JP2001515204A - Microfluidic system with electrofluid control and electrothermal control - Google Patents

Abstract

  (57) [Summary] Methods and devices are provided for transporting and / or monitoring a fluid in a multiport device. The multi-port device comprises a substrate with a defined channel arrangement. A first channel region having a first port and a second port for transporting a fluid between the first port and the second port is defined in the substrate. A second channel having a first port and a second port for applying a current to heat the fluid or for monitoring a fluid parameter between the first polo and the second port is also provided. Specified on the substrate. In some embodiments, the first channel crosses the second channel. The heating or monitoring aspects of the invention can be used with a variety of biological reactions, including the polymerase chain reaction and the ligase chain reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is related to US patent application Ser. No. 08 / 977,5, filed Nov. 25, 1997.
No. 28, claiming priority of US Provisional Patent Application No. 60 / 056,058, filed Sep. 2, 1997. This application also claims priority to US Provisional Patent Application No. 60 / 083,532, filed on April 29, 1998. Each of the above-referenced applications is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION The present invention relates generally to microfluidic systems. More particularly, the present invention provides techniques including methods and devices for the generation and control of heat in a fluid in a channel of a microfluidic system in an efficient manner. These methods and devices are useful in a wide range of analytical and synthetic procedures. By way of example only, the invention applies to the polymerase chain reaction (commonly referred to as PCR), but it will be appreciated that it has a broader scope. The present invention also provides techniques for monitoring and controlling various process parameters using resistivity and / or conductivity measurements.

[0003] There is still an increased interest in the manufacture and use of microfluidic systems for the collection of chemical and biochemical information. Generally, technologies associated with the semiconductor electronics industry (eg, photolithography, wet chemical etching, etc.) are used in the manufacture of these microfluidic systems.
The term "microfluid" refers to a system or device or "chip" having a channel or chamber. The channel or chamber is typically manufactured on a micron or submicron scale, for example, having at least one cross-sectional diameter in the range of about 0.1 μm to about 500 μm. An initial discussion of the use of planar chip technology for the manufacture of microfluidic systems is in Manz
Et al., Trends in Anal. Chem. (1990) 10 (5): 14
4-149 and Manz et al., Adv. in Chromatog. (1993
33) 1-66, which describe the manufacture of such fluidic devices, especially microcapillary devices, on silicon and glass substrates.

[0004] There are countless applications of microfluidic systems. For example, February 1, 1996
International patent application WO 96/04547, filed on the 5th, describes capillary electrophoresis, liquid chromatography, flow injection analysis, and the use of microfluidic systems for chemical reactions and synthesis. June 28, 1996
Filed on June 30, and filed as "HIGH THROUGHPUT SCREENING AS
SAY SYSTEMS IN MICROSCALE FLUIDIC DE
VICES ", J.M. No. 08 / 671,987 (Attorney Docket No. 17646-400), assigned to Wallace Parce et al. And the assignee of the present application, now has an effect on chemical systems, preferably biochemical systems. Discloses a wide range of applications for microfluidic systems in rapidly assaying large numbers of compounds. The phrase "biochemical system" generally refers to a chemical interaction involving a type of molecule commonly found in living organisms. Such interactions include the full range of catabolic and anabolic reactions that occur in living systems, including enzymatic reactions, binding reactions, signaling reactions and other reactions. Particularly desired biochemical systems include, for example, receptor-
These include ligand interactions, enzyme-substrate interactions, cell signaling pathways, transport reactions involving model barrier systems (eg, cells or membrane fractions) for bioavailability screening, and various other general systems.

[0005] Many chemical or biological systems also desire control over processing parameters such as, for example, temperature, reagent concentrations, buffers, salts and other materials. In particular, some chemical or biological systems require processes to be performed at controlled or varied temperatures. External heating elements are commonly used in providing such controlled temperatures in miniaturized fluid systems. Such heating elements typically include external resistive heating coils or materials, which provide heating to the fluid system in a conductive manner. This heating unit is itself mounted directly on the outer part of the chip in order to heat the whole chip and to provide that a uniform temperature distribution is present on the chip. However, this heating unit is cumbersome. This also complicates chip manufacturing and often affects chip quality and reliability. Further, resistive heating elements can often damage chips, devices, and the environment. In addition, resistive heating elements generally cannot efficiently control the heat supplied to the chip, which often causes undesirable large temperature gradients and fluctuations in the chip. Thus, resistive heating elements applied directly to the chip are very limited (eg, cannot be heated locally) and are unreliable in controlling process temperatures at the chip.

[0006] Larger scale temperature controllers can also be used to control the reaction temperature in the reaction vessel (eg, hot plate, water bath, etc.). These larger temperature controller elements are commonly used in biological and chemical laboratory environments to heat fluids in beakers, test tubes, and the like. Unfortunately, such controllers are not well-suited for providing accurate control of temperature in microfluidic systems. In fact, such an overall heating system that heats the entire material area of the microfluidic device cannot be used to selectively apply heating to specific areas (eg, specific channels and chambers) of the microfluidic device. . Further, these large temperature controllers (eg, hot plates) often require large resistive heating elements that conduct heat by conduction. These resistive heating elements have characteristic long response times, which, in some applications, often require a long time to heat or cool the material in the reaction vessel in contact with the resistive heating element. Related. Thus, hot plates can be very limited for use in heating fluids in microfluidic applications.

[0007] Other process parameters in microfluidic systems, such as fluid concentration, pH, etc., typically cannot be controlled by the prior art. Users of microfluidic systems often ascertain the concentration or pH of a fluid in a liquid source (eg, a bottle), but generally cannot monitor such fluid parameters by conventional techniques, while Fluids have been used in microfluidic systems. In fact, once a fluid enters a channel or process chamber of a microfluidic system, there is generally no simple or effective way to check these parameters. Thus, it is often difficult, if not impossible, to verify process consistency by monitoring these process parameters.

[0008] From the above, there is a strong need for a technique for selectively controlling various process parameters in a simple, effective, and safe microfluidic system.

SUMMARY OF THE INVENTION In accordance with the present invention, fluid temperatures, materials (eg, fluids) in a microfluidic system
Techniques are provided that include methods and devices for controlling process parameters, such as the concentration of glycerol. The present invention uses, for example, an electric current applied to a material for heating purposes. Since only a small amount of material is heated, the material can be successfully heated or cooled by controlling the application of current to the material for various chemical and biological applications (eg, PCR, etc.). In addition, the present invention provides techniques for monitoring process parameters such as temperature, fluid concentration, pH, etc. over the process steps. Accordingly, an in situ technique for monitoring these process parameters is provided.

[0010] In one aspect, the present invention provides a microfluidic system having a controlled temperature element. In particular, the system comprises a substrate having at least a first microscale channel disposed in the system, the channel typically comprising a fluid material disposed in the system. The system also includes an energy source operably coupled to the channel to increase a temperature of the fluid in the first portion of the channel by applying a first current or voltage through the first portion of the channel. Is provided. Current is applied through the first channel, or a second channel that intersects the first channel, as needed. The regions are selectively heated by providing regions having different (eg, narrower) cross-sectional areas to increase current density in these regions, thereby generating heat from the current. Typically, a sensor is also provided operably coupled to the channel to detect a temperature in the channel. Here, the energy source is responsive to the detected temperature in the channel.

[0011] In a related aspect, the invention provides a microfluidic system that has one or more thermal elements included in the microfluidic system. The system includes a first channel defined in the substrate, wherein the channel has a first end and a second end. A first end coupled between the first end and the second end of the channel;
Energy sources are provided. The first energy source is further configured with a current or voltage such that the fluid is pumped through the first channel. A second energy source is also provided for coupling to the fluid in the first channel such that a voltage from the energy source is heated such that a portion of the fluid is heated in the portion of the first capillary channel. You.

In yet another related aspect, the invention provides a microfluidic system, comprising a capillary channel having a first end and a second end, and a first end and a second end. And an energy source connected between them. The energy source provides a voltage across the fluid such that a portion of the fluid is heated in a portion of the capillary channel.

[0013] In a further related aspect, the invention provides a microfluidic system, comprising a capillary channel (including a fluid) defined in a substrate, wherein the capillary channel comprises a fluid in the region. Having a region that is selectively heated using a voltage bias applied to the fluid in the capillary channel.

In a still further related aspect, the invention provides a microfluidic system, comprising a substrate, a channel having a first end and a second end defined on the substrate. An energy source is coupled to the channel and the energy source comprises an AC source.
A first source comprising components and a second source comprising DC components.

[0015] In another but still related aspect, the present invention provides a microfluidic system having a temperature control device. In particular, the system comprises a substrate and a channel located in the substrate. The channel has a first end and a second end. An energy source is coupled to the channel to provide a current through the fluid in the channel that heats the fluid in the channel. A sensor may be further coupled to the channel to detect a temperature of the fluid in the channel. A controller coupled to the sensor and the energy source controls the temperature in the channel based on the desired set temperature.

The present invention also provides a multi-port microfluidic device, comprising a substrate having a first fluid-filled channel region defined therein. This board
At least a first port and a second port for transporting material between at least a first port and a second port, and for heating a fluid between the first port and the second port And a second channel region defined on the substrate for applying a current of

The present invention also provides a computer program product for operating a microfluidic system according to another aspect of the present invention. In particular, the computer program includes a computer-readable memory that includes code that instructs an energy source that heats the fluid to a selected elevated level and regulates current or voltage to a channel containing the fluid. Including.

The present invention also provides a method for controlling temperature in a microfluidic system. In particular, in one aspect, the invention provides a method for increasing the temperature in at least a first portion of a fluid-filled microscale channel disposed on a substrate to a first selected elevated temperature. The method typically includes applying a first selectable current through a first portion of the first channel, wherein the first portion of the first channel comprises a first portion of the first channel. Having an electrical resistance of Then, the at least one first selectable current or resistance is controlled to increase the temperature in the first portion of the first channel to a first selected elevated temperature.

In a related aspect, the invention provides a method of heating a fluid in a microfluidic system, the method comprising the steps of providing a substrate having a first end, a second end, and a region defined therebetween. Providing a disposed channel. Fluid is provided at least in the region of this channel. Electric current is applied to heat the fluid in this area through the fluid. Further, this current selectively heats the fluid in the region of the channel, while avoiding substantial heating of the fluid outside this region. In a still further related aspect, the invention provides a method for controlling the temperature of a fluid in a channel defined in a substrate in a microfluidic system. The method includes applying a power source to begin heating the fluid in the channel, adjusting a first parameter from the power source to provide a relatively constant second parameter. Here, the fluid is heated without a substantial increase in the temperature of the substrate.

In another aspect, the invention provides a method for monitoring a parameter of a process in a microfluidic system. The method includes providing a microfluidic system comprising a channel defined in a substrate. The material is transported to the channel and the conduction value of the material in the channel is determined. The measured conductivity in the measuring step is then correlated with a particular process parameter (ie, buffer concentration, pH, or temperature).

In a further aspect, the methods and systems of the present invention are useful for conducting aqueous reactions at very high temperatures. In particular, such reactions are performed by placing an aqueous reactant into a microscale channel located on a solid substrate. The microscale channel disposed on the substrate then has two ends, each end having an electrical port. The channel has at least a first electrical resistance integrated with the channel. Thereby, the application of a current through this channel results in the heating of the aqueous reactant to a temperature above 100 ° C.

In a related aspect, the invention provides a microfluidic system that includes a substrate having at least a first microscale channel provided in the substrate.
A sensor is operably connected to the channel to determine a temperature of the fluid in at least a portion of the channel. An energy source responsive to the determined temperature in the fluid in the first portion of the first channel is also provided. The energy source applies a first current through at least a first portion of the first channel that heats a fluid disposed within the first portion of the first channel to a first elevated temperature.

Another aspect of the invention is a method of controlling a temperature of a fluid in at least a first portion of a first channel disposed in a substrate. The method includes applying a first current through at least a first portion of a first channel. This first
Has a first electrical resistance. At least one of the first current and the first electrical resistance is controlled to increase the temperature of the fluid in the first portion of the first channel to a first selected elevated temperature.

[0024] Another aspect of the present invention is a method for performing a nucleic acid amplification reaction. The method includes providing an amplification reaction reagent within at least a first portion of a first microscale channel. The amplification reagent typically includes a template nucleic acid, a primer sequence, a nucleoside triphosphate, and a polymerase enzyme. The temperature is repeatedly cycled within at least the first portion to a temperature suitable for performing the melting, annealing, and extension reactions in the amplification reaction. Cycling the temperature comprises variably applying a current through the first portion of the first channel. The current then heats the fluid in the first portion of the first channel.

Another aspect of the present invention is a microfluidic device comprising a substrate. The microscale channel of the first embodiment is provided in the substrate. The first microscale channel has a first portion and a second portion. The first portion comprises an elevated temperature region that has an increased electrical resistance relative to other portions of the first channel when current is applied through the first microscale channel.

[0026] The present invention also provides a computer program product for operating a microfluidic system, the computer program product comprising a first channel responsive to a temperature detected in the channel. A computer readable memory containing code that instructs the energy source to regulate the current applied to the portion.

[0027] Relatedly, the present invention also provides a computer-executable process for controlling the temperature of a fluid in at least a portion of a microscale channel. The process includes detecting a temperature of a fluid in a portion of the channel and comparing a preselected temperature to a temperature in a portion of the channel. The applied current is then increased or decreased through the channel so as to raise or lower the temperature in the channel to a temperature approximately equal to the preselected temperature.

[0028] Other aspects of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides techniques for heating and cooling fluids in microfluidic systems, as well as techniques for monitoring various process parameters. In an exemplary embodiment, the invention also provides techniques for moving and heating fluid through a channel or tubular region of a microfluidic system. In particular, the present invention uses energy, such as electrical current, as a heat source to provide regional or global heating of a fluid in a microfluidic channel. The details of the invention are provided below with reference to the drawings, which have been briefly described above. Before discussing embodiments relating to fluid heating or cooling with novel heat sources and process monitor embodiments, the following section may make the content of the present invention easier for the reader to understand.

I. General of Microfluidic Systems As used herein, the term “microscale” or “miniaturization” generally ranges from about 0.1 μm to about 500 μm, with at least 1 μm. A structural element or feature of a device having two fabricated dimensions. Thus, devices that are said to be miniaturized or microscale are provided with at least one structural element or feature having such dimensions. When used to describe a fluidic device (eg, a passage, chamber or conduit), the term “microscale”, “miniaturized”, or “microfluid” generally refers to at least one internal cross-sectional dimension Refers to one or more fluid passages, chambers or conduits having a (e.g., depth, width, length, diameter, etc.), which is less than 500 [mu] m, and typically between about 0.1 [mu] m and about 500 [mu] m. . In the device of the present invention, the microscale channel or chamber is preferably about 0.1 μm and 200 μm.
m, more preferably between at least 0.1 μm and 100 μm, and often between about 0.1 μm and 20 μm. Thus, a microfluidic device or system made in accordance with the present invention typically comprises at least one microscale channel, and usually at least two intersecting microscale channels, arranged in a single body structure; And often with more than two intersecting channels. The channel intersection may exist in many forms, including a cross intersection, a “T” cross or many other structures, whereby the two channels are in fluid communication. The body structure may be a one-piece structure, or may be a collection of a plurality of separate parts that fit together to form a collective body structure.

Typically, the body structures of the microfluidic systems described herein, when properly combined or joined together (eg, the channels and / or (Or comprising a chamber) comprising a collection of two or more distinct layers forming a microfluidic device of the invention. Typically, the microfluidic devices described herein comprise a top portion, a bottom portion, and an interior portion, where the interior portion substantially defines the channels and chambers of the device.

FIG. 1 shows a two-layer body structure 10 for a microfluidic device. In a preferred aspect, the bottom portion of device 12 comprises a solid substrate that is substantially planar in structure, and the substrate has at least one substantially planar top surface 14. Various substrate materials can be used as the bottom portion. Typically, as the device is miniaturized, substrate materials can be made using known miniaturization techniques (eg, photolithography,
Wet chemical etching, laser cutting, air abrasion techniques, injection molding, embossing, and other techniques) and their compatibility. The substrate material is also
The choice is generally made for their compatibility with a range of conditions to which the microfluidic device can be exposed, including the extremes of pH, temperature, salt concentration, and application of an electric field. Thus, in some preferred aspects, the substrate material is a material typically associated with the semiconductor industry (eg, a silica-based substrate (eg, glass, quartz, silicon, or polysilicon)) as well as other substrate materials (eg, gallium arsenide). Etc.). Here, such a miniaturization technique is generally used. In the case of semiconductor materials, especially in applications where an electric field is applied to or within the device, it is often desirable to provide an insulating coating or layer (eg, silicon dioxide) over the substrate material. Although the preferred substrate is a planar structure, it is also understood that various substrate structures (concave or convex structures, eg, tubular structures such as capillaries) may be utilized.

In a further preferred aspect, the substrate material is a polymer material (eg, polymethyl methacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON ^™ ), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), polysulfone Such as plastics). Such polymer substrates can be prepared using the available miniaturization techniques described above or by well-known forming techniques (
Easily fabricated from miniaturized masters using, for example, injection molding, embossing, or stamping) or by polymerizing a polymeric precursor material in a mold (eg, US Pat. No. 5,512,512). 131). Such polymeric substrate materials are preferred for ease of manufacture, low cost and disposability, and generally inert to most extreme reaction conditions. Also,
These polymeric materials comprise a treated surface (eg, a derivatized or coated surface) and are described, for example, in US patent application Ser. No. 08 / 843,212, filed Apr. 14, 1997 (representative). Utility in microfluidic systems (e.g., enhanced), as described in human identification number 17646-002610, which is hereby incorporated by reference in its entirety for all purposes. (Providing a fluid flow direction). Further, such another substrate has various structures (
For example, it may be any of flat, tubular, concave, convex, etc.).

The channels and / or chambers of the microfluidic device are fabricated using the miniaturization techniques described above, typically as microscale grooves or depressions 16 in the top surface or portion 12 of the bottom substrate. You. The upper portion or substrate 18 also comprises a first planar substrate 20 and a second surface 22 comprises a first planar surface 2.
0. In a microfluidic device made according to the methods described herein, the top portion may also pass through the top portion (eg, the first planar surface 20).
To a second planar surface 22 opposite the first planar surface).

Next, the first planar surface 20 of the top surface 18 is bonded (eg, placed in contact therewith and bonded to the planar surface 14 of the bottom substrate 12, with grooves and / or grooves on the surface of the bottom substrate). Or cover and seal the depression 16), and the contacts of these two components form the channel and / or chamber (ie, interior portion) of the device. Holes 24 in the top portion of the device are oriented to communicate with at least one channel and / or chamber formed in the interior portion of the device from a groove or depression in the bottom substrate. In the completed device, these holes function as reservoirs to facilitate the introduction of fluids or materials into the channels or chambers of the internal portion of the device, and the electrodes may be placed in contact with the fluid in the device Provide a port. This allows for the application of an electric field along the channel of the device to control and direct fluid movement within the device.

[0036] In many embodiments, the microfluidic device comprises an optical detection window positioned across one or more channels and / or chambers of the device. The optical detection windows are typically based on the channel / channel in which they are located.
It is permeable so that it can transmit optical signals from the chamber. The optical detection window can simply be the area of the transmissive coating (eg, where the coating is glass or quartz, or a transmissive polymer material (eg, PMMA, polycarbonate, etc.)). Alternatively, if an impermeable substrate is used in the manufacture of the device, a permeable detection window made from the above materials may be manufactured separately from the device.

These devices may be used in a variety of applications, including, for example, performing high-throughput screening assays in drug discovery, immunoassays, diagnostic analysis, gene sequencing, and the like.
May be used. As such, the devices described herein often include multiple sample introduction ports or reservoirs for parallel or serial introduction and multiple sample analysis. Alternatively, these devices are coupled to a sample introduction port (eg, a pipettor), which introduces multiple samples in series to the device for analysis. Examples of such sample introduction systems are described, for example, in U.S. patent applications Ser. 17646-000410 and 17646-000510)
Which are incorporated herein by reference in their entirety for all purposes.

[0038] In a preferred aspect, the devices, methods and systems described herein use an electrokinetic material transport system, and preferably, a controlled electrokinetic material transport system. As used herein, "electrokinetic material transport system" includes a system that transports and directs a material into a structure comprising coupled channels and / or chambers by the application of an electric field to the material. This results in movement of material through and between the channels and / or chambers.

[0039] Preferred aspects of the invention generally use electrokinetic transport of materials in microfluidic systems, which is a system in which the heating and control aspects of the invention use other material transport systems. It will be readily appreciated that it is easily adaptable to For example, a pressure-based or pneumatic flow system using a pump and / or pressure source external to the microfluidic device can be used in connection with the heating, sensing, and control aspects of the present invention. Similarly, integrated microfluidic devices (eg,
Miniaturized pump and valve structures incorporated into the device) are also easily adaptable for use with these heating and control systems.

[0040] Such electrokinetic material transport and orientation systems include systems that rely on the electrophoretic mobility of charged species in an electric field applied to a structure. Such a system is more particularly called an electrophoretic material transport system. Other electrokinetic material directing and transport systems rely on the electroosmotic flow of fluids and materials within a channel or chamber structure, which results from the application of an electric field across such a structure. Briefly, when a fluid is placed in an etched glass channel or glass microcapillary in a channel having a surface bearing charged functional groups (eg, hydroxyl groups), these groups can be ionized. . In the case of the hydroxyl function, this ionization (eg, at neutral pH) results in the release of protons from the surface and into the fluid, which surrounds the bulk of the fluid at the fluid / surface interface or channel. Produces a certain concentration of protons near the charged outer cylinder. The application of a voltage gradient across the length of the channel causes an outer sheath of protons to move in the direction of the voltage drop (ie, toward the cathode) that draws a large amount of fluid along the voltage drop.

As used herein, “controlled electrokinetic material transport and orientation” refers to the electrokinetic system described above, which comprises multiple (ie, two, Active control of the voltage applied at the electrodes. In other words,
Such a controlled electrokinetic system simultaneously regulates the voltage gradient applied across at least two intersecting channels. Controlled electrokinetic material transport is described in Ramsey's published PCT application WO 96/04447, which is hereby incorporated by reference in its entirety for all purposes. In particular, the preferred microfluidic devices and systems described herein comprise a body structure comprising at least two intersecting channels or fluid conduits (eg, interconnected and enclosed chambers). These channels then have at least three non-intersecting ends. The intersection of two channels means that two or more channels are in fluid communication with each other, and
A "T-shaped" intersection, intersection, "wagon wheel" intersection of multiple channels or any other channel shape in which two or more channels are in such fluid communication. The non-intersecting end of a channel is the channel intersection with another channel (eg, "T
It is the point where the channel terminates, not as a result of the "shape" intersection). In a preferred aspect, the device has at least four non-intersecting ends,
It comprises at least three intersecting channels. In a basic intersecting channel structure, where a single horizontal channel intersects and intersects a single vertical channel, it operates controlled electrokinetic material transport to intersect other channels at the intersection. By providing a constraining flow, the flow of material through the intersection is directly controllably directed. For example, it has been assumed that it was desirable to transport the first material through the horizontal channel (eg, from left to right) and across the intersection with the vertical channel. A simple electrokinetic material flow of this material across the intersection applies a voltage gradient across the length of the horizontal channel (ie, applies a first voltage to the left end of the channel), and This can be achieved by applying a second lower voltage to the right end of the channel or by floating the right end (no current is applied). However, this type of material flow through the intersection results in a substantial amount of diffusion at the intersection, resulting from both the natural diffusion properties of the material being transported in the media used and the convective effects at the intersection.

In controlled electrokinetic material transport, material being transported across intersections is constrained by low levels of flow from side channels (eg, top and bottom channels). This is accomplished by applying a slight voltage gradient along the path of the material flow (eg, from the top or bottom end of the vertical channel to the right end). The result of this is a "narrowing" of the material flow at the intersection, which
Prevents diffusion of material into vertical channels. The material of the volume narrowed at the intersections then crosses the length of the vertical channel (ie, from the top end to the bottom end)
It can be injected into a vertical channel by applying a voltage gradient. To avoid any outflow of material from the horizontal channel during this injection, a low level flow is directed back to the side channel, resulting in "retreat" of the material from the intersection.

In addition to constricted injection schemes, controlled electrokinetic material transport is easily exploited to create apparent values, which does not include mechanical or moving parts. In particular, with reference to the intersection described above, the flow of material from one channel segment to another (eg, from the left arm to the right arm of the horizontal channel) is controlled from the vertical channel. Depending on the flow (eg, from the bottom arm to the top arm of the vertical channel), it can be efficiently regulated, stopped, and restarted. Specifically, in the "off" mode, this material
By applying a voltage gradient across the left and upper ends, the left arm is transported through the intersection to the upper arm. The constrained flow is directed from the bottom arm to the top arm by applying an equal voltage gradient along this path (from bottom end to top end). The measured amount of material is then distributed from the left arm to the right arm of the horizontal channel by switching the left-to-top applied voltage gradient from left to right. The amount of time and the applied voltage gradient indicate the amount of material thus dispensed.

Although described with respect to intersections in four ways for illustrative purposes, these controlled electrokinetic material transport systems are more complex in coordinated channel networks (eg, coordinated parallel channel arrays). Can be easily adapted to

The devices and systems described in detail herein are generally described in terms of the performance of some or one particular operation, which means that the flexibility of these systems increases It will be readily appreciated from this disclosure that it allows for easy incorporation of further manipulations into the device. For example, the described devices and systems may be provided with structures, reagents and systems for performing substantially any number of operations, both upstream and downstream, from the operations detailed herein, as appropriate. Is provided. Such upstream operations include sample handling and preparation operations (eg, cell separation, extraction, purification, amplification, cell activation, labeling reagents, dilution, aliquots, etc.). Similarly, downstream operations can include similar operations, including, for example, separation of sample components, labeling of components, assay and detection operations. Assays and detection procedures include probe interrogation assays, for example, free or constrained in channels or chambers of devices and / or probe arrays with a large number of different, separately arranged probes. Examples include, but are not limited to, nucleic acid hybridization assays utilizing individual probes, receptor / ligand assays, immunoassays, and the like.

The systems described herein generally detect additional equipment for controlling fluid transport and orientation within the device, the results of operations performed by the system, as described above. Or a sensing device for sensing, a processor (eg, controlling a control device according to pre-programmed instructions, receiving data from the detecting device, analyzing, storing, and interpreting the data, and easily accessible) A microfluidic device in combination with a computer to provide data and analysis in various forms.

Various controls can be utilized in combination with the microfluidic devices described above to control the transport and orientation of fluids and / or materials within the devices of the present invention. For example, in many cases, fluid transport and directing may be controlled in whole or in part using a pressure-based flow system that incorporates external or internal pressure sources to drive fluid flow. . Internal sources include miniaturized pumps (eg, membrane pumps, thermal pumps,
Lamb wave pump). See, for example, U.S. Patent Nos. 5,271,724; In such systems, fluid directing can often be achieved by incorporation of miniaturized valves. This valve restricts fluid flow in a controlled manner. See, for example, U.S. Patent No. 5,171,132.

As noted above, the systems described herein preferably utilize an electrokinetic material orientation and transport system. Thus, a controller system for use in combination with a microfluidic system is typically suitable for a plurality of electrodes that are placed in electrical contact with the fluid contained within the microfluidic device. A power supply and circuitry for simultaneously delivering voltages are provided. Examples of particularly preferred electrical controllers are U.S. patent application Ser. No. 08 / 888,064 and International Patent Application No. And 00061 OPC)
And these disclosures are incorporated herein by reference in their entirety for all purposes. Briefly, this controller uses current control in a microfluidic system. The flow of current at a given electrode is directly related to the flow of ions along the channel connecting the reservoir where the electrode is located. This is in contrast to the need to determine voltages at various nodes along a channel in a voltage control system. Thus, the voltage at the electrodes of the microfluidic system is set in response to the current flowing through the various electrodes of the system. This current control is less sensitive to dimensional variations in the process of making the microfluidic system in the device itself. Current control pumps and valves the material and buffer fluid of interest in a composite microfluidic system,
It further allows easier operation for dispersing, mixing and concentrating. Current control is also preferred to mitigate unwanted temperature effects in the channel.

In the microfluidic systems described herein, various detection methods and systems may be used depending on the specific operations performed by the system. Often, microfluidic systems use multiple different detection systems to monitor the output of the system. Examples of detection systems include optical sensors, temperature sensors, pressure sensors, pH sensors, conductivity sensors, and the like. Each of these types of sensors can be easily incorporated into the microfluidic systems described herein. In these systems, such a detector may be in a microfluidic device or in one or more channels, chambers or conduits of the device, such that the detector is in detection communication with the device, channel, or chamber. Or adjacent to it. As used herein, for a particular area or element, the phrase "in detection communication" generally refers to the detector in which the detector is a microfluidic device, a portion of a microfluidic device, or the content of a portion of a microfluidic device. This refers to the arrangement of detectors at positions where characteristics can be detected. For this purpose, this detector is intended. For example, a pH sensor placed in detection communication with a microscale channel may determine the pH of a fluid placed in this channel. Similarly, a temperature sensor located in sensor detection communication with the body of the microfluidic device can determine the temperature of the device itself.

A particularly preferred detection system is an optical detection system for detecting the optical properties of a material in a channel and / or chamber of a microfluidic device incorporated into a microfluidic system described herein. Prepare. Such optical detection systems are typically located adjacent to a microscale channel of a microfluidic device and communicate with the channel through an optical detection window located across the channel or chamber of the device. It is in detection communication.
Optical detection systems include systems that can measure the light emitted from the material in the channel, the transmittance or absorption of the material, and the spectral properties of the material. In a preferred aspect, the detector measures the amount of light emitted from the material (eg, a fluorescent or chemiluminescent material).

Thus, the detection system typically collects a signal based on the light transmitted through the detection window and collects the signal for transmission to a suitable photodetector. With optics. A mobile power source, field diameter, and focal length microscope objective can be readily utilized as at least a portion of this optical column. The light detector can be a photodiode, an avalanche photodiode, a photomultiplier tube, a diode array, or, in some cases, an imaging system (eg, a charge coupled device device (CCD), etc.). In a preferred aspect, a photodiode is at least partially utilized as a photodetector. The detection system typically includes an AD / DA converter to transmit the detected optical data to a computer for analysis, storage, and data manipulation (as described in detail below). Connected to the computer via

In the case of fluorescent materials, the detector typically comprises a light source that produces light at an appropriate wavelength to activate the fluorescent material, and a material contained in the channel or chamber through a detection window. Optics for directing the light source are provided.
The light source can be any number of light sources that provide the appropriate wavelength, including lasers, laser diodes and LEDs. Other light sources may be needed in other detection systems. For example, broadband light sources are typically used in light scattering / transmission detection schemes and the like. Typically, light selection parameters are well known to those skilled in the art.

The detector may be present in a separate unit, but preferably may be integrated with the controller system in a single instrument. Incorporating these functions into a single unit makes these functions possible by allowing the use of several or a single communication port to convey information between the controller, detector and computer. Facilitates the connection of the instrument to the computer (described below).

As described above and as described in greater detail below, either or both the controller system and / or the detection system are coupled to a suitably programmed processor or computer, Or the computer may be pre-programmed or
It operates in support of these instructions, receives data and information from these devices, and interprets, manipulates, and reports this information to the user. Thus, a computer is typically suitably coupled to one or both of these devices (eg, an AD / DA converter, if necessary).

The computer typically sets the parameters either in the form of instructions (eg, in a GUI) or pre-programmed (eg, pre-programmed for a variety of different specific operations). Include appropriate software to accept user instructions in the form of user input in the field. The software then translates these instructions into a language appropriate for instructing the operation of the fluid direction and transport controller to perform the desired operation. The computer then receives the data from one or more sensors / detectors provided within the system and interprets the data and provides the data in a format understood by the user, or (eg, flow rate, Either according to the programming (such as temperature, applied current monitoring and control, etc.), use the data to initiate further controller instructions.

II. Temperature Control in Microfluidic Systems As described above, the present invention generally relates to microfluidic systems,
This heats materials (eg, fluids, analytes, buffers and reagents, including samples) at desired locations (eg, in selected channels and / or chambers of a microfluidic device). Provides energy selectively. In particular, the present invention uses a power supply that passes an electric current through a fluid located in a channel and / or chamber of a microfluidic system to heat the material in a controlled manner. Accordingly, the present invention addresses the recognized problem in the art of resistive electroheating of fluids in electrically controlled systems,
The resistance of the experimenter is then utilized (eg, for heating in a microfluidic system to control operation) using resistive electroheating.

The methods and systems of the present invention provide several advantages over typical temperature control methods for fluid systems. For example, such systems provide the controllability and automation associated with accurate electrical control of temperature. Further, such a system offers the advantage of speed in changing the temperature of fluids and materials in the channel. In addition, these systems are easily integrated into state-of-the-art electrokinetic microfluidic systems. Finally, such methods and systems may be used in a separate microfluidic device of a given device (eg, in one or more separate channels in a given device), where other heating is not desired. Allows temperature control and / or precise regional control of heating without heating the region. In particular, in accordance with the methods and systems described in the present invention, heat is only generated in the fluidic elements where such heating is desired. Furthermore, such microfluidic devices are very small compared to the mass of the substrate (where they are assembled) such that heat is substantially localized, so that, for example, other Consumed by or from the substrate before affecting the fluidic device. In other words, a relatively large substrate functions as a heat sink for the separate fluidic elements provided therein. Thus, according to the present invention, one or more channels of an integrated microfluidic channel system (eg, multiple intersecting channels, or multiple channels closely grouped together on a single substrate or body structure). Can be selectively heated, while not substantially changing the temperature of the material in other channels on the substrate or the material intersecting the heated channels. "Do not substantially change temperature" means that the temperature of the material in the "unheated" channel does not change by more than about 40% (in degrees Celsius) from the temperature of the entire substrate (typically ambient). Is intended. For example, if the ambient temperature of the substrate before any heating is 20 ° C., heating of one portion of a channel in the substrate will increase the remaining portion of the channel by more than about 40% (ie, 8 ° C.). Occurs without. In a preferred aspect, the temperature of the remaining unheated channel portions does not change by more than 10%, and more preferably does not change by more than 5%. Of course, if a channel intersects a heated channel, a portion of the channel will be subjected to an elevated temperature (eg, at or near the intersection). However, most (eg, greater than 90%) of the channel portion is maintained at or near ambient temperature. Alternatively, the unheated channel has a temperature of about 1 at a temperature without any heating on the chip.
Maintained within 0 ° C, preferably within about 5 ° C, and more preferably within about 2 ° C.

In its simplest embodiment, the present invention provides a microfluidic device having at least one microscale channel having a fluid material disposed in the first microscale channel. A power supply electrically coupled to different points along the channel causes the fluid in the channel to increase the temperature of the fluid in a controlled localized manner (ie, localized within the fluid). Controllably delivering current through it. Numerous modifications can be made to the basic principles to apply the invention broadly to numerous applications, and many of these are described in detail below. For example, a heating current can be applied directly to a portion of the channel or chamber where elevated temperatures are desired, or an auxiliary heating channel (e.g., where the material to be heated is located) (Which crosses or is adjacent to the channel). Further, in many cases, the temperature in the channel is monitored, and the power supply changes the amount of power in response to the actual temperature. For example, if the actual temperature is lower than the desired temperature, the power supply may increase the current through the channel heating the fluid until the desired temperature is achieved. Thus, the power supply typically responds to the detected temperature in the channel for which temperature control is desired. An example of a temperature responsive controller in a channel is described in the following use of the term temperature set point.

The present invention preferably uses an electrokinetic mass transport system, as described above, since the power supply is used to directly control the temperature within the fluid contained in the microfluidic device. Used in microfluidic systems. In particular, the same electrical controllers, power supplies and electrodes can easily be used to control temperature as they control material transport. This is discussed in more detail below.

In an exemplary embodiment, the material is located in, and preferably through, a channel where temperature control is desired. This channel is typically
It has a desired cross-section (eg, diameter, width or depth) that enhances the heating effect of the current therethrough, and heat transfers energy from the current to the fluid. The channels described herein can be, for example, as described in detail above, such as, for example, amorphous materials (eg, glass, plastic, silicon) composites, multilayer materials, combinations thereof, and the like. , Can be formed of almost any type of substrate material.

To assist in understanding the present invention, it may be helpful to briefly describe embodiments of the present invention relating to the heating of the fluids described herein. Generally, current passing through a fluid in a channel generates heat by dissipating energy through the electrical resistance of the fluid. Power is dissipated as current passing through the fluid as a function of time to heat the fluid and enters the fluid as energy. The following mathematical expressions generally describe the relationship between power sources, currents, and fluid resistance.

POWER = I ² R where POWER = power dissipated in the fluid; I = current through the fluid; and R = electrical resistance of the fluid.

The above equation describes the power consumption (“PO”) versus current (“I”) and resistance (“R”).
WER "). In some embodiments involving moving fluid, a portion of the power source enters the kinetic energy that moves the fluid through the channel. Embodiments of the present invention use selected portions of a power supply to heat a fluid in a channel or selected channel region. This channel region is often narrower or smaller in cross-section than other channel regions in the channel structure. The smaller cross section provides higher resistance in the fluid, which increases the temperature of the fluid as current passes therethrough. Alternatively, the current can be increased along the length of the channel by increasing the voltage. This is also
Increase the amount of power dissipated in the fluid to increase in response to the temperature of the fluid.

FIG. 2 is a schematic diagram of an example of a microfluidic system 400 including a heat source according to the present invention. This diagram is merely an example, which should not limit the scope of the claims. One skilled in the art will recognize other variations, changes, and modifications. This figure shows a channel network or arrangement 405 for moving or heating a volume of material (eg, fluid) in a channel or capillary region 407 (which is located at the intersection of channels 413 and 421). Channel 413 may have similar lengths and widths as described herein, but others are also possible. Channel 41
3 is connected to regions 409 and 411. Regions 409 and 411 provide power to channel 413 for moving fluid between regions 409 and 411. The material may have an electrokinetic effect as described herein above, including, but not limited to, electroosmotic and / or electrophoretic forces.
Are moved between these areas. Power supply 403 is operably coupled to the channels and connected to regions 409 and 411, lines 415 and 41, respectively.
Power is supplied through 7. In particular, power supply 403 provides a differential voltage or electric field between regions 409 and 411 by applying a voltage differential to the electrodes in regions 409 and 411. This differential voltage drives the electrokinetic movement of the material. As shown, a differential voltage is applied along channel 413.

As used herein, the phrase “operably linked” means that one element may distribute a desired function to another element or derive a desired function from another element. Refers to a connection between two elements as may be possible. For example, in the case of an electrical element (eg, a power supply or an electrode) operably connected to a microscale channel,
The power supply is coupled to the channel to distribute the described functions of the power supply (eg, enter and pass current through the channel). Such connections typically involve known electrical connectors (eg, wires, electrical terminal pads,
Swipe terminals, immersion electrodes, etc.) are normally used. Similarly, a computer operably linked to a sensor can receive data from the sensor such that the computer can manipulate and / or analyze the data. On the other hand, the same computer operably linked to the electrical controller may convey instructions to the controller while receiving feedback information (eg, resistance, actual voltage, etc.) from the controller. Typically, the operable connection for the computer will generally be provided through a suitable commercially available data transmission cable.
Further, an optical detection system operably connected to the channel is typically within a sensor that is in communication with the channel. In particular, the detection system may detect an optical signal in the channel.

Preferably, power supply 403 also provides power to regions 423 and 425 for heating fluids and materials in region 407 in the channel arrangement. In particular, power supply 403 provides a differential voltage between regions 423 and 425, causing a current to flow between regions 423 and 425. The current is used to distribute energy to fluids and materials in region 407 for at least the purpose of heating. Channel 421 comprises a novel geometry designed to effectively heat the fluid in region 407 in an effective manner. As shown, the channels 421 include outer portions 421, each having a greater width or cross-sectional (eg, diameter) dimension than the inner portion or region 407. This has a correspondingly larger fluid or electrical resistance besides the outer portion 421. The exact dimensions of the wider and narrower portions can be optimized depending on the amount of current applied through the system, the amount of heat desired, the heat capacity of the substrate, etc., which is easily optimized experimentally. obtain. In any case, such dimensions are typically within the dimensions described for the microscale channels herein (eg, dimensions of at least one cross section between 0.1 and 500 μm). Enter.

Power supply 403 applies voltage and / or current in one of many ways to selectively control the temperature of the fluid or material in region 407 of the channel. For example, power supply 403 applies a direct current (ie, DC), which passes through channel 421 and enters channel region 407, which has a smaller cross-section that heats fluids and materials in region 407. This direct current can be selectively adjusted on a scale that complements any voltage or electric field. This optional voltage or electric field is applied to region 407
May be applied between regions 409 and 411 that move material in and out of the region. To heat the material in region 407, an alternating current (ie, AC) is applied by power supply 403 through channel 407 and channel region 407, which heats the fluid in region 407, without adversely affecting the movement of the material. It can be selectively applied. This alternating current used to heat the fluid may be applied between regions 409 and 411 and selectively adjusted to supplement a power or electric field that may move the fluid in and out of region 407. . The AC current, voltage, and / or frequency may be adjusted, for example, to heat the fluid without substantially moving the fluid. Alternatively, the power supply 403 applies current and / or voltage pulses or impulses, which pass through the channel 421 and into the channel region 407 and heat the fluid in the region 407 at the right time in certain cases. . The pulse can be selectively tuned to complement any voltage or electric field, which is applied between regions 409 and 411 to allow material inside and outside region 407 (eg, Fluid or other material). The pulse width, shape, and / or intensity can be adjusted, for example, to heat the fluid without substantially moving the fluid or material, or to heat the material while moving the fluid or material. Still further, this power supply may have a DC
, AC, and pulses may be applied.

A controller, such as a personal computer, commonly referred to as a PC, or computer 437, monitors the temperature of the fluid in the region 407 of the channel. The controller or computer, for example, receives current and voltage information from a power supply and identifies or detects the temperature of the fluid in region 407 in the channel. Area 4
Depending on the desired temperature of the fluid at 07, the controller or computer adjusts the voltage and / or current to be sensed inside the channel by a temperature sensor, which may be the power supply itself, as described herein. In response to temperature
Depending on the desired fluid temperature. The controller or computer can also be set to be "current controlled" or "voltage controlled" or "power controlled" depending on the application. The controller or computer 437 includes a monitor 439, which is often a cathode ray tube (CRT) display, a flat panel display (eg, an active matrix liquid crystal display, a liquid crystal display, etc.).
Computer circuits are often located in box 441, which includes a myriad of integrated circuit chips, such as microprocessors, memories, interface circuits, and the like. Box 441 also includes a hard disk drive, a floppy disk drive, a high capacity drive (eg, ZipDrive ^™ sold by Iomega Corporation), and other elements. Also shown is a keyboard 443 and a mouse 445, which provide a human interface to the computer box 441. A variety of techniques can be employed by the computer program to detect and monitor temperature as well as other processing parameters. Some of these techniques are described in more detail below.

In some embodiments, computer 437 is connected to a network, such as a local network or a wide area network. The local network may be configured as, for example, Ethernet or Token Ring. The local area network may be an “intranet”. One of these local area networks, or a combination thereof, is connectable to a wide area network, such as the "Internet", among others. The network may also be wireless, according to the application. The network is
Allow users to be off-site or allow multiple users to monitor, control, or view current microfluidic system processing.

The embodiment shown in FIG. 2, for example, provides a higher fluid temperature or a higher material temperature in the channel in region 407 than in peripheral region 421. FIG. 2A is a simplified diagram of a temperature distribution 450 along a channel. As shown, the vertical axis represents temperature (T) and the horizontal axis represents length along the channel, ie, distance (D). The fluid in the channel region 421 is maintained at the temperature T ₀ . The fluid in the channel region 407 is maintained at the temperature T _S. The fluid temperature in region 407 is lower than the channel cross-section in region 421, resulting from a higher current density (and higher resistance) in region 407 resulting from the cross-section of the channel in region 407. Higher than fluid temperature. Depending on the shape of the channel, the temperature profile from one end of the channel to the other end of the channel can be selectively varied. As can be appreciated, temperature control along the length of the channel allows the current to remain unchanged while changing the cross-sectional dimension of the channel.

FIG. 3 is a simplified diagram of a microfluidic system 500 with a heating source according to an alternative embodiment of the present invention. This diagram is merely an example, which should not limit the scope of the claims herein. One of ordinary skill in the art would recognize other variations, alternatives, and modifications. The microfluidic system 500
It includes a channel 502 having various temperature zones such as A, B, C, D, and E, and a fluid flowing therethrough.

Power supplies such as those described above include channel-shaped regions 505, 509, and 5
An electric current is provided between regions 501 and 503 to heat the fluid at 13. The power supply also provides a voltage difference between regions 501 and 503, which drives the transport of material through channel 502. The electric current is used to distribute energy to the fluid in region 509, at least in part, for the purpose of heating. The power source (or another power source) can also drive or move fluid between the regions 501 and 503.

The channel 502 comprises a novel geometry, which is designed to heat the fluid in the regions 505, 509 and 513 in an efficient manner. As shown, channel 502 comprises outer portions 507 and 511, each of which comprises
It has a greater width or greater cross-sectional dimension than the inner portion or region 509. In addition to this, regions 505 and 513 are attached to regions 501 and 503, respectively.
Have a smaller width or smaller cross-sectional dimension than each of the outer portions 507 and 511. As a result of the smaller dimensions, the passage of current through channel 502 results in increased current density inside these regions and results in heating of the fluid located inside these regions. Accordingly, the fluid also increases the temperature in regions 505 and 513. Material may also be transported from regions 501 to 503 while being heated in region 509, which allows for heating of only a portion of the material. In addition, additional areas of smaller dimensions are optionally provided along the length of the channel 502 to allow for "on the fly" as material is transported along the channel 502.
Provides heat circulation.

In certain embodiments, the fluid is heated in one area and cools in another.
In particular, the fluid in regions 507 and 511 will be filled with regions 505, 509, and
Cooler than the fluid at 513. Further, channel 502 can be connected to another channel, which allows fluid to flow from one region of channel 502 to channel 50.
2. Move to another area. Still further, the power supply between regions 501 and 503 can be varied depending on the application. For example, the power supply supplies energy in the form of current and / or voltage across regions 501 and 503. Current and / or
Or, the voltage can be DC, AC, pulsed, combinations thereof, and the like. Of course, the type of power used will depend on the application.

FIG. 4 is a simplified diagram of a microfluidic system 600 with a heating source, according to another embodiment of the present invention. This diagram is merely an example, which should not limit the scope of the claims herein. One of ordinary skill in the art would recognize other changes, alternatives, and modifications. This figure shows the area 601
6 illustrates a channel 605 with a progressively increasing cross-section from to a region 603. The power supply provides a voltage difference between regions 601 and 603. This voltage difference corresponds to the region 601
Cause or drive the current flowing through the fluid between and 603. The current density near region 601 is higher than the current density near region 603, which provides a higher temperature fluid near region 601. The power supplied between the areas 601 and 603 can be changed depending on the application. For example, the power supply
Energy is supplied in the form of current and / or voltage across regions 601 and 603. The current and / or voltage can be direct current, alternating current, pulsed, combinations thereof, and the like. Of course, the type of power used will depend on the application.

The material is also electrokinetically movable or transportable from region 601 to region 603, while the fluid is cooled from region 601 to region 603. The moving fluid and / or material, according to this embodiment, from region 603 to region 601
Increase the temperature. Alternatively, as the material is moved from region 601 to region 603, as the material is transported or moved from region 603 to region 601,
Reduce the temperature or allow it to cool. The movement or flow of material occurs using any of the techniques described herein and other techniques. Of course,
The particular combination of fluid transfer and heating or cooling will depend on the application.

FIG. 5 is a simplified diagram of a microfluidic system 700 with a heating source according to another embodiment of the present invention. This diagram is merely an example, which should not limit the scope of the claims herein. One of ordinary skill in the art would recognize other variations, alternatives, and modifications. The microfluidic system 700 includes a channel having a region 705 having a first cross-section and a region 709 having a second cross-section, where the second cross-section is larger than the first cross-section. Transition area 7
07 is arranged between the area 705 and the area 709.

Power is supplied between the region 701 and the region 703. In particular, power is applied to the fluid in channels 705 and 709 using electrodes applied to the fluid. Since the cross section of channel 705 is smaller than the cross section of channel 709, channel 7
The temperature of the fluid at 05 is greater than the temperature of the fluid at channel 709.
The power supply supplies energy in the form of current and / or voltage across regions 701 and 703. The current and / or voltage can be direct current, alternating current, pulsed, combinations thereof, and the like. Of course, the type of power used will depend on the application.

In combination with the cooling of the fluid, the fluid and / or material may also
To the area 709. Fluid flows between region 705 and region 709,
It may be moved or transported using any of the techniques described herein and others. During this transport period, the fluid or material cools. Alternatively, fluid or material is moved from region 709 to region 705 in combination with a process that heats the fluid in region 707. Of course, with fluid movement,
The particular combination with the heating or cooling treatment will depend on the application.

For an alternative embodiment, FIG. 6 shows a microfluidic system 80 according to the invention.
It is a simplified diagram of another specific example of 0. This diagram is merely an example, which should not limit the scope of the claims herein. One of ordinary skill in the art would recognize other variations, alternatives, and modifications. As shown in FIG. 6, an alternative embodiment of the present invention is illustrated in connection with a channel structure or network as shown in FIG. In addition, some identical reference numbers are used herein for easy cross-reference. In addition, the microfluidic system 800 includes a heating / cooling channel 801 located on the substrate 811, which is not directly fluidly connected to a channel or chamber where elevated temperatures are desired. In particular, this heating channel 801, also called a heating coil, is located on the substrate adjacent to the channel where the elevated temperature is desired. The heating coil provides thermal energy to channel 421 by conduction and / or convection.

The heating coil 801 comprises, inter alia, regions 803 and 805. Coil 8
01 may be a heating coil or a cooling coil according to the application. As a heating coil, coil 801 has a fluid therein. A voltage is applied between regions 803 and 805 to direct current through the fluid for heating purposes. In particular, the power supply
A voltage difference is provided between regions 803 and 805. Current flows between region 803 and region 805 and passes through a plurality of coils 809 (which may be flat) defined in substrate 811. The shape and dimensions of the coil can affect the ability of the current to heat the fluid. As current passes through the fluid, energy is transferred to the fluid for heating. Heat from the fluid is transferred through the substrate 811 to the channel 421, which is also filled with fluid. Thus, the fluid in channel 421 transfers heat from heating coil 801 through the substrate.

Alternatively, channel 801 can be a cooling coil. As a cooling coil, fluid traverses from region 805 through coil 809 to region 803. For example, region 805 can be a cooling source for the fluid, and region 803 can be a sink for cooling the fluid after passing through coil 809. The heated fluid in region 407 may be transmitted to channel region 421 where the fluid is cooled. In particular, heat from the fluid is dissipated through the walls of the channel 421, through the substrate 811 and into a heat sink or cooling fluid in the coil 809. The cooling fluid in coil 809 removes heat from the fluid in channel region 421 through either region 803 or region 805, depending on the application. The cooling fluid can be a variety of substances including liquids and gases. By way of example only, cooling fluids include aqueous solutions and the like. The cooling fluid can be moved between region 803 and region 805 using any of the techniques described herein and others.

In combination with a heating or cooling treatment, as described for the various embodiments specified above, the material may also move between any of the regions described in FIG. 6, or Be transported. For example, a fluid or material may be applied from region 409 to region 4
Flow to 11 or the opposite direction. Fluid can also flow from region 423 to region 425 or in the opposite direction. Fluid flow or fluid transfer may occur by any of the techniques described herein and others. Of course, the specific combination of fluid transfer and heating or cooling will depend on the application.

FIG. 7 is a simplified diagram of a microfluidic system with a heating or cooling source according to another alternative embodiment of the present invention. This diagram is merely an example, which should not limit the scope of the claims herein. If you are skilled in the art,
Other variations, alternatives, and modifications will be recognized. In this embodiment, microfluidic system 900 includes channel 9 having end regions 901 and 903.
02, and various other characteristics. Fluid or material moves via channel 902 from region 901 to region 903 or in the opposite direction.

As fluid or material moves from any region, the fluid can be cooled or heated by conduction from heating or cooling coils 906. As a heating coil,
Coil 906 provides hot fluid from region 907 to region 905 or in the opposite direction. Although illustrated as a coil surrounding channel 902, such as coiled around, in preferred aspects of a microfluidic device employing, for example, a flat structure, the coil is adjacent to or above channel 902. Alternatively, it will be readily understood that the thermal energy from the channel is conducted through the substrate to the channel, if disposed in an underlying layer. The power source preferably provides a voltage difference between region 905 and region 907 to provide current to the fluid for heating. This voltage difference is applied to the fluid using a pair of electrodes in contact with the fluid in regions 905 and 907. The heated fluid passing through the coil 906 transfers thermal energy to the fluid in the channel 902 due to the temperature gradient. Coil 906 can be defined on the substrate to include coil 906 and channel 902. Alternatively, coil 906 is made from a stand-alone structure, which is wound around channel 902. Preferably, coil 906 is sufficiently close to or in contact with channel 902 to transfer thermal energy from the fluid in coil 906 to the fluid in channel 902. The fluid in the coil 906 can be moved using a variety of techniques, including the techniques described herein.

As the cooling coil 906, the cooling fluid passes through the coil 906 and passes through the channel 902
Carries away thermal energy in the form of heat from Cooling fluid is removed from either region 905 or 907, depending on the application. The cooling fluid can be moved using a variety of techniques, including those described herein. Heat from the fluid in the channel 902 is removed from the fluid by a temperature gradient through the coil 906 and the substrate containing the channel 902 to the cooling fluid. That is, if necessary,
Utilizing a combination of conduction and convection, heat is removed from the fluid in channel 902.

In combination with the heating or cooling fluid in channel 902, the fluid
And the area 903. In particular, fluid is movable or transmitted from region 901 to region 903. Alternatively, the fluid may flow from region 903 to region 9
01 is movable or communicated. Fluid transfer occurs utilizing any of the techniques described herein and others. Of course, the specific combination of fluid transfer and heating or cooling will depend on the application.

In an alternative embodiment, the present invention provides a novel microfluidic system 1000 for continuously heating and cooling a moving fluid in a channel utilizing multiple heating sources, as shown by FIG. I will provide a. FIG. 8 is merely an example, which should not limit the scope of the claims herein. One of ordinary skill in the art would recognize other variations, alternatives, and modifications. System 100
0 represents channel regions 1021, 1025, 1033, 1035, and 103
7, but the main channel is located in the end region 1
019 to an end region 1009. The fluid is generally, according to the application,
Pass through the main channel from end region 1009 to end region 1019 or vice versa. A myriad of techniques can be used to move the fluid through the primary channels, including those described herein, but may be other. The system 1000 also includes a plurality of heating source channels 1023, 1027, 102
9 and 1031. Each of these heating channels has at least two end regions. For example, channel 1023 has end regions 1001 and 10
Seventeen. Channel 1027 has end regions 1003 and 1015. Channel 1029 has end regions 1005 and 1013. Channel 1
031 has end regions 1007 and 1011.

Each heating channel also has a region that intersects the main channel 1021. For example, channel 1023 intersects the primary channel between channel regions 1021 and 1025 at channel region 1024. Channel 1027 is channel region 1
Intersects the main channel between 025 and 1033 in channel region 1026. Channel 1029 intersects the primary channel between channel regions 1033 and 1035 at channel region 1028. The channel 1031 includes a channel region 1035
Intersect at the channel region 1030 with the primary channel between the two.

In certain embodiments, the heating of the fluid is applied to the intersection regions 1024, 1026,
28 and 1030. In particular, the power supply provides a voltage difference between each of the end regions of at least one of the heating channels to provide an intersection region 1024,
A current is provided to heat the fluid at at least one of 1026, 1028, and 1030. For example, the voltage difference between regions 1001 and 1017 causes current to flow through the fluid that heats the fluid in region 1024. Area 100
The voltage difference between 3 and 1015 heats the fluid in region 1026. The voltage difference between regions 1005 and 1013 heats the fluid in region 1028. Regions 1007 and 10
The voltage difference between 11 heats the fluid in region 1030. The voltage applied between any pair of end regions may be the same as the voltage of any other pair of end regions,
It may be different.

FIG. 9 shows a microfluidic channel system 110 having a cooling source according to the present invention.
FIG. 4 is a simplified cross-sectional view of the specific example of FIG. This diagram is merely an example, which should not limit the scope of the claims herein. One of ordinary skill in the art would recognize other changes, modifications, and alternatives. Fluid flows through a channel 1103, which is defined in a section 1107 of the substrate 1101 that projects outwardly from the surface 1104. The fluid in the channel 1103 may be heated utilizing any of the techniques defined herein. The protruding section 1107 can be created using various techniques such as masking, etching, milling, and the like. As shown, protruding section 1107 allows heat from the fluid in channel 1103 to pass through region 1107 to region 1105 (eg,
It is shaped like a "fin" to allow heat to dissipate (ambient, vacuum). Region 1105 is designed to transfer heat away from substrate 1107.

To illustrate the transfer of heat of the fluid in the channel 1103 in an embodiment of the present invention, FIG. 9A shows a more detailed view of the channel 1103 of FIG. This diagram is merely an example, which should not limit the scope of the claims herein. Channel 1103 has a width ("D") and region 1107 has a thickness ("t").
"). The fluid is heated in the channel 1103 by a heating source such as an electric current applied directly to the fluid along the length of the channel. Removing the current from the fluid removes the heat source from the fluid and removes the heat from the fluid through region 1107 to region 110
Due to the temperature gradient going out of 5, heat is dissipated from the fluid. The axis (“L”) from the central region of channel 1103 moves outward toward region 1105.

FIG. 10 is a simplified diagram illustrating a temperature gradient of a heated fluid in a channel 1103, for example, as shown by FIG. 9A. Temperature is shown on the vertical axis and the length along axis L is defined on the horizontal axis. This figure shows the region 120
1 is the fluid in the channel 1103 from the central region of the channel 1103 to the outer periphery of the channel 1103. This figure also illustrates a region 1203, which is defined by the thickness t of region 1107. External area 110
5 is defined by reference numeral 1205. A series of line plots 1211, 121
3, 1215, 1217 and 1219 indicate the temperature profile across the fluid through the channel region for different times, the thickness t and the surrounding region 110
5 is represented.

For example, referring to line plot 1211, the fluid in channel 1103 has a temperature (
"T _F "), which is higher than the ambient temperature ("T _a ") when _a current is applied to the fluid for the purpose of heating. Arrow and line plots 1213, 1215, 1217 when current is subtracted or removed from the channel fluid
, And, as indicated by 1219, the fluid temperature of the channel drops toward T _a. In fact, the temperature of the fluid is continuous to T ₂ from T _F, drops further to T _n, which is also T _a. As shown, the temperature gradient from T _F to T _a is reduced from the fluid in the channel to the region 120 until equilibrium is achieved as seen in line plot 1 219.
Drive out the heat to 5. A similar plot can be made for heat treatment of a fluid over time, from ambient temperature to _TF .

This example is performed with respect to an area 1105 that is surrounding (eg, air),
Region 1105 increases the transfer of heat from or to the fluid in the channel,
Alternatively, various fluids or materials may be filled or brought into contact with them to reduce. For example, region 1105 can be filled with or contacted with a highly conductive fluid at a relatively low temperature to further increase heat transfer from the fluid to region 1105. In addition, a highly conductive fluid may be flowed over the surface 1106 to provide convective forces, which transfer heat away from the fluid in the channel 1103. Specific examples of the fluid include water and oil, among others. Alternatively, region 1105 can be in contact with or filled with a heating source, which further drives heat into the fluid in channel 1103. The type of fluid used will depend on the application.

Although the devices, systems, and methods described herein generally will be useful instead of more convenient temperature control methods and systems, in certain embodiments, the present invention Used in conjunction with the system to provide complementary temperature control. In particular, in many embodiments, it is desirable to maintain the entire microfluidic system at elevated temperatures, for example, above ambient. For example, P
In many thermal cycling operations, such as CR, the base temperature is raised above the typical ambient temperature. However, the power requirements for heating all of the channels of a system utilizing the method of the present invention can be quite high. Thus, globally controlling the temperature of the system at the elevated base temperature can be performed utilizing conventional means, thereby minimizing the control and power requirements of such heating processes. Such conventional means include a common external temperature control system, such as a water tub, oven, hot plate, and the like. Alternatively, an integrated global temperature control system, such as integrated into the structure of the microfluidic device, may be utilized. Such systems include resistive heaters, such as thin film heaters, heating coils, Peltier heaters, and the like.

FIG. 11 illustrates a microfluidic system in accordance with the present invention that is external to a fluid and is used to generally raise and / or lower the temperature of a microfluidic device or system. 1 is a simplified diagram of a microfluidic system 200 with an energy source that heats and / or cools fluids or materials. This diagram is merely an example, which should not limit the scope of the claims herein.

As shown, the system includes various features, such as a microfluidic system 200, which has a plurality of channels 203, which are defined on a substrate 201. These channels are heated or cooled and contain the fluid or material to be transported. The fluid may be selectively heated at a particular location in the channel according to any of the techniques described above. Fluids can also be transported or moved inside the channel using any of the techniques described herein or other techniques. In some embodiments, the fluid is moved to a particular location in the channel and heated during the same procedure. In this embodiment, the fluid temperature is also increased or decreased overall during any of the steps or steps described herein or elsewhere.

A variety of methods can be used to increase or decrease the fluid temperature of the microfluidic system overall, but utilize an energy source or sink to affect this temperature change. The energy source may be a heat source, a chemical source, or any other source, as well as a power source. The energy sink can be a heat sink or a chemical sink. The energy source or sink can be full, time-varying, spatially varying, or continuous. By way of example only, the overall heat treatment can be provided by radiation, convection, or conduction. Heat sources can include photon beams, fluid jets, liquid jets, electromagnetic fields, gas jets, electron beams, thermoelectric heaters such as Peltier devices, resistive heaters such as thin film or coil heaters, water baths, or furnaces. Heat sinks include, inter alia, fluid jets, liquid jets, gas jets, cryogenic fluids, supercooled liquids, thermoelectric cooling means such as Peltier devices, or electromagnetic fields. The power supply can be an applied voltage or an applied electromagnetic means. Other types of energy sources may be used.

The overall heating or cooling system can be external, ie separate from the microfluidic device or integrated into the device structure. In a preferred aspect, the energy source is a heating element, either external to the device or integrated therein, such as a resistive thin film or coil heater or Peltier heater. In particular, the heating element is placed against the surface of a substrate in a microfluidic system, such as that of FIG. The heating element 205 transfers thermal energy to the microfluidic system by conduction, such as from the resistive element 207 in the heating element. The thermal energy provided to the microfluidic system generally increases the temperature of the microfluidic system to a desired temperature. More specifically, current flows through the heating element but is converted to thermal energy. Thermal energy is
It flows from a higher temperature region to a lower temperature region. Since the resistive element of the heating element is the hottest area, thermal energy flows by conduction from the resistive element toward the microfluidic system. Thermal energy or heat transfer from the heating element transfers to the surface of the microfluidic system and into the substrate. This thermal energy generally increases the temperature of the substrate in the microfluidic system. Accordingly, the fluid temperature inside the channels and / or chambers of the microfluidic system is also increased overall. Internal controls on the heating element or within the microfluidic device can be used to regulate the temperature of the microfluidic system. Of course, myriad other energy sources can be used to increase the fluid temperature of the microfluidic system in other applications as a whole.

A myriad of techniques can be used to control the power to the microchannel for the purpose of fluid movement. These techniques can also be used to selectively monitor and regulate the temperature of a microchannel or annular region. As noted above, one example of one of these techniques is the use of a processor or controller and / or computer software, such as those described above. Alternatively, exclusively, hardware, or preferably a combination of hardware and software, may implement these techniques. Details regarding a particular computer program capable of implementing the selected technique according to the present invention are described below.

In certain embodiments, the present invention provides techniques used to detect and control the temperature of a fluid being heated in a microchannel by current and voltage measurements. Other techniques may also be used, depending on the application. These techniques are, of course, determined by the application. This technique can be briefly described by the following sequential steps: (1) flowing the fluid in the channel of the microfluidic system (2) stopping the flow of the fluid in the microchannel (optional) (3) through the fluid Apply current (4) Measure the voltage drop across the fluid and corresponding to the current through the fluid (5) Calculate the power dissipated through the fluid from the voltage drop and current measurements (6) Calculating the actual temperature of the fluid based on the power dissipated through the fluid; (7) comparing the actual temperature of the fluid based on the desired temperature set point; and (8) comparing the actual temperature with the desired temperature set point. The current applied to the fluid is adjusted based on the difference between them.

The above-described successive steps are performed by using, for example, a computer program. Computer programs provide easy-to-use methods for performing the above-described steps of the microfluidic system. The computer program is executable in the form of computer software, firmware, hardware, or a combination thereof. The program performs the functions described above utilizing an interface coupled to the microfluidic system. The interface receives signals from the microfluidic system and provides signals for heating, for example, to a power source (which supplies current to the fluid). Details of the above continuous steps
This is described below with reference to the simplified flow diagram of FIG.

The process 1300 according to an embodiment of the invention begins at step 1301, which generally requires the use of a system described herein, and a microfluidic system, such as other systems. This process involves flowing fluid into microchannels or capillaries by electroosmotic and / or electrophoretic forces (step 1).
302) is not limited to these forces. These forces are described throughout this specification, but are not limiting. The fluid can be controlled by a controller or a computer. Optionally, by selectively applying energy to the fluid through the controller, the fluid flow decelerates, increases, or stops in the channel (step 1305). Alternatively, the fluid flow can maintain a constant flow in the channel.

To heat the fluid in the channel, an electric current is applied to the fluid, for example, by an electrode directly connected to the channel (step 1305). The controller or computer provides a signal to turn on the power supply to provide current directly to the electrodes. Upon application of an electric current, the fluid heats in the channel or in selected regions of the channel having a smaller internal perimeter. The current may be direct current, alternating current, or pulsed, depending on the application. A controller or computer may be coupled to the power supply to identify voltages and currents applied to the fluid, as shown in step 1307.

[0106] The amount of voltage and current can be converted to power that is dissipated into the fluid. A temperature is calculated based on the type of fluid (step 1311). For example, the fluid temperature may be found in a look-up table. These calculations can be performed, for example, by a computer software program on a controller or computer.

The controller, or more suitably, the computer program, compares the actual temperature from the calculation to the desired set point temperature (step 1313), where the set point temperature is
It is stored in the computer memory in advance. Depending on the difference between the actual temperature and the set point, a current may be applied to the fluid or reduced therefrom utilizing a controller (eg, a switch). For example, if the temperature of the fluid is equal to the set point (step 1315), the process returns to step 1307 via branch 1317. Alternatively, the method provides for regulating (eg, increasing, decreasing) the current to the fluid via branch 1319. This sequence can be indeterminately repeatable, as required by the application.

In a modification to the previous embodiment, the global or overall temperature of the microfluidic system is allowed to rise or fall during any one of the process steps described above. The overall fluid temperature is preferably raised or lowered entirely using the techniques described above, although other techniques may be used. Thus, fluid can move in the microfluidic system from one region to another. Fluid movement is
It may be combined with selective heating of fluid in selected portions of the microchannel and / or global fluid heating of the entire microfluidic system. In addition, the fluid of the microfluidic system can be static and can be heated entirely or selectively at specific locations within the microfluidic system.

[0109] In an alternative aspect of the invention, a technique is provided for controlling and detecting the temperature of a fluid in a microchannel by resistance measurement. Other techniques may be utilized depending on the application. These techniques, of course, depend on the application. The technology may be briefly described using the following steps: (1) flowing fluid in a channel of the microfluidic system; (2) stopping flow of fluid in the microchannel (optional); (3) applying a current to the fluid to heat the fluid in the microchannel; (4) measuring the resistance (or conductivity) of the heated fluid in the channel; (5) measuring the resistance ( (6) comparing the actual temperature of the fluid based on the desired temperature set point; and (7) the difference between the actual temperature and the desired temperature set point. , The current applied to the fluid is adjusted.

The above-described successive steps can be performed using, for example, a computer program. Computer programs provide an easy-to-use method for performing the above steps of a microfluidic system. The computer program is executable in the form of computer software, firmware, hardware, or a combination thereof. This program performs the above functions using an interface connected to the microfluidic system. This interface receives signals from the microfluidic system and for the purpose of heating, for example,
Provide a signal, such as to a power source (which supplies current to the fluid). Details of the successive steps will be described below with reference to FIG.

The process 1400 according to the present invention begins at step 1401, which generally requires the use of a microfluidic system, such as the systems described herein, and other systems. The process causes fluid to flow into the microchannels or capillaries by electroosmotic and / or electrophoretic forces (step 1403).
. These forces are described throughout this specification, but are not limited to hydraulics and the like.
The fluid can be controlled by a controller or a computer. Optionally, by selectively applying energy to the fluid through the controller, the fluid flow slows, increases, or stops in the channel (step 1405). Alternatively, the fluid flow may maintain a constant flow in the channel.

To heat the fluid in the channel, an electric current is applied to the fluid, such as by an electrode, etc., that is directly connected to the channel (step 1407). A controller or computer provides a signal to turn on a power or energy source to provide current directly to these electrodes. When a current is applied, the fluid is in the channel, or
Heat in selected areas of the channel, such as with a smaller internal perimeter. The current can be direct, alternating or pulsed, depending on the application. A controller or computer can be coupled to the fluid via electrodes or probes to measure the resistance or conductivity of the fluid, as shown in step 1409.

[0113] The measured resistance can be converted to the temperature of the fluid. Based on the type of fluid, a temperature is calculated (step 1411). For example, the fluid temperature can be found in a look-up table. These calculations can be performed by, for example, a computer software program in a controller or a computer.

The controller, or more suitably, the computer program, compares the calculated actual temperature to the desired set point temperature (step 1413), wherein the set point temperature is
It is stored first in the computer's memory. Depending on the difference between the actual temperature and the set point, by utilizing a controller, current may be added or reduced to the fluid. For example, if the fluid temperature is equal to the set point (step 1415), the process returns to step 1409 via branch 1419. Alternatively, the process provides regulation of the current to the fluid via branch 1417 and returning to step 1407. This successive step, as required by the application,
Indefinitely, it becomes repeatable.

In a modification to the previous embodiment, the overall temperature or the overall temperature of the microfluidic system can be raised or lowered during any one of the above-described process steps. The overall fluid temperature is preferably raised or lowered entirely, using the techniques described above, although other techniques may be used. Thus, fluid can be moved from one region to another in a microfluidic system. Fluid transfer may be combined with selective heating of the fluid in selected portions of the microchannel and / or overall fluid heating of the entire microfluidic system. In addition, the fluid of the microfluidic system can be static and can be heated globally or selectively at specific locations in the microfluidic system.

[0116] In an alternative embodiment, the temperature of the fluid is detected in the microchannel utilizing a fluorescent material according to the present invention. The method can be briefly outlined as follows: (1) providing a fluid with a tracer material in a channel of a microfluidic system; (2) applying a fluid to heat the fluid in the microchannel. Applying a current; (3) within the channel, the optical properties (eg, intensity) of the fluid with the tracer material
(4) correlating the optical properties with a reference; (5) determining the actual temperature of the fluid based on the optical properties; (6) comparing the actual temperature of the fluid with a desired set point; 7) Adjust the current applied to the fluid based on the difference between the actual temperature and the desired temperature set point.

The above continuous steps can be performed using a computer program or the like. The computer program provides a series of easy-to-use steps to perform the above steps of the microfluidic system. The computer program can be executed in the form of computer software, firmware, hardware, or a combination thereof. This program performs the above functions using an interface connected to the microfluidic system. The interface receives signals from the microfluidic system and provides signals to a power supply or the like that supplies current to the fluid for heating. Details of these steps are described below with reference to the simplified diagram of FIG.

A process 1500 according to the present invention begins at step 1501, which generally requires the use of a system described in the present invention, and a microfluidic system, such as another system. The process provides a fluid containing a tracer inside the system, as illustrated in step 1503. Tracers generally emit light in a manner that is proportional to the temperature of the tracer. More specifically, the tracer emits light in proportion to the temperature of the fluid. An example of this tracer is a temperature sensitive fluorescent dye. Of course, the type of tracer used will depend on the application.

The power source applies a current to the fluid (step 1505) to heat the fluid in the channel. The current is applied to the fluid using, for example, electrodes directly connected to the fluid in the channel. In particular, a controller or computer provides a signal to turn on a power or energy source to provide current directly to these electrodes. Upon application of an electric current, the fluid heats up in the channel or in selected areas of the channel having a smaller internal perimeter. The current can be direct, alternating or pulsed, depending on the application.

The sensor measures a property of the fluid (eg, optical intensity such as fluorescence) (Step 1).
507). In particular, a tracer emits a certain amount of light depending on its temperature.
The sensor reads the intensity level of the light emitted from the tracer. In a preferred embodiment, the sensors are optical sensors. Alternatively, the sensor may be a CCD (charge coupled device) device or a CCD camera, etc.
It has an array of CD elements. The light intensity value captured by the sensor is transmitted as an electrical signal to a controller or computer.

The controller correlates the intensity value with a reference (step 1511) to determine the actual temperature of the fluid (step 1509). The correlation is generated by, for example, a controller or a computer program executed by a computer. Of course, there are other ways to use the measured intensity value to determine the actual temperature of the fluid.

The controller, or more suitably, the computer program, compares the actual temperature to the desired set point temperature (step 1513), the latter being previously stored in the computer's memory. Depending on the difference between the actual temperature and the set point, the current may be applied to or reduced from the fluid utilizing the controller. For example, if the temperature of the fluid is equal to the set point (step 1515), the process proceeds to branch 1
The process returns to step 1507 via 519. Alternatively, the process provides additional current to the fluid via branch 1517 and returning to step 1505. This sequence of steps is indeterminately repeatable, as required by the application.

Embodiments that aim to control the temperature of the fluid may furthermore depend on the application,
It can be modified or controlled by an active feedback process or control. The activity feedback process generally receives signals such as, for example, temperature from a microfluidic process. The actual temperature is compared with the set point temperature and a difference is calculated. If the actual temperature is lower than the set point, the result based on the function of the temperature difference is
Used to provide additional current or voltage to the fluid. In a preferred embodiment, this function prevents substantial "overshoot" or "swing" of the actual temperature from the set point temperature. In addition, this function ensures that the set point is achieved in a valid manner. Examples of functions used to provide feedback control include, inter alia, proportional control, differential control, integral control, or combinations thereof.

For example, utilizing a proportional control, a feedback process according to the present invention provides active feedback to the process based on a multiplier. The output of the proportional controller is a fixed multiple of the measured "difference" or "error". That is,
A proportional controller is just a "multiplier". Terms often used in describing a proportional controller include proportional band and controller gain. Controller gain refers to the amount by which error multiplication is performed to obtain an output. The controller can be calibrated to a proportional band, rather than a gain, depending on the application.

The process according to the invention generally measures a signal, such as the signals described above, to identify the temperature of the fluid. This signal is transformed. The transformed signal is compared to a desired set point. The difference between the transformed signal and the signal corresponding to the desired set point is calculated. This difference is amplified by a constant that is a proportional gain. The amplified difference provides an output that drives the current to reduce the difference. This example is merely illustrative, and should not limit the scope of the claims herein.

In a modification to the previous embodiment, the overall temperature or the total temperature of the microfluidic system can be raised or lowered during any one of the process steps described above. Preferably, the total fluid temperature is raised or lowered generally using the techniques described above, but other techniques may be used. Thus, fluid can move from one region to another within a microfluidic system. Fluid transfer is combined with selective heating of the fluid in selected portions of the microchannel and / or global fluid heating of the entire microfluidic system. In addition, the fluid of the microfluidic system may be static and may be heated entirely or selectively at specific locations in the microfluidic system.

The above embodiments relate to the process of controlling the temperature of a fluid by measuring the actual temperature, which also simply sets the current or voltage or power applied to the fluid by the power supply. With just this, control becomes possible. For example, the power supply may be operable in a current controlled mode in which the amount of current applied to the fluid remains constant and the voltage varies depending on a change or perturbation in the resistance of the fluid. Or,
The voltage of the power supply may remain constant, but the current is varied depending on the change or perturbation of the resistance of the fluid. Still further, depending on the application, the power supply is operable in a mode where the combination of current and voltage remains constant. Of course, the mode used depends on the application.

Although the above description has been related to a flow diagram that can be implemented using computer software, the invention can also be implemented in many other ways. For example, computer software may use a field programmable gate array ("FPGA").
), Electrically erasable programmable read only memory ("EEPROM")
, Read only memory ("ROM"), random access memory ("RAM")
) Can be implemented in hardware such as a memory device. Another type of memory device is a compact disk read only memory ("CDROM"), hard disk, floppy disk, high capacity disk (eg, Iomega Co.).
ZipDrive ^™ sold by R. Corporation). Alternatively, the computer software can be implemented in a combination of hardware and software. Some of the functions described may be separate or combined, depending on the application. Of course, those skilled in the art will recognize other changes, modifications, and alternatives depending on the particular application. III. Embodiments of Process Monitoring Using Conductivity In certain embodiments, the present invention provides techniques for monitoring a variety of process parameters, such as the concentration of a fluid or material species, pH, temperature, and the like. . More specifically, these parameters are found by measuring the fluid conductivity.
That is, the fluid conductivity measurement is correlated with fluid parameters such as fluid concentration, pH, temperature, etc., and such parameters are compared to desired parameter set points. These techniques may be used alone or in combination with the heating applications and / or material transport systems described above. The method according to this embodiment can be briefly outlined as follows: (1) providing a fluid in a channel of a microfluidic system; (2) measuring the conductivity of the fluid in a channel of the microfluidic system. (3) Correlate the measured conductivity with reference parameter values such as fluid concentration, pH, or temperature in the lookup table; (4) For setpoint values, fluid from the lookup table Compare the values of the parameters; (5) Make adjustments to the process as needed.

The above continuous steps can be performed in the present microfluidic system. The program performs the above functions using at least an interface coupled to the microfluidic system. The interface receives a signal corresponding to the conductivity value from the microfluidic system and provides a signal as an intensity value to the controller. The controller reads the intensity value and determines the actual parameter value based on, for example, a reference table. Details of these steps will be described below with reference to the simplified diagram of FIG.

A process 1600 according to the present invention begins at step 1601, which generally comprises
Requires the use of microfluidic systems, such as the systems described herein, and others. This process involves flowing the fluid into the microchannel or capillary by electroosmotic and / or electrophoretic forces (step 1603).
). These forces are discussed throughout this specification, but are not limited to hydrodynamic forces and the like. The fluid can be controlled by a controller or a computer. Optionally, by selectively applying energy to the fluid through the controller, the fluid flow slows, increases, or stops in the channel (step 1605). Alternatively, the fluid flow can maintain a constant flow in the channel. To determine fluid conductivity, a controller or computer coupled to the fluid via an electrode or probe measures the resistance or conductivity of the fluid, as shown in step 1609.

The measured resistance or conductivity can be converted to fluid concentration, pH, temperature, or other fluid properties or parameters. Based on the type of fluid, a selected fluid parameter is calculated (step 1611). For example, the selected fluid parameter can be determined or calculated by using a reference table. These calculations can be performed, for example, by a computer software program on a controller or computer.

The controller, or more suitably, the computer program, compares the measured or calculated fluid parameters to a desired set point (step 1613)
The set point is stored first in the memory of the computer. Depending on the difference between the measured fluid parameter and the set point, the measured or other parameters of the system may be adjusted (step 1615). Alternatively, the system may be shut down temporarily to reset certain parameters. The additional conductivity value is determined by branch 16
17 is measured.

By way of example only, the conductivity of the buffer fluid determines the buffer type and pH value. The conductivity of the buffer is a function of the acid / salt concentration, pH, and temperature. Additionally, conductivity can also be utilized to determine buffer solution content, and other properties. A buffer consisting of 1 M sodium acetate buffer (NaOAc) at pH 5 made with hydrochloric acid (HCl) (eg, 1 M NaOAc adjusted to pH 5 by adding HCl) is sodium hydroxide (NaOH). ) Made with 1M NaOAc buffer at pH 5 (eg, 1M acetic acid (HOAc), which is adjusted to pH 5 by adding NaOH);
It has specific conductivity.

For a number of buffer solutions, the specific and unmatched unique properties allow, for example,
A lookup table of conductivity values for widely used buffers as a function of concentration, pH, temperature can be created and used to identify buffer fluids that are measured and used in microfluidic systems; Alternatively, it can be created and used as a quality assurance mechanism to ensure that a suitable buffer is utilized in the system. The particular value for a given buffer can be readily determined by routine experimentation.

These values are then optionally incorporated into look-up tables for a variety of widely used buffer solutions. The look-up table typically lists commonly used buffers in one row. The concentration, pH, and temperature are listed in the second, third, and fourth rows, respectively. This lookup table can be stored in the memory of the computer or controller for easy access. Thus, the present invention allows a user of a microfluidic system to identify and determine a number of parameters from the measured fluid conductivity values.

A number of advantages are provided by monitoring process parameters according to the present invention.
Achieved. For example, a user of a microfluidic system can easily determine whether a suitable fluid is being used in a selected microchannel by a simple conductivity measurement, but the measurement can be a variety of unique Correlated with a fluid parameter or property. In addition, the present invention allows users to have greater confidence in the data being generated in microfluidic systems, but this data is monitored using conventional equipment and monitoring equipment. It is often difficult to do. Furthermore, the present invention provides "high quality" data, which can be presented to potential customers of the data. Further, the present invention provides a technique for in situ process monitoring with simple conductivity measurements. Thus, in-situ monitoring technology can provide users with greater "trust" about the results of the operations being performed.

[0137] In a modification to the previous embodiment, the fluid can be selectively heated during the process monitoring step. A power supply that provides current through the fluid selectively heats a portion or portions of the fluid in the microchannel. Any of the techniques described herein, and others, can be used to heat a fluid. Fluids may even be made coolable using the present monitoring technology and by the techniques described herein. Therefore, the fluid, in combination with the monitoring technology,
It can be selectively cooled and heated.

In a further modification to the foregoing embodiment, the overall temperature or the total temperature of the microfluidic system can be raised or lowered during any one of the process steps described above. Preferably, the total fluid temperature is raised or lowered entirely using the techniques described above, although other techniques may be used. Thus, fluid can move from one region to another in a microfluidic system. Fluid transfer includes selective heat treatment of the fluid in a selected portion of the microchannel, and / or
Alternatively, this is combined with the overall fluid heating of the entire microfluidic system. In addition, the fluid of the microfluidic system may be static and may be heated entirely or selectively at specific locations in the microfluidic system.

Although the above description relates to a flow diagram that can be implemented with computer software, the present invention can also be implemented in many other ways. For example, computer software may use a field programmable gate array ("FPG
A "), electrically erasable programmable read only memory (" EEPROM
)), Read-only memory (“ROM”), random access memory (“RA
M ”) or other hardware such as a memory device. Another type of memory device is a compact disk read only memory ("CDROM"), hard disk, floppy disk, high capacity disk (eg, Iomega).
Such as ZipDrive ^TM sold by the Corporation), and the like. Alternatively, the computer software can be implemented by a combination of hardware and software. Some of the functions described may be separable or combinable depending on the application. Of course, those skilled in the art will recognize other changes, modifications, and alternatives depending on the particular application. IV. Amplification and Detection of Nucleic Acids In certain embodiments, the present invention provides a process for treating nucleic acid material to effect a complete or partial change from a double-stranded form to a single-stranded form; Related to the process of amplifying or detecting nucleic acids involved in the denaturation process. Preferably, the process for treatment of the nucleic acid material occurs by continuously changing the temperature of the nucleic acid material by thermal cycling. Thermal cycling according to the present invention occurs by applying an electrical current to nucleic acid material in a microcapillary of a microfluidic system. The present invention utilizes any of the techniques described herein to apply a current to a nucleic acid material, but is not limited to such techniques.

[0140] Double-stranded DNA (commonly referred to as "deoxyribonucleic acid") and DNA / RNA and RNA / RNA complexes in the well-known double helix configuration are stable molecules in vitro. This requires aggressive conditions to separate the complementary strands of the nucleic acid. Typically, the methods used for strand separation require the use of a temperature of at least 60 ° C, and often an elevated temperature of 100 ° C or higher, or an alkaline pH of 11 or higher. The present invention provides a method for obtaining the elevated temperatures often required for strand separation.

Details of strand separation can be found, for example, in US Pat. No. 4,683 to Mullis et al.
, No. 202. Mullis et al. Hybridize target nucleic acid sequences or mixtures thereof contained in nucleic acids with specific oligonucleotide primers, separating the complementary strands of the nucleic acids, extending the primers with a nucleic acid polymerase, Amplification by using the above extension product for further synthesis of the desired nucleic acid sequence by forming a complementary primer-extension product and then hybridizing with the particular oligonucleotide primer that reappears And a process for detecting. Repeating the process cycle with the elevated temperatures required to denature the nucleic acid strands also results in overall denaturation of the polymerase enzyme, which is generally very expensive. As a result, these thermal PCR processes typically used a thermostable polymerase (eg, Thermus aquaticus polymerase (Taq)) to preserve polymerase activation throughout the amplification process.

This process can be performed iteratively to generate large quantities of the required nucleic acid sequence, even from a single molecule of starting material. Separation of the complementary strands of the nucleic acids is preferably achieved by thermal denaturation in a continuous cycle. This is because the thermal process provides a simple reversibility of the denaturation process to reform the double-stranded nucleic acid to continue the amplification cycle. According to the present invention, thermal denaturation in a continuous cycle is caused by continuous heating of a region of the channel utilizing the techniques described herein. Since the area being heated is for a small amount of fluid, heating and cooling occur at a sufficient rate and rate to efficiently perform the denaturation process.

The technique for continuous cycle heat denaturation according to the present invention can be briefly outlined as follows: (1) providing nucleic acid material to a channel; (2) adjusting the temperature of the nucleic acid material to a first temperature. Applying a current to the fluid to increase the temperature: (3) removing the current from the fluid to reduce the temperature of the nucleic acid material to a second temperature; and Step 3 is repeated continuously.

The sequence of steps described above provides a continuous thermal cycle in a certain local area of the channel. This allows heat to be efficiently transferred to the fluid itself, which allows for efficient heating and cooling of the nucleic acid material. In addition, the present invention provides for local heating of the material itself, which is generally the case when continuous addition of thermolabile enzymes is to be avoided, e.g., for thermophilic organisms such as primer-extension steps. It may not require the use of certain thermostable polymerase enzymes from the body.

Furthermore, because heat is applied to selected regions of the channel, the destruction of the DNA structure itself, for example, from high temperature broken phosphodiester bonds is limited.
Further, the invention provides for heating nucleic acid material in a closed volume channel (eg,
(Above 90 ° C.), which substantially inhibits evaporation of the fluid, which is often associated with previously used techniques. Still further, the present invention is useful in applications such as the Human Genome Project and the routine diagnostic industry where reagent economy, assay format design, and speed of the DNA denaturation / reconstitution process are important, for example, reagent economics. The reaction can take place in very small amounts to provide

As mentioned above, the temperature control methods and systems described herein reduce the temperature of fluids and other materials inside microscale channels without simultaneously heating the entire substrate on which the channels are located. Can be raised quickly. The advantage of rapid and / or local heating is that many biochemical processes, especially the polymerase chain reaction (
When used in conjunction with an amplification process such as PCR), it offers additional advantages. For example, the rapid and / or local heating capabilities of the devices and systems of the present invention allow performing thermal amplification without the need for a thermostable polymerase. Although thermostable polymerases have been proven to be effective in the thermal amplification process in the past, these enzymes have a number of problems associated with their use. For example, in addition to their high cost, the most commonly used Taq (Thermus aquaticus) polymerase, for example, has incorrectly incorporated nucleotides (eg, improperly incorporated based on template sequence) ) Suffer from relatively low fidelity (eg, up to 1%).

In general, the methods of the present invention provide the ability to amplify nucleic acid sequences without requiring the use of a thermostable polymerase enzyme, ie, Taq. Eliminating the need for such polymerases offers the cost advantages of reagents and enzymes used in the amplification process, as well as the advantages of polymerization fidelity and extension rate. In particular, a wide range of non-thermostable polymerase enzymes are commercially available (eg, T4 DNA polymerase) and are much less expensive than thermostable species. In addition, these non-thermostable polymerases typically incorporate nucleotides into the extension product at higher speeds and higher fidelity than thermostable polymerases.

In a first aspect, the methods, devices and systems of the present invention take advantage of the extremely localized heating capabilities of these methods and devices. In particular, the thermal cycling steps of the reagents for the amplification reaction (e.g., to melt and anneal the template and primer) are performed in certain regions of the channel or channel network, while the extension reaction involving the polymerase enzyme involves: Performed in a different region of the channel or channel network, this region is not subjected to the elevated temperatures of the thermal cycling process or is performed within the thermal cycling region during the non-heating time of the thermal cycling process. Reagents, such as templates, primers, extension reagents, polymerases, etc., are transported between regions or reacted, as described herein, using, for example, controlled electrokinetic transport. Injected into the region. As the heating is localized, the polymerase enzyme stored or maintained in different areas of the device is substantially unaffected by the elevated temperatures inside the reaction area of the device.

However, in a related aspect, the methods, devices and systems take advantage of the extremely high speed at which such systems can heat fluids inside microscale channels. In particular, the resistive heating method and system of the present invention can heat a fluid in a microscale channel to a temperature close to 100 ° C. or more in about seconds and milliseconds. This is compared to the few minutes required to achieve such a temperature in a conventional thermal cycler.

This rapid and precise control over temperature allows the reaction mixture containing the template, primer and enzyme to reduce the melting temperature of the template and primer to
It is possible to reach it very quickly. In a preferred aspect, the mixture is maintained at the melting temperature for a period of time sufficient to cause the template and primer strands to melt apart without denaturing the polymerase enzyme. In particular, and without being bound by any particular theory, the more complex tertiary structure of proteins (particularly polymerase enzymes) is more thermally denatured than for the simpler secondary structure of double-stranded nucleic acids. Is thought to occur at a much slower rate. this is,
It is thought to be due to the effect of stabilizing the more complex structure of the protein.

In practice, depending on the structure of the protein or nucleic acid (eg, number of disulfide bonds, relative GC content, etc.), there is some variation in the rate of heat denaturation between different proteins or different nucleic acids. However, nucleic acids are typically thermally denatured (eg, melt away) at temperatures near the boiling point (eg, 100 ° C.) within seconds or milliseconds, while being non-thermostable Sex proteins must typically allow seconds, tens of seconds, and in some cases, minutes to thermally denature at such temperatures.

Thus, heating the amplification mixture to the melting point of the nucleic acid of interest for approximately a few seconds, eg, less than 20 seconds, preferably less than 10 seconds, more preferably less than 5 seconds, and often less than 1 second. Thereby, thermal amplification can be performed effectively without denaturing the non-thermostable polymerase enzyme used during the reaction. As noted earlier,
This results in faster extension of the primer along the template due to the use of higher efficiency enzymes, as well as higher fidelity amplification.

The PCR process described above is not intended to be limiting. The PCR process is also described, for example, in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, "PCR Technology: Principles and Applications for DNA Amplification (Errich, eds. Freeman Press, New York, NY, 1
992), "PCR Protocol: Guide to Methods and Applications (Innis et al., Eds.
Academic Press, San Diego, CA, 1990), Ma
ttila et al., Nucleic Acids Research, 19: 4967 (1991), Eckert and K.
unkel, PCR method and application example, 1:17 (1991), PCR (McPhe
rson et al., IRL Press, Oxford), each of which is hereby incorporated by reference in its entirety for all purposes. reagent,
Devices and instructions for using them are described, for example, in Perkin-Elm.
er Corp. Promega Corp., Promega. Commercially available from such as. Other amplification systems include ligase chain reaction, QB RNA replicase, and RA
An amplification system based on N transcription is mentioned.

[0154] Further described with respect to performing a PCR amplification reaction, the methods, devices and systems described herein may be used with virtually any method, including cycle sequencing reactions, non-reciprocal amplification or transcription reactions, and the like. Useful in performing temperature controlled or cycled nucleic acid reactions.

Thus, as described in greater detail above, the present invention provides for the use of an electrical energy source (eg, a power supply) and is located within the microscale channel and within each portion thereof. Control the temperature of the fluid. Use of such an energy source includes, for example, applying an electric current from the energy source through a fluid located in the microchannel or a portion thereof. As mentioned above, this application uses electrodes at one end and the other end of the channel that are operably connected to an energy source. The applied current can be an AC current, a DC current, any current,
Alternatively, any or all of these may be combined.

The invention is further illustrated with reference to the following non-limiting examples.

[0157]

【Example】

Example 1-Thermal Denaturation / Reconstitution of Nucleic Acids A number of experiments were performed to demonstrate the principles and operation of embodiments of the present invention. These experiments are only a few examples that can be used to demonstrate the utility and effect of the present invention. Therefore, these experiments should not limit the scope of the claims described herein. One skilled in the art will recognize other changes, modifications, and alternatives. Examples of certain useful applications of the methods and systems of the present invention involve processing nucleic acid material and denaturing processes of these types to completely or partially change from double-stranded to single-stranded form In the process of amplifying or detecting nucleic acids.

This experiment was performed to illustrate the thermal denaturation of nucleic acids by a heating cycle according to the invention. Nucleic acids were heated in microchannels having a length of about 5 mm, dimensions of about 70 × 10 μm, and rectangular cross sections. The microchannel has been defined in a substrate made of glass, but is not limited to such a material. This channel was similar, for example, to that illustrated by FIG. Fluids containing nucleic acids were introduced into microchannels using the fluid transfer techniques described herein. The nucleic acids in this experiment included the DNA sequences, GACACAGCTACTCCT and AGGAGTAGCTGTGTC. Add the intercalating dye to the fluid,
A fluorescent signal was provided depending on whether the nucleic acid was double-stranded or denatured (eg, melted apart). That is, the fluorescent signal from the intercalating dye is greater when the nucleic acid is double-stranded and smaller when the nucleic acid is denatured. In this experiment, the intercalating dye used is a product sold under the trade name CyberGreen ^™ , which is available from Molecular Probes, Inc. ,
Manufactured by Eugene, Oregon. Other types of dyes can be used depending on the application.

As noted, a fluid containing DNA and dye was introduced into the channels of the substrate of the microfluidic system. Current was applied to the channel, which caused a decrease in the intensity of the fluorescent signal. Removing the current increased the intensity of the fluorescent signal. FIG. 17 shows a simplified relationship between the intensity of the fluorescent signal and time according to this experiment. This diagram is merely an example, which should not limit the scope of the claims herein. As shown, the vertical axis represents intensity measured in several arbitrary units. The intensity is generally positive and increases along the positive length of the vertical axis. The horizontal axis represents time, which generally relates to a selected time period when current is applied to the fluid for heating. The dashed line near the bottom of the figure represents the time when current was applied to the fluid, and the magnitude of the current is shown on the vertical axis on the right hand side of the figure.

The measurements show that when current was applied to the fluid, the intensity was reduced, indicating that the DNA in the fluid changed from double-stranded to single-stranded. As the current is removed from the fluid, the intensity decreases, which tends to indicate that the DNA in the fluid has been converted from single-stranded to double-stranded DNA (eg, has been re-annealed). The melting temperature of the DNA in this experiment was about 46 ° C. in a solution of 500 mM borate. The cycling properties of the changing fluorescent signal indicate that reversible denaturation of DNA from single stranded to double stranded is possible with the present invention. Thus, the present invention provides for selective heating and cooling of a fluid containing DNA, which can be used in a variety of reactions.

Although the experiments described above are generally described with reference to a process of continuously heating and cooling a particular type of nucleic acid material, it is not intended that the type of nucleic acid material be limited. For example, the nucleic acid material can be of almost any type that can be heated and cooled for various reactions, such as, for example, PCR, LCR, and the like.

Depending on the application, the present invention provides a fluid having one of many flow and temperature characteristics in a channel of a microfluidic system. In certain embodiments, the fluid moves through the channel at a relatively constant temperature, which is not regulated by the heat source. Alternatively, the fluid travels through the channel at elevated temperatures throughout the length of the channel or in a particular region of the channel, which often depends on the geometry of the particular region. Alternatively, the fluid is moved into the channel and then stops in the channel and is completely or partially heated, according to the present embodiment. Alternatively, the fluid moves at a variable speed through the length of the channel. The temperature of the fluid may also heat and / or cool over the entire channel or at a particular area of the channel. Of course, channel flow and temperature characteristics are highly dependent on the application (eg, PCR, LCR, etc.).

In a related embodiment, thermal control of nucleic acid melting and re-annealing is performed using an applied current. This example uses a molecular label consisting of a fluorescent donor-acceptor pair attached to opposite ends of a self-hybridizing nucleic acid sequence. This molecular label self-hybridizes and places the donor and acceptor moieties in close enough proximity to quench fluorescence. Upon denaturation or melting, and / or upon hybridization to the target sequence, the donor and acceptor are separated and no fluorescence quenching occurs, resulting in a fluorescent reaction mixture.

In this experiment performed, molecular labels (Oswel DNA) were placed in 1 × PCR buffer.
(Service, Southampton, UK) was placed in a microfluidic channel, which had a central portion with a narrower cross-sectional dimension. Heating was controlled by applying a current (DC) through the channel, and the temperature was determined from the conductivity of the solution. The fluorescence of the solution in the narrow part of the channel is increased by first increasing the current / temperature from 20 ° C. to about 95 ° C.
When it decreased to, it was measured over time. Control runs were also performed in a standard fluorescent cuvette using a water bath to provide temperature control and a thermocouple to monitor temperature. The fluorescence plot is shown in FIG. 18 (diamond: increasing temperature in microscale channel, open circle: decreasing temperature in microscale channel,
Square: control). As can be seen from the plot of FIG. 18, thermal control of nucleic acid melting by electrical means tracks in much the same way as conventional heating techniques, both during raising and lowering temperature manipulation.

Example 2 In Situ Nucleic Acid Amplification by Electrical Thermocycling in a Microfluidic System Electrical temperature control and monitoring were also used in conventional biochemical applications. In particular, the methods and apparatus described above were used in performing a polymerase chain reaction (PCR) inside a microfluidic channel.

A. Hardware and Software for AC Thermal Circulation As described above, general thermal control experiments were performed by applying a controlled direct current through a microchannel. In performing the PCR reaction, an AC power supply was used to prevent electroosmotic movement of the amplification mixture through the amplification channel.
FIG. 19 illustrates a circuit diagram of an AC power supply connected to a reservoir at the end of an experimental amplification channel in a microfluidic device. The power supply is also shown with a DC power supply that can be used to transport materials in a microfluidic system in an electro-osmotic manner.

A power supply can generate high voltage, high frequency current through an amplification channel in a microfluidic device, where it heats the fluid, but a low voltage, constant frequency signal measures conductivity through the channel. Used for The temperature-conductivity relationship was used to determine the temperature of the fluid inside the channel according to the following relationship.

• (· T) = · _amb [1 + ·· T] where: is the specific conductivity of the fluid; T is the increase in temperature above ambient temperature; Fluid conductivity dependence on temperature in ° C. (. = 0.0194 / ° C. for PCR buffer).

The low frequency signal was generated by a lock-in amplifier, while the heating signal was generated by amplifying the output from the function generator and stepping up the voltage through a series of transformers. The active filter is built into the system and
Over the high frequency currents going to the input of the lock-in amplifier.

All amplification reactions were performed in a microfluidic device (structure shown) connected to the power supply in FIG. The microfluidic channel in which the reaction was performed had a uniform cross-sectional dimension, a length of approximately 9 mm, a width of 70 μm, and 10 μm.
m depth. Although simple straight channels were employed for illustrative purposes, more complex channel networks were used to amplify the amplification process as described in other analytical or preparative reactions (eg, see Example 3 below). It should be appreciated that this may be integrated.

The electrical connection between the power supply and the microfluidic test channel of the device was made via platinum electrodes placed into the reservoir at the end of the test channel, which electrodes are shown in FIG. As shown, the power supply was connected to the appropriate lead.

The AC temperature control system is a computer (IBM PC compatible machine, Pentium manufactured by Intel (> / = 200 MHz)) using LabVIEW software.
Microprocessor / monitor) to monitor and control the temperature and acquire fluorescence data from a microscope / photomultiplier tube (PMT) observing the system. FIG. 20 shows the organization of the LabVIEW subroutines, their state of communication with each other (LavVIEW programs are generally Virtual
FIG. 6 is a flowchart illustrating an example of a sub-VI, which is called an Instrument or VI, and thus a sub-VI is equivalent to a subroutine). Arrows indicate communication between subroutines, while shadowing and size indicate the speed of relative communication (darker / greater = faster communication).

Subroutine 2002 consists of all of the variables describing the system (experimental parameters).
And it is employed to store all of the data (living variables) that change on a continuous basis during the experiment and need to be shared between various subroutines. In general, the experimental parameters were DAQ device, ADC channel, ADC channel, DAC output channel, DAC shutter channel, lock-in sensitivity, function generator amplitude, reference temperature, reference conductivity, file name, and comments. Living variables are temperature set point, lock-in voltage set point, lock-in voltage output,
% Output, binary data (data read from 0 to 2 ¹² (4096) full scale from ADC input), cycle number, and state cluster (temperature, time, and shutter state).

In subroutine 2004 (subroutine of “data reading and temperature control”), data / voltage from the microscope / PMT and the lock-in amplifier is read, and data (“global variables and The "parameter" subroutine) is updated. In addition, this subroutine controlled the temperature by changing the output from the function generator. This% output is the percentage of the amplitude displayed on the front panel of the function generator entering the amplifier transformer circuit.

In the subroutine 2006 (live data viewer subroutine), the following data was continuously displayed. Temperature, temperature set point,% output, and
Fluorescent signals read for channel A and channel B. Data is obtained from subroutine 2002 ("global variables and parameters" subroutine) at a user specified rate. When the entire experiment is complete, this subroutine prompts the user to save the file to disk.

In the subroutine 2008 (“PCR temperature profiler” subroutine),
The temperature set points and voltage set points are changed in the global variables and parameters subroutine 2002 to generate a user entered temperature profile. Next, the user selects a state, which includes the temperature set point, hold time, and shutter state (open or closed). Each state can be selected for a series of additional states that can be programmed to repeat the initial stringent conditions of melting, final elongation, and any number of times, eg, annealing, elongation, melting, etc. When cycling is complete, the program changes the temperature set point to 20 ° C to maintain the system in a safe low voltage state.

Subroutine 2010 (“Shutter (background)” subroutine)
In, the shutter state is toggled between an open state and a closed state by changing the voltage on the "DAC out" pin. The "shutter (background)" subroutine is called by the PCR temperature profiler subroutine 2008, as described above. The other "Shutter" subroutine 2012 is used by the user to manually open and close the microscope shutter.

Subroutine 2014 (“Data Reviewer” subroutine) is used to open a data file from a previously saved experiment and plot data in the same format as Live Data Viewer subroutine 2006. . As shown, both the data reviewer subroutine 2014 and the shutter subroutine 2012 are each independent of the operation of the entire system.

B. Experimental Procedure A conventional lambda PCR protocol was used to demonstrate the performance of PCR operations in microfluidic devices utilizing controlled electrothermal or resistive heating according to the present invention.

The 500 nucleotide segment of lambda DNA (48,500 bases in total) was
Used as target sequence for amplification. The PCR buffers and primers used were from Perkin Elmer Corp. (Norwalk, Conn) as the GeneAmp PCR Reagent Kit. Primer # 1 had the following sequence: 5′-GATGA GTTCG TGTCC GTACA ACTGG-3 ′, while the sequence of Primer # 2 was: 5′-GGTTA TCGAA ATCAG CCACA GCCGCC-3 ′. In these experiments, an intercalating dye (Sybr Green I) was prepared using ds
Used to indicate an increase in the amount of DNA or amplified product.

The fluorescence signal is expected to be constant over the first few cycles of the reaction until a sufficient number of copies have been made to overcome background fluorescence. The cycle at which the signal begins to rise is an indicator of the number of DNA template copies in which the reaction has begun; the earlier the signal rises, the more template copies are present. The fluorescence then begins to increase exponentially with the number of cycles as DNA replication doubles with each cycle. Soon, fluorescence begins to plateau as the amount of primers and dNTPs decreases and it becomes increasingly difficult to anneal the primers to the targeted DNA sequence.

The results of two PCR experiments are shown in FIG. 21 (A and B). As can be seen, these figures show the characteristic exponential growth of the product representing a successful amplification reaction. The conditions for each PCR experiment are shown in the table below, with the differences between the two experiments highlighted in bold.

[0183]

[Table 1]

One of the most significant differences between the two experiments was that the device used in the second experiment (Run B) contained an expanded collar applied to the reservoir, with a higher mineral oil volume (20 μL) can be overlaid on the solution to prevent evaporation and conductivity drift. These conductivity drifts have caused temperature changes. The other major difference is that the first run used a two temperature cycle, while the second run used a three temperature cycle at two different temperatures for the annealing and elongation steps.

Example 3-Integrated nucleic acid amplification and analysis in a microfluidic system The microfluidic device shown in Figure 22 was used to perform multiplex operations in biochemical assays performed on a microfluidic device. This includes complex (blood) sample preparations, specialized reactions (polymerase chain reaction, PCR
), And the ability to integrate functions such as precision analysis (DNA size separation) in a single format.

In this experiment, microfluidic device 2210 was prepared by preparing whole blood and
The NA template was loaded, a PCR reaction was performed, and then used to size the resulting PCR product by gel separation. Channels 2230 and 2240 were filled with sieving matrix gel 2250. In addition, the wells 2260 and 2270 were filled with gel at the end of the separation channel 2230. For the first part of the experiment, approximately 2% purified from whole blood by conventional methods (centrifugation).
000 lymphocytes (white blood cells) were added to 20 μL of the PCR reaction mixture and placed in the sample well 2280 of the chip 2210. The wells are overlaid with mineral oil and the chips are cycled using a thermocycler. After the cycle, the PCR products were separated by a passage through the second chip through channel 2230. FIG. 23 shows an electropherogram of the portion of the HLA locus (approximately 300 bp) where the amplified peak is approximately 34 seconds and is found simultaneously with the 270 bp to 310 bp fragment in the PhiX 174 standard ladder.
For the second part of the experiment, the PCR reaction mixture without the DNA template was placed in the well 2280 of a fresh microfluidic device (of the same design) and 5% whole blood (here, red blood cells were lysed). Is placed in another well. Lymphocytes (white blood cells) were electrophoresed through the channel into wells containing the PCR reaction mixture until 20-100 lymphocytes were present in the PCR wells. All devices were thermally cycled to separate DNA as in the previous example. The results are shown in FIG. Amplification was achieved for both purified and electrophoresed lymphocytes, but the amount of product for the purified lymphocytes was greater than the amount of product for the electrophoresed lymphocytes. Sufficient PCR cycles were performed to ensure that the reaction reached a plateau stage. This is because the number of starting replicas was different. These experiments demonstrate the ability to integrate several steps of a complex biochemical assay on a microchip format.

Although the foregoing invention has been described in some detail for purposes of clarity and understanding,
After reading this disclosure, it will be apparent to one skilled in the art that various changes in form and detail may be made without departing from the true scope of the invention. For example, all the techniques described above can be used in various combinations. All publications and patent documents cited herein are in their entirety for all purposes, just as each individual publication or patent document is individually indicated as an extension. It is incorporated herein by reference.

[Brief description of the drawings]

FIG. 1 is a simplified schematic diagram of an embodiment of a microfluidic system;

FIG. 2 is a schematic diagram of a microfluidic system with a heat source according to the invention;

2A is a schematic diagram of a temperature profile in the system of FIG. 2;

FIG. 3 is a schematic diagram of a microfluidic system with a heat source according to another embodiment of the present invention;

FIG. 4 is a schematic illustration of a microfluidic system with a heat source according to yet another embodiment of the present invention;

FIG. 5 is a schematic diagram of a microfluidic system with a heat source according to yet another embodiment of the present invention;

FIG. 6 is a schematic diagram of a microfluidic system with a heat source according to yet another embodiment of the present invention;

FIG. 7 is a schematic diagram of a microfluidic system with a heat source according to yet another embodiment of the present invention;

FIG. 8 is a schematic diagram of a microfluidic system with multiple heat sources according to the present invention;

FIG. 9 is a schematic diagram of a microfluidic system with a heat or cooling source according to the present invention;

FIG. 9A is a schematic illustration of a microfluidic system with a heat or cooling source according to the present invention;

FIG. 10 is a simple plot of temperature in the microfluidic system of the above figure;

FIG. 11 is a schematic diagram of a microfluidic system with an overall heat and cooling source according to the present invention;

FIG. 12 is a schematic diagram of a microfluidic system with an overall heat source according to the present invention;

FIG. 13 is a simplified flow diagram of a temperature control and temperature detection method according to the present invention;

FIG. 14 is a simplified flow diagram of another temperature control and temperature detection method according to the present invention;

FIG. 15 is a temperature control and temperature detection method using a tracer material according to the present invention;

FIG. 16 is a simplified flow chart for monitoring process parameters according to the present invention; and

FIG. 17 is a simplified plot of experimental results for the step of thermally cycling nucleic acid material according to the present invention.

FIG. 18 is a temperature plot in a microscale channel, as determined from the measured conductivity of the solution in the channel, which is above 100 ° C. and increasing to about 150 ° C.

FIG. 19 is a circuit diagram of a power supply coupled to a microfluidic device for monitoring and / or controlling fluid temperature in a microscale channel by resistive electrothermal and conductivity-based temperature measurements in accordance with the present invention.

FIG. 20 is a flow chart for a computer program used to control and monitor the temperature in a microfluidic device according to the present invention.

FIG. 21A shows two plots (FIGS. 21A and 21B) of increasing fluorescence as a function of cycle number in a PCR amplification reaction. Here, the fluorescence is related to the amount of the double-stranded DNA amplification product.

FIG. 21B shows two plots (FIGS. 21A and 21B) of increasing fluorescence as a function of cycle number in a PCR amplification reaction. Here, the fluorescence is related to the amount of the double-stranded DNA amplification product.

FIG. 22 is a plan view of a microfluidic device for performing integrated amplification and analysis operations.

FIG. 23 is an electropherogram for the assay.

FIG. 24 is an electropherogram for the assay, wherein leukocytes are electrophoresed.

────────────────────────────────────────────────── ─── Continued on the front page (31) Priority claim number 60 / 083,532 (32) Priority date April 29, 1998 (April 29, 1998) (33) Priority claim country United States (US) ( 81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, K, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG U.S.A., U.S.A., UZ, VN, YU, ZW (72) Inventor Kopf-Sill, Ann A. U.S.A. 94028, Portola Valley, Minoca Road 30 (72) Inventor Perth, Jay. Wallace USA California 94306, Palo Alto, Los Robles Avenue 754 F-term (reference) 2G042 AA10 BD19 CA10 GA01 4B029 AA23 BB20 CC01 4B063 QA01 QQ42 QQ52 QR08 QR32 QR35 QR38 QR42 QR62 QR66 QS16 QS24 QS25 4G057 AD01

Claims (70)

[Claims] 1. A microfluidic system, comprising: a substrate having at least a first microscale channel disposed on the substrate; and a system for determining a temperature of a fluid in at least a portion of the channel. A sensor operably connected to the channel, and passing through the at least a first portion of the first channel to heat a fluid disposed within the first portion of the channel to a first elevated temperature. An energy source responsive to a determined temperature of the fluid in the first portion of the first channel for applying a first current. 2. The system of claim 1, wherein the first portion of the first channel has a cross-sectional area that is less than a cross-sectional area of a second portion of the first channel. 3. A second micro-scale channel disposed on said substrate and said second channel intersecting said first channel at said first portion of said first channel.
And a first portion of the first channel. 4. The system of claim 3, wherein the energy source applies a current through the second channel and the first portion of the first channel. 5. The system of claim 3, wherein the first portion of the second channel has a smaller cross-sectional area than the second portion of the second channel. 6. The system of claim 1, wherein the energy source comprises a power source operably connected to the first channel. 7. The first channel has a first end and a second end,
7. The system of claim 6, wherein the power supply is coupled to the first end and the second end of the first channel. 8. The power supply is coupled to the first terminal and the second terminal of the first channel via a first electrode and a second electrode, respectively.
The system according to claim 6. 9. The system of claim 6, wherein the power supply delivers a current to the first channel portion, wherein the current is selected from DC, AC, or any current. 10. The method according to claim 1, wherein the power supply is configured to supply AC power through a first portion of the first channel.
7. The system of claim 6, wherein the DC, DC, or AC and DC currents are applied as needed. 11. A material transport system operably connected to the first channel for transporting the material through the first portion of the first channel for increasing a temperature of the material. The system of claim 1, comprising: 12. The material transport system comprises an electrokinetic material transport system, wherein the electrokinetic material transport system comprises a power source operably coupled to the first channel, wherein the power source comprises 12. The system of claim 11, providing a second current through the fluid-filled channel that affects electrokinetic material transport in the first channel. 13. The system according to claim 11, wherein said first current is alternating current and said second current is direct current. 14. The system according to claim 11, wherein the electrokinetic material transport system and the energy source comprise the same power supply. 15. The sensor according to claim 15, wherein the sensor comprises a system for measuring a relative resistance at the at least a first portion of the first channel, wherein the relative resistance indicates a temperature at the at least a first portion. The system of claim 1, wherein: 16. The sensor and an energy source for applying a first current through the first portion of the first channel to increase a temperature in the first portion of the first channel. Providing a power supply operably coupled to the first channel and determining the relative resistance through the first portion of the first channel;
The system according to claim 15. 17. A processor operably connected to the sensor and a temperature of the first channel, the temperature being increased based on the temperature at the at least first portion, and the temperature at the at least first portion. And monitoring the first
Means for instructing an electrical controller to increase or decrease the selectable current of the electronic controller. 18. The system of claim 1, further comprising an overall temperature controller operably coupled to the substrate, wherein the overall temperature controller maintains the temperature of the substrate at a selected level. 19. The system of claim 18, wherein said selected level is a level above or below ambient temperature. 20. The system of claim 18, wherein said overall temperature controller is selected from a resistive heating element, a Peltier thermoelectric heater, a controlled temperature gas jet, and a controlled temperature fluid bath. 21. The system of claim 18, wherein said overall temperature controller is a resistive heating element or a Peltier thermoelectric heater located adjacent to said substrate. 22. A material transport system operably connected to the first channel for transporting the material through the first portion of the first channel that increases a temperature of the material. The system of claim 1, comprising: 23. The material transport system comprises an electrokinetic material transport system, wherein the electrokinetic material transport system comprises a power supply operably coupled to the first channel, wherein the power supply comprises 23. The system of claim 22, wherein a second current is provided through the fluid-filled channel that affects electrokinetic material transport in the first channel. 24. The system of claim 1, wherein said first portion of said first channel comprises a reagent for performing a nucleic acid amplification reaction. 25. The system according to claim 24, wherein the nucleic acid amplification reaction is selected from PCR and LCR. 26. The system of claim 1, further comprising a computer operably connected to the energy source and a sensor, wherein the computer is in the first portion of the first channel. A suitable programming to instruct an energy source to apply the first current through the first portion of the first channel in response to a temperature detected by the sensor of the first channel. . 27. A method for controlling a temperature of a fluid in at least a first portion of a first channel disposed in a substrate, the method comprising controlling a temperature of the first channel through at least the first portion of the first channel. Applying a first current to the first portion having an electrical resistance of 1; increasing a temperature of the fluid in the first portion of the first channel to a first selected elevated temperature. At least one of the first current and the first resistance
Controlling one of the methods. 28. The method according to claim 28, wherein the controlling step determines a temperature of the fluid in the first portion of the first channel, and the fluid temperature to a temperature substantially equal to the first selected elevated temperature. 3. Increasing or decreasing the first current, respectively, to increase or decrease the current.
7. The method according to 7. 29. The step of determining the temperature includes determining a relative conductivity of the fluid in the first channel, and correlating the temperature with the relative conductivity. 28. The method according to 28. 30. The method according to claim 30, wherein the controlling step includes providing the first portion of the first channel having an increased resistance as compared to a second portion of the first channel. Item 28. The method according to Item 27. 31. The method of claim 30, wherein the first portion has a smaller cross-sectional area than the second portion of the first channel. 32. The controlling step includes applying the first current through a second microscale channel disposed on the substrate, wherein the second channel comprises the second channel. Intersects the first fluid channel at a first portion of the first channel, the first portion of the second channel intersects the first channel at the first portion of the first channel, and 28. The method of claim 27, wherein the method is in fluid communication. 33. The method of claim 32, wherein the first portion of the second channel has a smaller cross-sectional area than the second portion of the second channel. 34. The method according to claim 34, wherein the controlling step comprises:
28. The method of claim 27, further comprising cycling the fluid temperature between at least a second selected temperature. 35. The controlling step comprises: cycling a temperature of the fluid between at least the first selected temperature, the second selected temperature, and a third selected temperature. 35. The method of claim 34, comprising. 36. The method of claim 34, wherein said fluid comprises a reagent for performing a nucleic acid amplification reaction. 37. The method of claim 27, further comprising maintaining the temperature of said substrate at a second selected temperature. 38. The method of claim 37, wherein said second selected temperature is higher or lower than ambient temperature. 39. The method of claim 27, further comprising transporting the material in the fluid through the at least a first portion that increases the temperature of the material. 40. The method of claim 39, wherein said transporting step comprises electrokinetically transporting material in said fluid through said first portion of said first channel. . 41. Electrokinetically transporting the material comprises electroosmotically transporting a fluid containing the material through the first portion of the first channel. 41. The method according to claim 40. 42. The electrokinetically transporting the material comprises electrophoretically transporting the material through the first portion of the first channel. The method described in. 43. The step of electrokinetically transporting the material through the first portion of the first channel, wherein the step of electrokinetically transporting the material comprises a second current through the first portion of the first channel. 41. The method of claim 40, comprising applying electromechanically to transport the material through the first portion of the first channel. 44. The method of claim 43, wherein said second current is DC. 45. The method of claim 43, wherein said first current is an alternating current. 46. The method of claim 27, wherein the fluid in the first portion of the first channel is selected from a sample, an analyte, a buffer, or a reagent. 47. The method of claim 27, further comprising maintaining a second portion of the first channel at a temperature lower than the first selected elevated temperature. 48. The method according to claim 48, wherein the controlling step does not substantially increase the temperature of the fluid in another channel disposed on the substrate.
28. The method of claim 27, comprising increasing the temperature of the fluid in a portion of the fluid. 49. The first current flows to the first portion of the first channel via at least a first electrode and a second electrode operably connected to the first channel. 28. The method of claim 27, wherein the applied first electrode and the second electrode are also operably connected to a power supply. 50. A method of performing a nucleic acid amplification reaction, comprising providing an amplification reaction reagent in at least a first portion of a first microscale channel, wherein the amplification reagent comprises a template nucleic acid, a primer A sequence comprising a sequence, a nucleoside triphosphate, and a polymerase enzyme; cycling the temperature at least in the first portion to at least a suitable temperature for performing the melting, annealing, and extension reactions in the amplification reaction Cycling the temperature, wherein cycling the temperature comprises variably applying a current through the first portion of the first channel, wherein the current is applied to the first channel. Heating the fluid in the first portion of the method. 51. The method of claim 50, wherein said polymerase provided in said providing step is a thermostable polymerase enzyme. 52. The method of claim 50, wherein said polymerase enzyme provided in said providing step is a non-thermostable polymerase. 53. The method of claim 52, wherein said cycling comprises increasing the temperature of said fluid in said first portion of said first channel in less than 20 seconds to a melting temperature of a template nucleic acid. The described method. 54. The method of claim 52, wherein said cycling comprises increasing the temperature of said fluid in said first portion of said first channel in less than 10 seconds to a melting temperature of a template nucleic acid. The described method. 55. The method of claim 52, wherein said cycling comprises increasing the temperature of said fluid in said first portion of said first channel to a melting temperature of a template nucleic acid in less than 5 seconds. The described method. 56. A microfluidic device comprising: a substrate; a first microscale channel disposed in the substrate, the first microscale channel having a first portion and a second portion. And wherein the first portion comprises an elevated temperature region, wherein when the current is applied through the first microscale channel, the elevated temperature region is compared to other portions of the first channel. A first microscale channel having increased electrical resistance therethrough. 57. The device of claim 56, wherein the elevated temperature region of the first channel comprises a smaller cross-sectional area than other portions of the first channel. 58. The device of claim 56, further comprising at least a second channel disposed on said substrate, said second channel intersecting said first channel at said elevated temperature region. 59. A computer program product for operating a microfluidic system, wherein the computer program product is disposed in a first portion of a first channel in response to a temperature detected in the channel. Computer program product comprising a computer readable memory comprising code for instructing an energy source to regulate an electric current applied to the first portion of the first channel containing the fluid. 60. The computer readable memory further includes code for instructing an energy source that modulates an electric field across the fluid in the first channel to electrokinetically move material in the fluid. A computer program according to claim 59. 61. The computer of claim 59, wherein the program is installed on a computer operably connected to an energy source operably connected to at least the first portion of the first channel. program. 62. The apparatus of claim 62, further comprising code for accepting a user input temperature profile, wherein the program heats fluid in the first portion of the first channel according to the user input temperature profile. 60. The computer program of claim 59, wherein the computer program directs an energy source that regulates the current applied to the first portion. 63. A computer-implemented process for controlling the temperature of a fluid in at least a portion of a microscale channel, the method comprising: detecting a temperature of a fluid in the portion of the channel; Comparing the temperature in the portion of the channel with the temperature; and increasing the current applied through the channel to increase or decrease the temperature in the channel to a temperature approximately equal to the preselected temperature Reducing or reducing the process. 64. The detecting step includes determining a relative conductivity of a fluid in a first portion of the channel, and correlating the relative conductivity of the fluid with a temperature of the fluid. 64. The process of claim 63. 65. Use of a power supply to control the temperature of a fluid in at least a portion of the microscale channel, wherein a first current is applied through the portion of the microscale channel to bring the channel to a desired temperature. Use by increasing the temperature of the fluid in the first portion of the method. 66. The use of claim 65, wherein the current comprises an alternating current. 67. The use of claim 65, wherein said current comprises a direct current. 68. The use of claim 65, further comprising the use of a second current to move fluid through the portion of the channel. 69. The first current is applied to a portion of a channel via at least a first electrode and a second electrode operatively connected to an energy source and operably connected to the first channel. 66. The use of claim 65, wherein 70. The use of claim 65, wherein the temperature of the fluid in the portion of the first channel is monitored using fluid conductivity.