reactor chip 140 may have multiple reactors 40, each reactor 40 having its own sample reservoir 10, microchannel network 120, and product reservoirs 30.

Figure 2 shows one embodiment of a reactor chip with thermal reactor 40 and peripheral components. In this configuration for a PCR reaction (as an example), a sample reservoir 10 contains a low concentration of a target DNA sequence .

(received in the sample reservoir 10 by an earlier step). The electrodes90 placed at the two ends of the microchannel network 120 are connected to a power source 50 (which may be on- or off-chip) for applying an electrical field along each microchannel. As shown, the electrodes 90 are placed in the sample reservoir 10 and the product reservoir 30 near the ends of the reactor 40 containing the microchannels 110 (see figure 3). When the reaction is to be initiated the circuit switch 20 is closed, exposing the upstream (sample) reservoir 10 and downstream (product) reservoir 30 to an electric potential difference due to the power source 50. This induces electroosmotic flow (i.e. electro-osmotic pumping) through the microchannels 110 of the microchannel network 120 and the target DNA sequence is transported into the microchannels 110. Then because of the viscous effect, the moving counterions drag the surrounding water molecules to move with them, resulting in a bulk liquid motion, the electro-osmotic flow. (This invention also includes the variation where flow is driven by a gradient in fluid pressure instead of electro-osmotic effect.) As shown, an on-board electronics member 60 may also control the amount and the time of electrical power supplied to the electrodes 90. Instead of being an on-board system, variations are discussed later where the electronics member 60 is partially or entirely external to the reactor chip and consists of a number of electrical and electronics components. Figure 3 shows the internal workings of one possible embodiment of a reactor 40 with 3 microchannels 110 in its microchannel network 120 (a reactor 40 could also have a single microchannel 110 for its microchannel network 120). One or more embedded heating elements 80 arranged non-overlapping consecutively along the length on one or more sides of the microchannels 110 (integrally part of the internal surface wall) provide direct, in-channel, heating. The heating elements 80 may expose the sample to a series of temperature pulses in a pre-determined number of cycles (at least one), in a preferred embodiment mimicking the ideal thermal cycle shown in Figure 1 for PCR reactions . Cooling is achieved within the microchannel 110 by way of natural conduction through and convection from surface(s) of the reactor 40 (being typically the top and/or bottom surface of the reactor chip 140). The convection may be active or passive, in the latter case not requiring an additional active cooling system (like many of the other devices in the field). As the sample is transported through the microchannel network 120 it may be exposed to a predetermined number of full temperature cycles (typically in the order of 10 to 20 cycles depending on the applied voltage and battery power) each cycle approximately doubling the amount of DNA for a DNA PCR. Once through the microchannel network 120 the streams from separate microchannels 110 will be collected in one or more product reservoir 30 to await further analysis. The microchannel network 120 comprises typically 3 portions: a planar upper substrate 260, channel network elements 250, and a planar lower substrate 270. These 3 portions may be formed integrally from a single piece, or may be the result of composition of individual pieces. A preferred embodiment of the latter is discussed later (see Figure 10). The channel network elements 250 are typically a number of thin parallel longitudinal elements formed between the upper substrate 260 and the lower substrate 270. The microchannels 110 are defined by the spacing between the upper substrate 260, the lower substrate 270, and the channel network elements 250 coupled together.

Each heating elements 80 is constructed typically of one or more thin metal films (such as platinum, copper, or other metals used in microelectrical circuits); these films may be as thin as about one micron. Each heating element 80 is preferably the electrical resistive type; it is possible, though not as efficient, to use Peltier type heaters 80 also. Peltier type heater 80 requires more complicated devices/additional equipment, and is much less efficient thermodynamically as the simple metal film resistive heater 80. A heating element 80 in a microchannel 110 is defined to be one or more heat transmitting elements (films) providing heat to a longitudinal section of the . microchannel 110. For example, if a microchannel 110 were round in cross-section, then a possible heating element 80 may comprise two metal films equal in length and spanning the same longitudinal section of the microchannel 110, but located 180 degrees apart with each film subtending 90 degrees of the circular cross-sectional region. This is defined to be a singje heating element 80 since the films cover the same longitudinal section of the microchannel 110. Each heating element 80 (also known as heater 80) has length from about 10 to 100 percent of the total microchannel 110 length. The preferred range of coverage of such a single heating element 80 is 90 to 100 percent of the longitudinal span of a microchannel 110.

In a preferred embodiment, there would only be a single heating element 80 with a single heat transmitting (e.g. a film) for each microchannel 110 in the microchannel network 120; the heat transmitting element is preferably located directly opposite the side of the microchannel 110 (typically adjoining a chip surface) which allows cooling.

In a fashion similar to the integrated electrical circuits (for creating electroosmotic movement), each heating element 80 embedded in the microchannel 110 is connected by wires (preferably printed on the chip or thin film wire adhered on the chip surfaces) to an on-board electronics member 60 on the chip that controls the amount and the time of electrical power supplied to each resistive heater 80. One or more sensors may be placed along the microchannel for detecting the thermal characteristics of the channel and connected to the on-board electronics member 60. Such sensors could be one of a variety of sensors, such as resistance temperature detector (RTD), a thermistor, or a thermocouple. A sensor may be embedded in a side of the microchannel or placed to run in part within and along a section of the microchannel. In this way, the temperature at every material location in each microchannel 110 of a reactor 40 chip can be independently detected and controlled with great accuracy. This chip also provides optional connection between the heating elements 80 and a power source 50 (e.g., the battery power system). The power source 50 may drive both the electro-osmotic flow and the heating element 80. Similarly, the on-board electronics member 60 may also be the same as that controlling the amount and the time of electrical power supplied to the electrodes 90 for inducing electro-osmotic fluid flow through the microchannels 110 (or to drive the fluid pressure gradient in a different variation). As mentioned earlier, in variations of this, the electronics member 60 may reside externally (partially or entirely) to the reactor chip 40.

The cross-sectional shape of the microchannel 110 may be of any general shape, whether square, round, oblong, or trapezoidal, for example, and are practically limited by the method and means of manufacture A microchannel network 120 having a single microchannel 110 with an oblong cross-section results in a reactor 40 of high throughput for as the regions between multiple microchannels 110 are eliminated. The radial cross-sectional surface area of a single microchannel 110 is typically in the range of about 25 square micrometers to 250,000 square micrometers for considerations of cooling. A preferred embodiment has cross-section height of between 100 to 200 micrometers.

The material forming the upper and lower substrate 260 270 may be silicon, glass, plastics, or ceramics, or combinations thereof. The upper and lower surfaces (i.e. those above and below the plate into which the microchannels 110 are etched in one embodiment of the reactor) are typically made of a low thermal conductivity (between 0.1 and 20 W/mK) material such as glass or plastic, which is non-reacting with respect to the sample. The material between the upper and lower surfaces (i.e. the section of the substrate into which the microchannels 110 are to be etched) would also preferably be made of glass or plastic, however a silicon based material may be used. In order to assure that the sample solution does not react with the electrical components, such as the heating element 80, a passivation layer (e.g. Silicon oxide) may be added also to the wall of the microchannel 110.

Concerning the thickness of the channel wall between the microchannel 110 and an external side of the substrate 260 270 which provides cooling (typically the upper substrate 260), the thickness of this substrate 260 (or more appropriately the distance between the top or bottom of the microchannel 110 and the solid-air interface) has an impact on the heat transfer; however the significance of its importance is dependent on the particular application.

In general a thicker upper substrate 260 will reduce the rate of heat lost from the microchannel 110 to the atmosphere (i.e. heat loss from the microchannel 110 will be less significant) and as a result the heating can be done more rapidly. As a trade off however this same affect tends to slow the cooling process (i.e. the microchannel 110 can not get rid of its heat very rapidly). So as the microchannel 110 cover is increased in thickness the heating takes less time but the cooling takes longer (and thus the microchannel 110 cover thickness is decreased the heating takes longer but the cooling is faster). As a result the effect of cover thickness on the total cycling time is not significant.

Having said that, as the cover thickness increases the total cycle time does tend to slightly increase as well. However this is offset by an increase in heating efficiency (i.e. there is more "insulation" so there is less need to add as much heat). The final thickness will depend on the user's demands (for example if the emphasis is on cycle time then a smaller cover plate will be used, while if the emphasis is on efficiency then a thicker one will be used).

Typically, for PCR thermal cycling it is possible to adopt as the minimum cover thickness of about 400 microns (below this thickness the device may not be mechanically sufficiently strong). A preferable upper boundary is about 3 mm since cooling tends to get very slow above this point.

The design makes it possible for such a chip to use at most a standard 9V battery 50 for very high flow rate applications and as little as a typical watch battery for less demanding cases.

Because the flow in microchannel 110 is typically so slow, the convection heat transfer between the liquid sample and the microchannel 110 wall is very small. The main heat transfer mechanism is the heat conduction. Once the heat is conducted to the outer wall, it will be transferred to the air via convection heat transfer from the top and the bottom surfaces of the glass chip (being preferably the reactor cover plate). Because of the small heating power (about 10-15 mW/channel) typically used in the chip, the convection to the air from the top and/or the bottom chip surfaces is sufficient to generate rapid cooling.

As mentioned above, the invented reactor uses a combination of an electro- osmotic pump and localised, in-channel heating to heat it. The use of small microchannel(s) 110 with in-channel heating significantly reduces the amount of power required to perform the high speed thermal cycling. The PCR cycle shown in Figure 1 can be performed at powers as low as 10 to 15 mW per microchannel 110 (each microchannel 110 having an effective volume of 1.2 μL). The microchannel 110 design and low voltage electro-osmotic pump also have significant advantages in that there are no moving parts, the sample may either be run continuously or on a batch basis, and most importantly no external handing is required. The ultra low power and voltage requirements allow it to be operated using as little as a simple watch battery 50 in some cases, while a common 9V battery 50 would be capable of powering a high flow rate (on the order of 1 μL/min) reactor 40 comprised of from one to as many as individual microchannels 110 as needed. Such a reactor 40 could be integrated as part of other more complex lab-chip devices for genetic or biochemical processes. These devices would control the heating characteristics and the flow rate, and provide the peripheral components such as the power source 50, the reservoirs (10, 30), etc.

Figure 10 provides a cross-sectional side view of a preferred embodiment of the reactor chip with printed circuit board 220 ("PCB"). Power is provided to the reactor chip via a power connector 210; the sample solution is provided to the sample reservoir 10 (not shown) by the fluid inlet connector 240. Bond wiring 230 preferably provides electrical connection from the PCB 220 to the lower substrate 270 for both the heating element(s) 80 and the temperature sensing elements (e.g. RTD). The microchannel network 120 is defined by the top reactor plate 260, the lower substrate 270, and the channel network elements 250, each channel network element 250 comprising an upper and a bottom channel element part. Each upper channel element part may be carried by the top reactor plate 260 (typically glass); and each bottom channel element part preferably flip-chip glued to the lower substrate 270 (also typically glass). In a variation each channel network element 250 may be integral to the lower substrate 270 (and not comprising two parts). The heating element(s) 80 may be integral to the channel network elements 250, or embedded in the lower substrate 270 (flip-chip glued to the lower substrate 270); similarly, the temperature sensing elements may be integral to the channel network elements 250, or embedded in the lower substrate 270. Passivation with SiO₂ is done on exposed surfaces (e.g. for the upper substrate 260, the lower substrate 270, and any heating elements 80 or temperature sensing elements). A carrier 280 may underlie the entire reactor 40 or reactor chip 140 to provide a base; the carrier 280 may also be integral with the lower substrate 270 in a variation.

A preferred embodiment of a computer controlled system which implements feedback control of the temperature of a microchannel network 120 (one or more microchannels 110) is illustrated in Figure 11. The electronics member 60 is shown as 3 components: a power amplifier and controller 160, a temperature measurement electronics member 180, and a computer 190. A high frequency AC function generator 150 produces a high frequency wave form (typically a 20 to 50 kHz sine wave) as the power supply. The power is supplied to the power amplifier and controller 160, which interprets a control signal from the computer 190 and amplifies the high frequency signal from the function generator 150 to adjust power delivered by the heating elements 80. The system first reads the change in resistance of the embedded RTD's in microchannel(s) 110 and converts it to an analog voltage signal via the Wheatstone bridge circuits in the temperature measurement electronics member 180. The analog voltage signal representing the temperature value is filtered to remove any high frequency noise (approximately 20 to 50 kHz) that may be induced by the power supplied.

The analog signal is digitized by an A/D converter in data acquisition hardware (e.g. a card) on the computer 190 The digital reading is converted to a temperature value via a previously determined calibration relationship. A PID control algorithm based on the following equation is then used to determine how much power should be supplied to each heating element 80 of the microchannel(s) 110,

W = P(T_s -T) + D _t {T_s -T)+

-T)dt

where P, D and I are the proportional, derivative and integral gains respectively, T_s is the temperature set point and T is the measured temperature. In essence the proportional control adjusts the power based on the difference between the current temperature and the set point, the derivative control minimizes/eliminates the overshoot and the integral control forces the difference between the set-point and the average temperature to zero. The amount of power that should be supplied to the heating element(s) 80 is then converted to a digital value and sent to the power amplifier and controller 160. In the power amplifier and controller 160 the serial signal is then received by an micro-controller which outputs a serial word to a digital potentiometer (e.g. MCP41010 by Microchip) which adjusts the output gain by a (preferably) separate amplifier circuit. The amplifier circuit is used to amplify the power/voltage of the high frequency signal (typically 20kHz and over) from the function generator 150 to that required by the heating elements 80. The combination of the amplifier circuit, microcontroller and associated electronics functions as a computer controlled high frequency power source. High frequency power should be used to minimize any electrolysis that may occur in highly concentrated buffers. The amplifier output is sent to the reactor chip and applied across the embedded heating elements 80. The biological or biochemical sample may comprise a plurality of a thermostable DNA polymerase, a plurality of nucleotides, a nucleic acid template, and at least one primer which hybridizes to the nucleic acid template; alternatively, instead of primer, the sample may include Mg++. The nucleic acid template above may be a DNA template. In a different reaction, the sample may comprise reverse transcriptase, a plurality of nucleotides, a nucleic acid template and Mg++. The nucleic acid template may be mRNA.

This invention also includes a method for thermocycling a biological or biochemical sample during a reaction using devices as described above with in- channel heating element 80 embedded in the microchannel(s) 110 of the devices. The method consists of directing the sample to flow through the microchannel network 120, and cyclically heating and cooling the sample at pre-determined temperatures while directing the sample to flow through the microchannel network 120. During the flow, the sample would be heated using the heating element 80 and cooled by conduction and convection through at least one surface of the device. The convection may be active, or preferably, passive.

Typically, for a PCR reaction, the sample is heated to a first temperature and maintained at the first temperature for a first dwell time; it is then cooled to a second temperature and maintained at the second temperature for a second dwell time; and finally heated to a third temperature and maintained at the third temperature for a third dwell time, wherein the third temperature is higher than the second temperature but lower than the first temperature. There are a pre-determined number of cycles for a reaction, in one variation between about 5 and 100 cycles are performed, and in others, between 10 and 20 cycles.

The method may be used to amplify DNA, effect protein folding and unfolding, effect sequencing of a protein or peptide, or effect denaturation of enzymes.

As mentioned earlier, this method is usable for other biological or biochemical samples during a reaction. Furthermore, although the devices and systems specifically illustrated herein are generally described in terms of performance of a few or one particular operation, it will be readily appreciated that the flexibility of these systems permits easy integration of additional operations into these devices. Operations (biochemical or biological) that are performed prior to the operations specifically described herein include, but are not limited to, cell separation, extraction, purification, amplification, cellular activation, labelling reactions and dilution. Similarly operations that are performed after the operations specifically described include, but are not limited to, separation of sample components, labelling of components, assays and detection operations.

Numerical Verification Simulations have been conducted using a model of the invention to investigate the characteristics of various configurations. The configurations below are not exhaustive of the variations for the invention.

The nomenclature of the discussion to follow is first indicated below.

A_c cross sectional area of the microchannel 110 [m²]

Bi Biot number, hD_h/k_s

D non dimensional electroosmotic scaling term, D = 2en₀φ_maxD_h/μv_eo

D_h hydraulic diameter [m] K non dimensional Debye-Huckel parameter, K = κD_h

P non dimensional pressure P = pD_h/μv_e0

Pe Peclet number, Pe = pC_pD_hv_eo/k

P_eo Power required to generate electroosmotic flow

Q Volume flow rate [μL/min] Re Reynolds number, Re = pv_eoD_h/μ

T temperature [K\

V non dimensional velocity, V = v/v_eo

X,Y,Z non dimensional coordinate directions, X = x/D_h, Y = y/D_h, Z = z/D_h

c_p specific heat [J/kg K] e electron charge, 1.6021 x 10^"19 C h convective heat transfer coefficient [W/m K] k thermal conductivity [W/m² K] k ^■ Boltzmann constant 1.3805 x 10^"23 J/mol K l_z length of the channel in the z direction [m] n₀ average ionic concentration [1/m³] p pressure [Pa] q volumetric heat generation [W/m³] t time [s] v velocity [m/s] v_eo electroosmotic mobility, v_e0 = εε₀ζφ_max/μlz x,y,z coordinate directions [m]

Greek Symbols

Φ non dimensional applied electric field strength, Φ = Ψ non dimensional electrostatic potential in the double layer, Ψ = eψ/k_bT Z non dimensional zeta potential, Z = eζ/k_bT

ε dielectric constant of the medium ε₀ permittivity of a vacuum, 8.854 x 10^"12 C/Vm φ applied electric field strength [V]

K Debye-Huckel parameter, K = (2n₀e²/εε₀k_bT)^1/2 λ_t total conductivity of the microchannel [1/Ωm] μ viscosity [kg/ms] θ non dimensional temperature, θ = (T-T₀)VeoP_hCph/D_hq p density [kg/m³] τ non dimensional time τ = tv_eo/D_h ψ electrostatic potential in the double layer, [V] ζ zeta potential [V]

Subscripts h heating element f fluid o atmospheric conditions s solid

Superscripts non dimensional parameter

Figures 4a and 4b show a top and a side cross sectional view of the microchannel 110 thermal cycling reactor 40 to be modeled in this study. To promote the symmetry of the problem and minimize the size of the computational domain two assumptions were made. Although only five individual microchannels 110 are shown in Figure 4a, the actual reactor 40 would consist of a large array of microchannels 110. As such it was assumed that the majority of microchannels 110 would be sufficiently isolated from the sides of the reactor 40 that the temperature distribution would be symmetric about both the edge and vertical symmetry boundaries shown in Figure 4b. Furthermore it was also assumed that natural convection occurjed on both the top and bottom surfaces and that the convective heat transfer coefficients were sufficiently similar that the temperature profile was also symmetric about the horizontal symmetry boundary (Figure 4b). Flow Model

In general when a solid surface, such as the microchannel 110 wall, comes into contact with an aqueous solution a charge develops which is characterized by the surface electrostatic potential, or ζ-potential. If the liquid contains a small number of ions, the electrostatic charges on the surface will attract counterions in the liquid with a net force balanced by the thermal energy of the ions. The rearrangement of these charges in the fluid, known as the electrical double layer, or EDL, is described as,

V²ψ - iC ² sinh(Ψ) = 0 , (1)

The proper boundary conditions for Eq. (1) are the non-dimensional ζ-potential, Z, at the liquid solid interface (X = 0, Y = 0) and the appropriate symmetry conditions along the vertical and horizontal symmetry axes shown in Figure 4b. Insulation type boundary conditions were applied at the channel inlet and exit (Z = 0, Z = l_z/D_h).

The applied electric field resulting in electroosmotic flow can be described by

V²Φ = 0. (2)

Note the few assumptions that have been made in using Eq. (2) to describe Φ. The description of the applied electric field by the homogenous Poisson equation is a simplification valid only in cases where the bulk conductivity of the aqueous solution does not change significantly along the length of the microchannel 110. Secondly, by decoupling the equations for the EDL and applied electric field, the charge distribution near the wall is assumed to be unaffected by the externally applied field, which is generally valid so long as double layer thickness is not large, or equivalents the ionic concentration of the solution is not very low. In most practical cases this condition is met. To induce flow in the positive z direction a boundary conditions of Φ = 0 and Φ = -1 were applied at the inlet (Z = 0) and exit (Z = l_z/D_h) of the microchannel 110 respectively. Insulation type boundary conditions were then applied along the solid liquid interface and as before symmetry conditions were used along the symmetry axes.

Eq. (3a) and (3b) then represent the non-dimensional Navier-Stokes momentum equation, modified to account for the electrokinetic body force, and the continuity relation, dV

Re + (Γ - V)F = -V + ΨV + D sinh(Ψ) VΦ , (3a) dτ V - V = 0. (3b)

Knowledge of the physical situation being modeled here allows for significant simplification of the above equations. To induce very high Reynolds number flow extremely large voltage gradients would have to be applied which are well beyond practical on-chip capabilities. Thus consider only the cases where Re « 1 and the transient, convective and inertial terms on the left hand side of Eq. (3a) can be ignored. From Figures 4a and 4b it is apparent that the flow is dominantly uniaxial in the z direction, thus the x and y momentum equations are ignored. Finally in electroosmotic flow both sample and product reservoirs 10 30 are typically maintained at atmospheric pressure and thus for uniaxial flow the pressure gradient term can be ignored. This assumption is only valid in cases where surface properties are constant along the flow axis. Changes in the magnitude of the ζ-potential along this axis can lead to significant changes in the magnitude of the electroosmotic forcing term in Eq. (3a), inducing pressure gradients within the microchannel 110 in order to satisfy continuity.

To properly define the flow equations, no slip boundary conditions were applied at the solid liquid interface and symmetry conditions were applied along the symmetry axes shown in Figure 4b. At both the microchannel 110 inlet and outlet the condition δV/δz = 0 was applied.

Temperature Model

The temperature distribution must consider not only the fluid region but the surrounding solid zone as well. As a result the convection-diffusion problem in the fluid region must be coupled with the purely diffusionary problems in both the heating element 80 and solid substrate 70 (the term "substrate" is used here to denote all surrounding sides of a microchannel 110 including the lower substrate 260, upper substrate 270, and the immediate channel elements 250). Within the fluid zone the non-dimensional energy equation takes the form shown below in Eq. (4a),

— + v - VΘ = — θ . (4a) dτ Pe_f

The solid zone itself can also be further subdivided into the substrate 70 region, where the temperature distribution is governed by the simple transient diffusion equation dτ— P±e-_s V» . ^' (4b)

and the heating element 80 zones which incorporates the uniform heat generation source term on the right hand side, dθ 1 ~_{2 /1} . . ,

— = V 0 + 1 . (4c) dτ Pe_h

As eluded to earlier, a convective heat transfer boundary condition was placed on the lower surface of the computational domain, Figure 4b, described by Eq. (5),

The average convective heat transfer coefficient, used in determining the Biot number, was modeled using the "short vertical square cylinder" relation with the microchannel 110 length as the characteristic dimension. In general h was the order of 6-10 W/mK. As mentioned earlier a consequence of the symmetry assumption along the horizontal symmetry boundary is that the convective heat transfer coefficient must be the same on the top and bottom surfaces of the microchannel 110. Since the average h over the entire surface of the reactor 40 was used it is expected that while slight discrepancies may exist within temperature profile in the x- y plane, the overall results will still be quantitatively correct.

Symmetry boundary conditions were placed on the three symmetry axes in Figure 4b. It was assumed that the microchannel 110 inlet and outlet conditions matched the upstream and downstream reservoir 10, 30 temperatures, which were assumed to be sufficiently large and isolated from the reactor 40 so that their temperatures would be maintained reasonably close to the atmospheric temperature. θ(τ=0) = 0 was applied as an initial condition indicating that the system was initially at the atmospheric temperature.

In this study, constant thermal properties, evaluated at an intermediate temperature, were assumed. Another, more fundamental, assumption implied by the above model, specifically Eqs. (3) and Eq. (4a), is incompressibility.

Numerical Algorithm The flow and temperature models were solved using the finite element method by discretizing the computational domain using 8-noded trilinear "brick" type elements. The flow domain was discretized first and a solutions for Ψ, Φ and V were obtained. This study introduces an independent level of grid refinement within a band of thickness 3/K from the solid liquid interface. Once a solution to the flow field was obtained, the original computational grid was extended to encompass the temperature field. Further grid refinement along the z axis was used in the regions near the beginning and end of the heating element 80 zones, where temperature gradients were found to be strongest.

The thermal cycling in the reactor 40 was simulated by independently controlling each heating element 80 using" an on/off algorithm. At each time step the average temperature of each heating element 80 was monitored to see if it had reached the desired temperature plateau. If the average temperature of the heating element 80 was below the source term, shown in Eq. (4c), was included (indicating an on phase) and if it was above the source term was omitted (indicating an off phase). Once all the heating elements 80 had reached the desired temperature the system was maintained at that level for the desired dwell time using the same on/off algorithm as above. The temporal derivatives in Eqs. (4) were discretized using an implicit Euler method and the system was solved at each time step using a biconjugate gradient stabilized method.

Discussion

It was assumed that the channel substrate 70 was glass and that the heating elements 80 were made of silicon similar to those discussed by others. The fluid was assumed to be an aqueous solution of with an ionic concentration of 10^"3 M. All thermophysical properties were evaluated at an intermediate temperature and held constant throughout the simulations. In general it was found that the distance between microchannels 110, the upper/lower cover thickness and the heating element 80 x-y plane dimensions did not significantly affect the overall results, when varied within the range of interest, and thus these values were held constant throughout all simulations at 200μm, 50 μm, 25μm and 100 μm. The ζ-potential at the liquid glass interface was fixed at -56mV.

1 ) Flow Simulations

Figure 5 shows the electroosmotic velocity profile within the computational domain for a 200 μm by 200 μm microchannel 110 with an applied driving voltage of 4V. Under these conditions the velocity profile is nearly flat over the entire microchannel 110 as opposed to the traditional parabolic profile for pressure driven flow. This velocity profile provides advantages in terms of transport in that all species are convected with the same velocity, independent of their location in the microchannel 110 cross section.

In Figure 6 the relationship between applied electroosmotic driving voltage, Φ_max, and volume flow rate, Q, is shown for microchannels 110 of different size and aspect ratio. Of most interest on this chart is the scale of the volume flow rates obtained at the given voltages, which vary from approximately 1 to 40 nL/min per microchannel 110. The total voltages, rather than the lengthwise gradient, are given on the independent axis to emphasize the fact that the applied potential is of the order of that which can be produced by a micro-device, however it is important to point out that the Q vs. Φ_max relationship given here is only applicable to the 40mm long microchannel 110 used in the study. To extrapolate these results to different lengths the volume flow rate must be scaled by the relative microchannel 110 length (i.e. a microchannel 110 half as long would have approximately doubled the flow rate). Also of interest in Figure 6 is the linear increase in the volume flow rate with increasing Φ_max, and the significant increase in volume flow rate with microchannel 110 cross sectional area. The latter of these does not come without a price however in that increasing the microchannel 110 cross sectional area necessarily decreases the resistance leading to an increase in the required electroosmotic pumping power, as described by Eq. (6) for this application,

2

/ ^}

P_eo however is significantly lower than the power which will be required by the reactor to perform the thermal cycling and as such is not considered a serious limitation.

2) Temperature Simulations

Figures 7a and 7b show the temperature contours in the microchannel 110 reactor 40 along both the horizontal symmetry axis and at a cross section located at the midpoint between the microchannel 110 entrance and exit. The gray boxes indicate the heating element regions, while the thick dark lines show the solid-liquid boundary. Darker regions represent colder temperatures and contour labels indicate temperature in degrees Celsius. By comparing the two figures it is apparent that the more significant temperature gradients occur along the z (lengthwise) axis, whereas temperature is relatively uniform within the x-y cross section. This is a result of the extremely high length to width ratio of the microchannel 110 yielding a much smaller diffusion length scale in the x-y plane than along the z-axis. This result can also serve as a justification to reinforce the applicability of using the average convective heat transfer coefficient to model the natural convection, rather than accounting for the differences between the top and bottom surfaces, since the relatively small temperature gradients in that exist in the x-y plane are not likely to influence the global result.

3) Minimum Power Requirements

The most critical parameter of interest to this invention is the amount of power required to perform the thermal cycling in the reactor 40. First of all it was determined that a minimum power requirement exists below which the system will come to a steady state prior to reaching the desired plateau temperature prohibiting the device 40 from performing the cycle. This minimum power requirement was most significantly influenced by the thermal conductivity of the microchannel 110 material, in that microchannels 110 constructed from materials with a high relatively high thermal conductivity, like silicon, have a significantly higher minimum power requirement than those with lower thermal conductivities, like glass. Longer microchannels 110, where a greater portion of the fluid is more isolated from the atmospheric temperature reservoirs 10, 30, and lower heat transfer coefficients, where convective heat transfer is minimized, and other conditions which resulted in minimizing heat loss from the device 40 were also found to be effective methods of reducing the minimum power requirement. In general changes resulting in a lower minimum power requirement resulted in a significant increase in the cooling times and thus were detrimental to the goal of minimizing the overall cycle time.

4) Influence of reactor power on cycle time

Figure 8 compares the microchannel 110 centerline temperature of identical simulations conducted for at total reactor 40 power of 10 mW/channel, consistent with the minimum heating element power requirement of the reactor 40 configuration detailed in Figures 4a and 4b, and 15 mW/channel. A significant decrease in the cycle time is obtained when the power is increased over that of the minimum power requirement. Figure 9 also highlights this trend by showing the decrease in initial heating time from 25°C to 95°C (used in this study as a benchmark) with increasing heating element power. As is also apparent in Figure 9 is that increasing the reactor 40 power is only effective to a point, above which further increases are simply wasted in not significantly reducing the heating time. Combining this maximum effective value with the minimum power requirement from the previous section is a crucial result in providing a qualitative range over which the reactor 40 will operate most effectively with the minimum power.

It will be appreciated that the above description relates to the preferred embodiments by way of example only. Many variations on the apparatus and method for delivering the invention will be obvious to those knowledgeable in the field, and such obvious variations are within the scope of the invention as described and claimed, whether or not expressly described.

All publications referred to are incorporated by reference in their entirety.

References [1] M.U. Martin, A.J. de Mello, A. Manz, "Chemical Amplification: Continuous Flow PCR on a Chip", Science 280 (1998) 1146-1148.

[2] J.H. Daniel, S. Iqbal, R.B. Millington, D.F. Moore, OR. Lowe, D.L Leslie, M.A. Lee, M.J. Pearce, "Silicon Microchambers for DNA Amplification", Sensors and Actuators A 71 (1998) 81-88. [3] L.C. Waters, S.C. Jacobson, N. Kroutchinina, J Khandurina, R. S. Foote, J.M. Ramsey, "Multiple Sample PCR Amplification and Electrophoritic Analysis on a Microchip", Anal. Chem. 70 (24) (1998) 5172-5176.

[4] M.A. Northrup, B. Benett, D. Hadley, P. Landre, S. Lehew, J Richards, P. Stratton "A Miniature Analytical Instrument for Nucleic Acids Based on Micromachined Silicon Reaction Chambers", Anal. Chem. 70 (5) (1998) 918-

922.

[5] AT. Woolley, D. Hadley, P. Landre, A.J. de Mello, R.A. Mathies, M. A. Northrup, "Functional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DNA Analysis Device", Anal. Chem. 68 (23) (1996) 4081-4086.

Claims

CLAIMSWe claim:

1. A microchannel thermal reactor for carrying out a reaction process on a sample comprising:

(a) a sample reservoir for containing the sample prior to the reaction process;

(b) a product reservoir for containing the sample after undergoing the reaction process;

(c) a microchannel defined by a microchannel network for containing the fluid during the reaction process, the microchannel having a wall and a first and a second terminal end, the first terminal end in fluid communication with the sample reservoir and the second terminal end in fluid communication with the product reservoir; and

(d) a heating element integrally embedded in the wall adapted to receive energy from a power source and deliver thermal energy to the fluid contained in the microchannel during the reaction process, wherein during the reaction process the sample is heated and cooled to predetermined temperatures for a pre-determined number of thermal cycles.

2. The thermal microchannel reactor of claim 1 , further comprising a temperature sensing element for sensing temperature of the sample contained in the microchannel during the reaction process.

3. The thermal microchannel reactor of claim 1 , wherein the sample is cooled to a pre-determined temperature by conduction and convection, convective cooling being assisted actively by a device.

4. The microchannel thermal reactor of claim 1 , wherein the sample is cooled to the second pre-determined temperature by conduction and passive convective cooling.

5. The microchannel thermal reactor of claim 1 , wherein the radial cross-sectional surface area of the microchannel is in the range of about 25 square micrometers to about 250,000 micrometers.

6. The microchannel thermal reactor of claim 1 , wherein the substrate is a material with thermal conductivity in the range of about 0.1 to 20 watts per meter kelvin.

7. The microchannel thermal reactor of claim 1 , wherein the material of the microchannel network comprises a material selected from the group consisting of glass, silicon, plastics, or ceramics, or combinations thereof.

8. The microchannel thermal reactor of claim 1 , wherein the maximum thickness of the substrate surface used for conduction and convection is about 3 millimetres.

9. The microchannel thermal reactor of claim 8, wherein the minimum thickness of the substrate surface used for conduction and convection is about 300 micrometres.

10. The microchannel thermal reactor of claim 1, wherein the heating element extends to cover from about 10 to about 100 percent of the length of the microchannel.

11. The microchannel thermal reactor of claim 1 , wherein the heating element comprises at least two heat transmitting elements.

12. The microchannel thermal reactor of any one of claims 10 and 11 , wherein the heating elements are of a type selected from the group comprising Peltier heater and resistive heater.

13. The microchannel thermal reactor of claim 12, wherein the heat transmitting elements are resistive heaters and each heater is a metallic film.

14. The microchannel thermal reactor of claim 13, wherein the metallic film is constructed of a material chosen from the group comprising platinum and copper.

15. The microchannel thermal reactor of claim 1, wherein the reaction is a polymerase chain reaction for reproducing or amplifying nucleic acid sequences in a biological sample, and a thermal cycle comprising:

(a) heating the sample to a first temperature and maintaining the first temperature for a first dwell time;

(b) cooling the sample to a second temperature and maintaining the second temperature for a second dwell time; and (c) heating the sample to a third temperature and maintaining the third temperature for a third dwell time, wherein the third temperature is higher than the second temperature but lower than the first temperature.

16. The microchannel thermal reactor of claim 15, wherein the first, second, and third temperatures lie in the range of about 40°C to about 100°C.

17. The microchannel thermal reactor of claim 1 , wherein the sample comprises a plurality of a thermostable DNA polymerase, a plurality of nucleotides, a nucleic acid template, and at least one primer which hybridizes to the nucleic acid template.

18. The microchannel thermal reactor of claim 1 , wherein the sample comprises a thermostable DNA polymerase, a plurality of nucleotides, a nucleic acid template and Mg++.

19. The microchannel thermal reactor of any of claims 17 and 18, wherein the nucleic acid template is a DNA template.

20. The microchannel thermal reactor of claim 1 , wherein the sample comprises reverse transcriptase, a plurality of nucleotides, a nucleic acid template and Mg++.

21. The microchannel thermal reactor of claim 20, wherein the nucleic acid template is mRNA.

22. The microchannel thermal reactor of claim 1 , wherein the microchannel wall is coated with a passivation layer.

23. The microchannel thermal reactor of claim 1 , wherein the microchannel network comprises:

• a top reactor plate;

• a channel network coupled to the top reactor plate; and

• a bottom reactor plate coupled to the channel network; wherein the channel network comprises two longitudinal channel elements disposed in parallel and defining thereby the microchannel.

24. The microchannel thermal reactor of claim 23, wherein each channel element comprises an upper channel element part and a lower channel element part, the upper channel element part being attached to the top reactor plate and the lower channel element part being attached to the bottom reactor plate.

25. The microchannel thermal reactor of claim 1 , wherein the reaction process takes place while the sample is in continuous flow.

26. The microchannel thermal reactor of claim 1 , wherein the reaction process takes place while the sample is stationary in the microchannel.

27. A thermal reactor chip for carrying out a reaction on a sample comprising:

(a) the microchannel thermal reactor of claim 2; and

(b) a sample transport member for transporting the sample from the sample reservoir to the product reservoir.

28. The thermal reactor chip of claim 27, where the transport member comprises an pressure application element for applying a fluid pressure gradient between the sample reservoir and the product reservoir.

29. The thermal reactor chip of claim 27, where the transport element comprises an electrode disposed in each of the sample reservoir and the product reservoir, wherein a voltage difference may be applied across the said electrodes for inducing electro-osmotic flow of the sample from the sample reservoir to the product reservoir.

30. The thermal reactor chip of claim 27, wherein the voltage difference is applied by power supplied by an on-chip voltage source switchably connected to the electrodes.

31. The thermal reactor chip of claim 29, wherein an electronics member controls the amount and duration of voltage difference applied across the electrodes.

32. The thermal reactor chip of claim 29, wherein the electronics member: • controls the amount of energy received by the heating element from the power source; and

• is responsive to the temperature of the sample contained in the microchannel sensed by the temperature sensing element.

33. A system for carrying out a reaction on a sample comprising:

• the thermal reactor chip of claim 27; and

• a power source for controllably providing energy to the heating element, the power source being responsive to temperature of the sample contained in the microchannel.

34. The system of claim 33, the power source is comprised of:

• a function generator for delivering an electrical energy; and

• an electronics member, electrically connected to the temperature sensing element and responsive to temperature of the sample contained in the microchannel, for receiving the electrical energy from the function generator and variably supplying an electrical current to the heating element.

35. The system of claim 34, wherein the electronics member is comprised of:

• a power amplifier and controller connected to the function generator responsive to a control signal for supplying the electrical current to the heating element;

• a temperature measurement electronics member connected to the temperature sensing element for converting the temperature sensed to an analog temperature signal; and

• a computer comprising an electronic processor and input-output devices for carrying out the steps of:

• receiving the analog temperature signal from the temperature measurement electronics member;

• determining the amount of energy to be supplied to the heating element based on the analog temperature signal using a PID control algorithm; and

• sending a control signal representing the amount of energy to be supplied to the heating element to the power amplifier and controller.

36. The system of claim 35, wherein the temperature measurement electronics member comprises:

• a wheatstone bridge circuit for generating the analog temperature signal from the temperature sensed; and

• a band-stop filter for eliminating high frequency noise in the analog temperature signal if the function generator delivers an AC electrical current.

37. A method for carrying out a reaction on a biological or biochemical sample using the microchannel reactor of claim 2, comprising the steps of:

(a) providing a sample to the sample reservoir;

(b) directing the sample to flow into the microchannel network;

(c) heating and cooling the sample at pre-determined temperatures for a predetermined number of thermal cycles; and

(d) directing the sample to flow to the product reservoir.

38. The method of claim 37, wherein convection cooling occurs passively.

39. The method of claim 37, wherein the reaction is a polymerase chain reaction and each thermal cycle comprises:

(a) heating the sample to a first temperature and maintained at the first temperature for a first dwell time;

(b) cooling the sample to a second temperature and maintained at the second temperature for a second dwell time; and

(c) heating the sample to a third temperature and maintained at the third temperature for a third dwell time, wherein the third temperature is higher than the second temperature but lower than the first temperature; and

40. The method of claim 39, wherein the method is used for a purpose chosen from the group consisting of amplifying DNA in a sample; effecting protein folding and unfolding; sequencing of a protein or peptide; and effecting denaturation of enzymes.