JP2001515216A - Microstructure for manipulating fluid samples - Google Patents

Abstract

(57) SUMMARY The present invention is a microfluidic device and method for separating a desired substance, such as a nucleic acid, in a fluid sample from other substances. In a preferred embodiment, the device comprises a micromachined chip (20). The tip has an inlet port (28) and an outlet port (30), and an extraction chamber (26) in fluid communication with these ports. The chamber (26) has an internal adsorption surface that captures a desired substance from the fluid sample as the fluid sample flows continuously into the chamber. The trapped substance is then eluted by forcing the elution solution through the chamber (26), thus releasing the substance from the inner surface into the elution solution. The flow-through design of the device allows the target substance to be concentrated from a relatively large volume of the fluid sample into a much smaller volume of the elution solution. The inner surface is preferably formed by an array of columns (32), which are formed integrally with the walls of the chamber (26) and extend into the chamber (26). Column (32) provides a large surface area to capture the desired material. The device also preferably includes an integral heater to increase elution efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION

RELATED APPLICATION INFORMATION This application is filed with US application Ser. No. 08/910, filed Aug. 13, 1997.
, 434 and US application Ser. No. 09 / 115,454, filed Jul. 14, 1998.

TECHNICAL FIELD [0002] The present invention relates generally to the processing of fluid samples, and in particular, to new methods and microfluidics for manipulating substances containing macromolecules, such as proteins, nucleic acids, and other complex molecular components, in fluid samples. Related to the device.

BACKGROUND OF THE INVENTION In many fields of molecular biology and biomedical diagnostics, effective methods and techniques for extracting, separating, and concentrating target substances, such as nucleic acids, from biological samples have been developed. There is a great need for it. Illustrative in these areas include the processing of medical, industrial, and environmentally large samples such as food, water, blood, biological tissue, expectorants, urine, industrial fluids and soil. These samples are used for genetic analysis. Genetic analysis includes, for example, sequence and pathogen identification and quantification, nucleic acid mutation analysis, genomic analysis, organ suitability for transplantation, gene expression studies, pharmacological monitoring, DNA for drug discovery Library creation and verification.

[0004] Conventional bench-top techniques for extracting nucleic acids from fluid samples mix the sample with disturbing salts (chaotropic salts) and silica particles. Silica particles include, for example, glass or diatomaceous earth particles. This technique is described in US Pat. No. 5,234,809 to Boom et al. And US Pat. No. 5,155,018 to Gillespie et al. DNA, RNA, and many proteins naturally attach and bind to the surface of silica. The disrupting agent causes lysis of the cells in the sample and increases the binding of the nucleic acid to the silica. This also denatures the protein that inhibits adhesion to silica. These salts also soften the secondary structure of DNA and RNA, thus providing a good chemical environment for their attachment.

[0005] A sample containing nucleic acids is mixed with the silica particles, causing the fluid sample to be disturbed, for example by vibration, so as to attach the nucleic acids to any surface. The nucleic acid adheres to the silica particles to form a silica nucleic acid pellet. These pellets are then physically separated from the fluid, typically by centrifugation and disposal of the supernatant, such as by aspiration. The pellets are then washed to remove contaminants, for example using a vortex mixer (vortex mixer) and precipitated again. Also, several additional washing steps are performed. Finally, the nucleic acid is eluted from the pellet using an aqueous solution buffer. These treatments are typically
It is performed manually on a bench top using a standard reaction vessel such as a 1.5 mL conical tube.

[0006] Another benchtop technique for separating and purifying nucleic acids uses hydroxylated silica as a binding matrix. This technique is described in Woodard et al., US Pat. No. 5,693,785. The hydroxylated silica described above allows nucleic acids to bind in aqueous solutions at room temperature without the use of disruptors. According to this method, the DNA-containing solution is incubated with the hydroxylated silica to effect DNA binding. This mixture is then centrifuged to obtain a pellet of bound DNA and the hydroxylated silica. The pellet is then resuspended in the elution solution, typically at 37 ° C. DN
By heating to a temperature that is not destructive to A, DNA elutes from the hydroxylated silica.

As an alternative to traditional bench top measurement techniques, attempts have been made to develop automated microfluidic devices, which perform some of the sample preparation and processing functions. . Microfluidic systems, compared to benchtop measurements,
It has the potential for lightness, improved reproducibility, reduced contamination, reduced reagent consumption, reduced operator intervention, and lower measurement costs. The technology of micromachining enables the production of microfluidic devices, for example using semiconductor substrates, to manipulate, react and detect samples in volumes from microliters to picoliters. For example, U.S. Pat. No. 5,639,423 to Northrop et al. Discloses a microfabricated reactor for performing a biochemical reaction, particularly a D-like reactor such as the polymerase chain reaction.
A reactor for performing a NA-based reaction is disclosed.

However, despite advances in microfluidic technology, microfluidic devices that can effectively extract nucleic acids from relatively large volumes of fluid samples and that concentrate nucleic acids into smaller volumes for subsequent analytical processing. Needs are still there. Particularly in diagnostic applications, it is frequently necessary to extract and concentrate low concentrations of nucleic acids from relatively large volumes of samples, for example several milliliters, such as pathogenic organs or cancer cells. This type of treatment is, for example, toxic E. coli 0157 in food (1 per 10 grams of food).
Toxic at the organism level), HIV in blood (less than 200 viral organisms per milliliter of blood), Bacillus anthracis in bacterial warfare scenarios (100 per liter of air)
Necessary for the detection of cancer cells in blood or urine (less than 10 cells per milliliter of fluid), and pathogens in urine of sexually transmitted diseases (eg gonorrhea and chlamydia) (less than 100 organisms per milliliter of fluid). .

Existing microfluidic devices for extracting and analyzing nucleic acids unfortunately focus on picoliter, nanoliter and microliter sample volumes. For example, Anderson et al., "Microfluidic Biochemical Analysis System"("Microfluidic Bioc
chemical Analysis System ", Transducers '97, 1997 International Conference
on Solid-State Sensors and Actuators, Chicago, June 16-19, 1997, pg.477
-480) discloses a microfluidic device for sample preparation and sample analysis.

[0010] The device disclosed by Anderson includes a 5 to 20 microliter chamber with a glass wall for separating nucleic acids from a fluid sample. In operation, a user first mixes a fluid sample with a perturbing salt to release DNA. The lysed sample is then injected into the chamber and incubated for 10-20 minutes to allow the nucleic acids in the fluid sample to bind to the glass wall. Next, the fluid sample is drained from the chamber and the chamber is washed several times to remove contaminants. Finally, the nucleic acids are eluted from the chamber by filling the chamber with the elution solution and incubating at 50 ° C. for 20 minutes.

[0011] Other existing microfluidic devices rely on electrophoresis to separate nucleic acids from fluid samples. Such devices generally include a chamber that includes a separation groove or an electrode disposed on an opposing surface of the chamber. Also, the chamber typically includes a suitable barrier, such as a filter or membrane, which provides a path for current flow, but is kept away from nucleic acids and other macromolecules. When an appropriate electric field is applied, nucleic acids present in the sample move toward the positive electrode and are trapped on the membrane. Sample impurities that escape the membrane are subsequently washed out of the chamber. When the voltage is reversed, the nucleic acids are released cleanly from the membrane. A microfluidic device for purifying such nucleic acids is described in International Publication No. WO 97/02357, issued Jan. 23, 1997 by Anderson et al., And also described by Sheldon et al. International Publication Number WO98 / 10277 issued to
No.

A major drawback of the microfluidic device is that the microfluidic device is relatively inefficient in extracting nucleic acids from a fluid sample, and the nucleic acids may be transferred from a relatively large volume of fluid sample into a small volume of eluted fluid. It is not concentrated quickly and effectively. In particular, the existing microfluidic devices are limited to macromass processing approaches where a limited and defined amount of fluid sample is held in a chamber and kept warm or electrophoresed. As a result, the volume of fluid that can be processed by such devices is limited to the maximum capacity of the chamber, typically in picoliters, nanoliters, and microliters.

[0013] Such small sample volumes are impractical for many real-world diagnostic applications. In particular, detection of low copy nucleic acids such as DNA requires a sample volume of several milliliters or more for accurate detection. Existing microfluidic devices are:
It is typically limited to picoliter, nanoliter, and microliter throughputs and is not useful for diagnostic applications involving many nucleic acids. Of course, one possible approach is to make a large device to accommodate a large volume of fluid sample. However, if this device is fabricated using integrated circuit chip technology, the ultra-fine fabricated device will have to accommodate the sample volume required to detect low concentrations of nucleic acid, and thus be very difficult. Must be big. Such a large chip is not only expensive but also contrary to the purpose of miniaturization, and is not particularly suitable for various types of disposable medical diagnostic applications or environmental diagnostic applications.

(Objects and advantages of the present invention) Accordingly, a microfluidic fluid for effectively extracting a desired substance in a fluid sample from other substances and providing an eluent highly concentrated with the desired substance It is an object of the present invention to provide an apparatus and method. In particular, it is an object of the present invention to provide a flow-through device in which nucleic acids can be separated from a relatively large volume of a fluid sample, and in which the nucleic acids can be concentrated into a much smaller volume of eluent fluid. It is a further object of the present invention to provide an apparatus with an integrated heater to increase the efficiency of the extraction and elution processes.

[0015] These and other objects and advantages of the present invention will be apparent in light of the following description and accompanying drawings.

Overview The present invention provides a novel method and apparatus for manipulating a fluid sample. An exemplary use of the present invention is to separate desired substances, such as nucleic acids, in a fluid sample from other substances, and to provide a highly concentrated eluent of the desired substance. The desired substance is, for example, a nucleic acid or a target cell or organism or a cell or a protein or a nucleic acid or a carbohydrate or a virus particle or a bacterium or a chemical or a biochemical.

According to a first embodiment, the device comprises a body, preferably a micromachined tip, an inlet port for introducing the fluid sample formed therein into the body, An extraction port for extracting the fluid sample from the body, an extraction port for extracting the desired substance from the fluid sample as the sample flows through the body, and an extraction port in fluid communication with the outlet port. And a chamber. The inlet port and the outlet port are arranged such that the fluid can flow continuously through the extraction chamber. The chamber has an internal adsorption surface that has a sufficiently large surface area and a sufficiently high binding affinity for the desired substance, for example, for nucleic acids, such that the desired fluid sample flows through the chamber. , Such as nucleic acids.

[0018] A preferred method of using the apparatus to separate a desired substance in a fluid sample from other substances comprises continuously forcing the fluid sample into the chamber, whereby the fluid sample flows through the chamber. The desired material to the interior surface of the chamber. Fluid samples include, for example, pumps, electroosmotic forces, electrophoretic forces, air pressure,
The chamber is forced to flow using any suitable fluid actuation source, such as vacuum or gravity. The trapped material is then eluted from the device by flowing the elution fluid into the chamber, thus releasing the material from the internal surface into the eluent fluid.

[0019] In contrast to conventional techniques, the volume of fluid sample flowed into the chamber is preferably
Larger than the volume capacity of the chamber. In particular, the flow-through device of the present invention is capable of concentrating a target substance of a relatively large volume of fluid sample, for example, a few milliliters or more, into a much smaller volume of eluate fluid, for example, 25 microliters or less. 10 to 10
An extraordinary enrichment factor of 0 is easily achieved. Also, the apparatus preferably includes a heater, such as a resistor connected to the wall of the extraction chamber, for heating the chamber. Heating the chamber is optionally used to increase elution efficiency and to facilitate capture of the desired material.

In a preferred embodiment, the internal adsorption surface for capturing a desired substance is formed by an array of internal microstructures. The internal microstructure preferably has a high aspect ratio, is integrally formed on at least one wall of the extraction chamber, and extends into the chamber. High aspect ratio columns provide a large surface area to which the desired material binds. Thus, the fluid sample flows gently or laminarly through the chamber, resulting in efficient extraction of the substance. Therefore, the microstructure enables a simple one-stage extraction process. In contrast, conventional techniques require a two-stage extraction process. In this process, a fluid sample and silica particles are first mixed by creating a turbulent flow by vibration or the like, and then physically separated by a technique such as centrifugation.
The microstructure of the present invention provides a very effective thermal interface from the chamber to the fluid.

Preferably, the columns are arranged in an arrangement that minimizes interaction of the fluid with the column surface as the fluid flows through the chamber. In a preferred embodiment, the columns are in rows, and the rows of columns are each spaced a fixed distance from each adjacent row of columns. Further, preferably, adjacent rows are offset from each other such that the columns of each row are not even slightly aligned with the adjacent columns. Further, in a preferred embodiment, the rows are arranged such that the columns of each row are not aligned with at least two previous or two subsequent rows. This non-collinear array is applied to successive rows of patterns, and the chamber contains one pattern or a repeating pattern. For example, this pattern is repeated every three to ten columns. Alternatively, the non-collinear arrangement of columns may be random from row to row. This offset of the rows increases the interaction of the fluid with the column surface by subdividing the fluid flow. This fluid stream segmentation occurs as any part of the stream comes into very short intervals of the column as the fluid flows through the extraction chamber.

In an alternative embodiment, the internal adsorption surface is formed by a solid support contained within the extraction chamber. Solid supports suitable for capturing the target material include, for example, beads, fibers, membranes, glass wool, filter paper, and gel.

To ensure effective capture of the desired substance, one or more internal surfaces of the chamber are coated with a material that has a high binding affinity for the substance. Suitable materials include, for example, silicon, silicon derivatives such as silicon dioxide, polymers, polymer derivatives such as polyamides, nucleic acids, certain metals, polypeptides, proteins, polysaccharides, or the like as desired. There are other substances with binding affinity.

The device of the invention is preferably used in combination with a cartridge, said cartridge having a pretreatment site for a fluid sample and / or a post-treatment site for eluted nucleic acids. As an example, in order to pre-treat a fluid sample, the pre-treatment site includes a cell lysis region, a precipitation region, and a purification region, and the post-treatment site includes a capillary electrophoresis region, an isoelectric focusing region, a nucleic acid expansion region, and the like. It comprises a hybridization region and an array of hybridization regions of the eluted material. In these embodiments, the device is inserted into the cartridge such that the inlet port is in fluid communication with the pre-treatment site and / or the outlet port is in fluid communication with the post-treatment site.

The cartridge optionally includes or is coupled to one or more fluid actuation sources, such as a pump, a pneumatic source, or a suction device, for forcing a fluid sample into the extraction chamber. The cartridge includes or is coupled to processing electronics having, for example, one or more microprocessors, a multiplexer, a power control circuit, and a sensor circuit to control operation of the cartridge.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention deals with situations where fluids are controlled and moved on a micro-scale, or conversely, manually operated. Microfluidic systems useful for fully automated biochemical analysis require specific functionality, such as volume measurement, fluid mixing, heating or liquid phase separation, if incorporated into a microfluidic circuit. These microfluidic systems must be designed to overcome small-scale effects, such as low Reynolds numbers, that make conventional macroscopic design approaches ineffective.

Fluid mixing is a common and significant event in most biochemical analytical protocols. On the conventional macroscopic scale, there are a variety of effective means for mixing two or more fluids, including pipettes, vortexers and suction / dispensing robotics. However, it is well known that mixing fluids is difficult in microfluidic systems. This problem stems from the fact that in most microfluidic systems, the Reynolds number is generally very small (less than 100) and the flow is virtually always laminar. The Reynolds number is density × speed × groove width / viscosity. In microfluidic systems, the Reynolds number is small because the groove width is very small. In a macroscopic system, before the onset of turbulence, the Reynolds number must be about 2300 or more. In microfluidic systems, mixing occurs almost completely by diffusion, since the fluid flow is virtually never turbulent. Mixing by diffusion can be very slow. An example is a “zigzag” groove 300 μm deep and 600 μm wide. This groove allows the fluid to mix completely with a flow length of only 100 mm.

In biochemical analysis, other methods are required besides mixing. For example, a widely accepted method for extracting proteins and other hydrophobic chemicals from aqueous solutions containing biological compounds is liquid phase separation. Proteins are usually present in high concentrations in aqueous biological solutions such as serum, plasma (plasma) and tissue homogenates, and are structurally composed of hydrophobic and hydrophilic domains. Both the hydrophobic domain and the hydrophilic domain determine the secondary structure and the tertiary structure as a function of a solvent that decomposes them. Usually, the hydrophobic domain is much larger than the hydrophilic domain, but the hydrophobic domain self-bonds to the core of the spherical structure, and when the hydrophilic domain is exposed to the solvent, a stable structure is formed in the polar solvent. can get. This satisfies the difference in Gibbs free energy states between folded and unfolded structures as a function of solvent properties. However, conditions can be created in the aqueous phase, and in most cases are limited solely by extreme salinity and pH or the presence of denaturants or detergents, but low Gibbs free energy states for proteins include, for example, alcohol and It can be achieved in various non-polar solvents such as alkanes and chloroform.

To extract proteins from a polar-based biological sample, selective resolution of proteins, transfer or diffusion of proteins from a polar solvent to a non-polar solvent is used. When a non-polar liquid, such as chloroform, is added to a water-based solution, the fluids (liquids) do not mix and remain separated into two immiscible phases. The denser non-polar phase rises and remains as a separation layer on top of the polar phase. The small number of proteins in the polar phase undergo changes in the tertiary structure, and they prefer the low energy state associated with dissolution in the non-polar phase,
Crosses the boundary between two phases. Over a very long period, the polar phase will be free of protein. When the immiscible fluids are vigorously stirred, the solution becomes an emulsion. In such emulsions, the non-polar phase forms a number of small droplets. These droplets are surrounded by a polar phase and are equally distributed throughout the polar phase. This dramatically increases the effective surface area between the two phases for protein interaction with the non-polar phase and diffusion of the protein into the non-polar phase. The rate of protein transfer to the non-polar phase can be increased by changing the conditions within the polar phase that reduce the structural stability of the polar phase. High salinity, the presence of detergents, phenols and / or contaminants can reduce the stability of the folded state of the protein in the polar phase.

After leaving the emulsion gently without stirring for a while, or (2)
With the aid of relatively increased centrifugal force (if there is a density difference between the two phases), the two phases separate again at the end, with the non-polar phase forming a layer on top of the polar phase. Form. The non-polar phase is highly enriched with protein and the polar phase is depleted of protein. If the original concentration of protein is very high, residual protein may still be present in the polar phase due to back diffusion. Thus, a new non-polar phase solvent is added and the separation is repeated. Similar phase separation methods have been used for many years to separate proteins from water-based biological solutions.

The present invention is a highly useful method for mixing fluids quickly and effectively and for making artificial emulsions that provide a means for performing the same protein extraction function as conventional stirring-based liquid phase separation methods. Provided is a microdevice suitable for producing a large microfluidic interface. New process technologies are becoming available to create ultra-micromachined microfluidic grooves to provide a structure that allows efficient mixing of fluids. With a method known as deep reactive ion etching (DRIE), it is possible to form very deep, but surprisingly narrow, grooves. Rapid diffusion mixing is achieved using this process by flowing two fluids through a deep vertical groove and joining them together. This results in two thin vertical sheaths
These sheaths mix by diffusion over a much shorter distance than conventional grooves. A microgroove structure utilizing the DRIE process is designed to support the liquid phase separation process. As shown in FIGS. 1-5, the fluid channels are formed in various external forms on the solid substrate. FIG. 2 shows a device in which two deep grooves 70, 72 merge into one common groove 74. The groove is formed by etching the silicon substrate using DRIE. Each groove has a depth 73 greater than its width 75 and provides a surface area ratio greater than 3: 1. The fluid flowing through the separated grooves 70, 72 merges at the common groove 74 to form two co-current thin fluid sheaths. Grooves that are deeper in width provide a larger interfacial area during flow convection for diffusion mixing of the two fluids.
FIG. 1E is a scanning electron micrograph of a similar device where six deep grooves merge into a single common groove. Fluids passing through the separate grooves merge at the common groove to form six co-current fluid sheaths.

FIG. 3 shows another apparatus having two deep grooves 70, 72 for passing two immiscible fluid streams (a polar solvent and a non-polar solvent). The grooves 70 and 72 meet at the contact area 80. Because the fluids are immiscible, they flow co-currently between the regions 80 and then enter the corresponding grooves 76, 78, respectively. While the fluids are in the contact area 80, the immiscible fluids do not mix themselves, but one fluid has an advantageous partition coefficient for the other fluid, and the molecules of one fluid are Diffuses between fluids.

FIG. 4 shows a device with a network of grooves 70, 72, 76, 78, which merge to form a composite contact area 80 and increase the fluid contact time. Increased contact time increases particle diffusion. Region 85 is a fluid path that is counter-rotated (rotated 180 degrees) to increase the total path length on a single substrate.

The grooves of the device have a ratio of the area of the inner surface to the outer surface of more than 3: 1. The term "internal surface area", as used herein, refers to the total surface area inside a groove, including the surface area of internal structures such as pillars and microcolumns. . In its simplest embodiment, the device of the present invention comprises a single groove and the "internal surface area" is the sum of the surface area of the two sidewalls and the surface area of the bottom. In this embodiment, the depth of the groove is at least as large as the width to ensure a minimum surface area ratio.

The term “outer surface area” as used herein refers to the area on the outer surface, the area of the substrate surface that is removed to create the internal structure. In the single groove example, "outer surface area" is the surface area of the upper surface of the groove. When used here,
The term "substantially large" as applied to the ratio of internal surface area to external surface area is greater than about 3: 1, preferably greater than about 5: 1, and more preferably greater than about 10: 1. , Most preferably greater than about 20: 1. Typical dimensions of the grooves are 10 μm to 1,000 μm in depth and 5 μm to 50 μm in width. One of the preferred methods is deep reactive ion etching of a silicon substrate, although many different methods can be used to form the trench.

Two or more separate grooves are arranged to intersect (each groove passes a non-mixable fluid) and merge into a single groove. The maximum value of this merging distance depends on many factors, including the polarity of each fluid, the hydrophobicity / hydrophilicity of the inner surface of the groove, the degree of immiscibility of the fluid, the surface tension, and the two parallel flows. There is fluid stability, relative flow velocity and viscosity of the thin fluid sheath. Assuming a relatively stable fluid sheath, turbulent mixing is ruled out due to the low Reynolds number. After this distance, the merged flute is preferably split into two or more flutes and again separated into two or more immiscible fluid streams.

During and during the time the two independent streams merge and meet at the interface, protein and other hydrophobic solutions at the interface diffuse across the interface into the stream of non-polar fluid. . When the two streams come into contact for the first time, the protein is distributed evenly throughout the polar fluid. However, as the fluid moves through the common groove, the protein concentration in the polar fluid at the interface of the two fluid streams begins to decrease as the protein moves across the interface into the non-polar fluid. And
A depletion zone forms at the polar fluid interface, forming a concentration gradient across the width of the polar fluid stream. The rate at which the depletion zone is replenished is a function of the concentration gradient formed and the solute diffusion coefficient, and varies with different proteins. Since the width of the polar stream is typically very small, the rate of replenishment of the depletion zone is very fast. Thus, as the fluid moves through the contact area, more protein is absorbed into the non-polar flow. After the fluid stream is in the common channel, the protein remaining in the polar stream equilibrates back to a uniform distribution. However, the overall concentration of protein decreases.

When many such diffusion regions are arranged in a series, high levels of protein are removed from the polar stream, absorbed by the non-polar stream, and dissolved into the non-polar stream.
Numerous diffusion regions are incorporated into microfabricated small elements such as silicon chips. The typical outline structure shown here is capable of at least 50 diffusion regions per mm ² .

Many variations on this concept are possible. For example, depending on the stability of the fluid streams in contact with each other, it is possible to have a very long diffusion zone without an equilibrium zone.
In this case, the fluid flow is "flat" on the surface of the element. On the other hand, if the fluid flow stability is very low, very small microcolumns or "" It is possible to provide "pillars". FIG. 5 shows an apparatus having pillars 91. Pillars 91 are located in the common groove to increase fluid flow stability. 1F-1G are scanning electron micrographs showing many examples of such devices. It should be noted that the apparatus can be used to reverse operation to perform further downstream processing, i.e., to separate the fluid flowing in the common channel into a number of separate streams. For example, the apparatus of FIG. 1E can be used to separate fluid flowing in a common channel into six separate streams.

FIG. 1H shows a hydrodynamic focusing device according to the invention useful for flow structures. The device includes an inlet port and two side grooves located opposite the port. The inlet port and the side groove merge to reach a common groove. In operation, sample fluid is added to the inlet port, and reagents or focusing fluids flow through the side channels. Fluid flowing through the side channels causes the sample fluid to flow into the central stream of the common channel, thus focusing the sample fluid. The apparatus also includes an optical detector positioned adjacent to the common groove to determine a property of the sample fluid.

The ability to change the surface of the grooves in the device described above provides greater flexibility, which can increase fluid flow stability and prevent physical mixing. For example, if the groove surface in the region through which the non-polar fluid passes is hydrophobic, the tendency of the polar fluid to inadvertently flow into the non-polar fluid is significantly reduced as a result of small cumulative differences in flow rate and viscosity. .

In addition to an apparatus for liquid phase separation, the present invention provides a method and apparatus for separating a desired substance in a sample fluid from other substances. The desired substance may be, for example, a nucleic acid, target cell, organism, protein, carbohydrate, virus particle, bacterium, chemical or biochemical.

A typical use of the device of the present invention is for the extraction, purification and concentration of nucleic acids from a sample fluid. The term "nucleic acid", as used herein, is an artificially or naturally occurring nucleic acid, such as DNA or RNA or PNA, in any possible form, i.e., double-stranded nucleic acid or single-stranded nucleic acid. And nucleic acids in the form of combinations thereof. The term "sample fluid" as used herein includes gases and liquids, and preferably refers to liquids. The sample fluid is particles,
An aqueous solution containing cells, microorganisms, ions, large and small molecules such as proteins and nucleic acids. In particular applications, the fluid sample may be, for example, a bodily fluid such as blood or urine or a suspension such as a finely divided food. The fluid sample may be pre-treated, for example, mixed with a drug, centrifuged, or pelletized, or may be in an unprocessed state.

A preferred embodiment of the present invention is shown in FIGS. FIG. 6 is a schematic cross-sectional view of a microfluidic device 20 that extracts a desired substance such as a nucleic acid from a fluid sample and provides a high-concentration eluent of the substance. The apparatus 20 includes a body therein defining an inlet port 28, an outlet port 30, and an extraction chamber 26, which extracts nucleic acids from the fluid sample as the fluid sample flows through the body. The chamber 26 has an inlet port 28 and an outlet port 30 in fluid communication. These ports are preferably located on the side facing the chamber 26 so that fluid flows continuously through the chamber.

The body is preferably a micro-machined chip comprising a base substrate 22 and a top substrate 24 coupled to the base substrate 22. Substrates 22 and 24 comprise a suitable substrate material such as, for example, silicon, glass, silicon dioxide, plastic, ceramics, and the like. In a preferred embodiment, the extraction chamber 26 is formed in the base substrate 22 and the fluid ports 28, 30 are formed in the top substrate 24. However, in alternative embodiments, many different forms are possible. For example, chamber 26 may be partially or completely formed in top substrate 24, and fluid ports may be formed on the bottom or sides of base substrate 22. Some of these alternative embodiments are described below. The extraction chamber 26 has a sufficiently large surface area to capture nucleic acids as the fluid sample flows through the chamber, and an internal adsorption surface that has a binding affinity for nucleic acids. In a preferred embodiment,
The internal adsorption surface is provided by an array of internal microstructures integrally formed on the walls of the chamber 26 and extending into the chamber, preferably by high aspect ratio columns 32.
It is formed. For simplicity, only 25 columns are shown in the schematic diagram of FIG. However, it must be understood that the device of the present invention includes more columns. In general, it is preferred to make the device using at least 100 columns, and more preferably to make the device using 1,000 to 10,000 columns. The number of columns depends, among other things, on the amount and concentration of nucleic acids in the sample, the dimensions of the chamber, the spacing of the columns, the flow rate of fluid through the chamber, and the like. The specific techniques for manufacturing the device are described below.

FIG. 8 shows part of a row of columns extending from the bottom wall 23 of the extraction chamber. Columns 32 preferably have an aspect ratio (height to width or diameter ratio) of at least 2: 1 and more preferably have an aspect ratio of at least 4: 1. This high aspect ratio column 32 provides a large surface area for capturing nucleic acids. As the fluid sample flows through the chamber, the nucleic acid contacts and adheres to the surface of column 32. To extract nucleic acids, the extraction solution is forced through the chamber, releasing nucleic acids from the surface of the column 32 into the extraction solution. In a preferred embodiment, the column 32 has a height preferably equal to the depth of the extraction chamber of at least 100 μm. In an alternative embodiment, the depth of the extraction chamber is shallow,
At depths below 100 μm, fluid flow through the chamber becomes too slow.

FIG. 9 is a schematic diagram of an arrangement of the columns 32 arranged in the chamber 26. Fluid enters chamber 26 through inlet port 28 and flows between columns 32 to outlet port 30. The columns 32 are preferably arranged in an arrangement that optimizes the interaction of the fluid with the column surface as the fluid flows through the chamber 26. Optimization of the column arrangement results in higher fluid flow rates through the chamber without losing the effectiveness of the extraction.

In a preferred embodiment, the columns 32 are arranged in a number of rows, each in a row being spaced from a column adjacent to that row. That is, the aligned columns preferably have a uniform center spacing. For example, FIG. 9 shows ten equally spaced columns 32 in the horizontal direction. In addition, adjacent rows are preferably offset from each other so that the columns of each row are not aligned with the columns of the adjacent row. For example, each column of the column in FIG. 9 is horizontally offset from an adjacent column.

In a preferred embodiment, the rows are offset so that the columns of each row are not aligned with the columns in at least two previous and / or next rows.
The non-linear arrangement of the columns may be performed in a subsequent row configuration, with the chamber containing a single pattern or a repeating pattern. The pattern is, for example, 3 to 10
It may be repeated for each column. The non-linear arrangement of the columns may be random from row to row. In general, two adjacent columns in the above arrangement should not be offset from each other such that the columns of the first column are exactly at the midpoint between the columns of the second column. Rather, at present, the center-to-center spacing between columns is 5
Preferably, adjacent columns are offset by a distance greater than or less than 0%. This arrangement allows for an asymmetrically split flow pattern through the chamber and ensures that the tributary of the fluid flow interacts as strongly as possible with the surface of the column.

Referring to FIG. 9, a specific example of a proper arrangement of columns is given. In each row, the center-to-center spacing between adjacent columns is 15 μm. The columns are arranged in a pattern that repeats every five rows. In particular, the top five rows are each offset 6 μm from the previous or next row. The lower five rows (sixth to tenth rows) repeat the upper five patterns, and the sixth and top rows are arranged in a straight line. For example, column 32A is in line with column 32B. Of course, this is an example of a suitable arrangement of columns, and is not intended to limit the scope of the invention. From this description, preferably within the general guidelines described above,
It will be apparent to those skilled in the art that the columns can be arranged in many other patterns.

FIG. 10 is a plan view of two adjacent columns in a row. Column 32 preferably has a cross-sectional shape and size that maximizes fluid contact with the surface of the column while allowing for fluid to flow smoothly through the chamber. In a preferred embodiment, this is accomplished by making a column with a six-part streamlined elongated cross section, for example, as shown in FIG. In particular, column 3
2 each have a ratio of cross-sectional length L to cross-sectional width W of preferably at least 2: 1,
More preferably at least 4: 1. Furthermore, the length L of the cross section is preferably in the range from 2 to 200 μm, and the width W of the cross section is preferably in the range from 0.2 to 20 μm.

The gap distance S between adjacent columns in a row is preferably chosen to be as small as possible so that fluid can flow between the columns without undue resistance. Generally, the gap distance S ranges from 0.2 to 200 μm, but more preferably ranges from 2 to 20 μm. The range of 2 to 20 μm is presently most preferred as the fluid substantially contacts the surface of the column without causing excessive resistance to fluid flow through the chamber. The center-to-center spacing between adjacent columns is the sum of the cross-sectional width W and the gap distance S, and is preferably in the range of 0.2 to 40 μm.

The length of the extraction chamber 26, which is the vertical dimension in FIG. 9, is preferably 100
To 5,000 μm, more preferably at least 1,000 μm.
m. The width of the extraction chamber 26 is preferably in the range of 100 to 3,000 μm. Fluid ports 28 and 30 each preferably have at least 10
It has a width or diameter of 0 μm. At present, the chamber 26 has a minimum of 1,000 μm to allow sufficient room for the array of columns 32 and the fluid ports 28, 30.
It preferably has a length of m. In particular, it is presently preferred to limit the arrangement of columns 23 to the central region of chamber 26, leaving an open space at the end of chamber 26 where fluid ports 28, 30 are added. This arrangement increases the uniformity of the fluid flowing into chamber 26 before the fluid flows between columns 32.

Referring again to FIG. 6, the chamber 2 such as the column 32 or the chamber wall
The inner surface of No. 6 is coated with a material having a strong binding affinity to the nucleic acid. Suitable materials include, for example, silicon derivatives such as silicon and silicon dioxide, polymer derivatives such as polymers and polyamides, nucleic acids, certain metals, polypeptides, proteins, polysaccharides.

The properties of glass silicates (SiO ₂ ) can attack and solidify nucleic acids. When the silicon is oxidized, the surface will have similar chemical properties. Such non-permanent (non-covalent) attachment (adsorption) to a surface is typically based on weak dipolar or hydrogen bonding or ionic interactions between the surface and the component being captured. I have. These interactions are reversible through changes in the ionic properties of the solvent and / or surface, heat, or other physiochemical means. Many substances can be tailored to have various interactions between the solute and the solvent in the solution. The polymer can have a group of active surfaces that provide a particular mutual force, and can have a copolymer or dopant that provides ionic or hydrogen bonding capabilities. Certain polymers can have reversible polarity or tunable conductivity. Artificial or natural polypeptides and proteins similarly exhibit the ability to have various interactions with solute molecules. Metals such as gold are well known for their ability to capture DNA and, due to their electronic properties, can alter their interaction with solutes. The inner surface of the chamber 26 may be coated with a material having a strong binding affinity for a target nucleic acid, such as a specific sequence RNA from a virus or a specific sequence DNA from a bacterium, for example. This is accomplished by coating the inner surface with a specific nucleic acid sequence that is complementary to the target nucleic acid sequence. These surfaces are coated during device manufacture or shortly before use.

The microfluidic device 20 preferably includes a heater that heats the extraction chamber 26. The heater is designed to release nucleic acids from the chamber very efficiently, so that large quantities of nucleic acids are released into a small volume of eluent fluid. Heaters are used to facilitate nucleic acid capture. One advantage of using a heater in a small volume microchamber is that minimal energy is required to heat the device.

Generally, the heater comprises a suitable mechanism for heating the chamber 26, which includes a resistive heater or an optical or electromagnetic heater that transmits visible or infrared light. If the body of device 20 is made of an electrically conductive material, preferably silicon, a heater may be provided with a power supply and electrodes to apply a voltage to a portion of the body forming chamber 26. Can be easily provided. In addition, a material having high thermal conductivity shortens the heating time, reduces the required power, and makes the temperature uniform. This embodiment is described in more detail below.

In a preferred embodiment, the heater comprises a resistive heating element 34 coupled to the bottom wall of chamber 26. As shown in FIG. 8, the resistive heating element 34 is preferably a thin film of metal or carbon or polysilicon patterned on the bottom surface of the substrate 22. As an alternative, the heating element may comprise a stacked heating source. The stacked heating source is, for example, an etched foil film heating element attached to the substrate 22. Electrically conductive bonding pads 38A, 38B are patterned on the substrate 22 and make electrical contact with the opposite ends of the heating element 34.

The bonding pads 38 A, 38 B are connected to a power supply by electrical leads for applying a voltage to the heating element 34. Preferably, the control of the power supply is performed by a suitably programmed controller such as, for example, a computer or microprocessor or microcontroller. The controller is programmed to control the chamber 26 by varying the amount of power supplied to the heating element 34 using a number of predetermined time / temperature graphs.

The microfluidic device preferably includes one or more temperature sensors that communicate with the controller to measure the temperature of the extraction chamber 26. Generally, a temperature sensor is a suitable device for measuring temperature, such as a thermocouple, a resistance thermometer, a thermistor, an IC temperature sensor, a quartz thermometer, and the like. Alternatively, the temperature coefficient of resistance of the heating element 34 is used as a means of monitoring the chamber temperature and controlling the heat input by measuring a resistance value indicative of the temperature.

In a preferred embodiment, the temperature sensor comprises a strip 36 of electrically conductive material, which is patterned on the substrate 22. Strip 36 is made of a material having an electrical resistance that varies depending on the temperature of the material, and by monitoring the resistance of strip 36, the temperature of chamber 26 is monitored. Also, electrically conductive bonding pads 40A, 40B are patterned on the substrate 22 to make electrical contact with the opposing ends of the sensor strip 36.

In an alternative embodiment, substrate 22 has additional bond pads 42 patterned on the substrate to provide bulk contact to substrate 22. This bulk contact is used to charge the interior adsorption surface of the chamber at a voltage that attracts and / or elutes the nucleic acids. Suitable metals that form the bond pads with the resistive heating elements, sensor strips, include aluminum, gold, silver, copper, and tungsten.

[0063] The bonding pads 40A, 40B are connected to the controller by electrical leads. The controller is preferably programmed to adjust the amount of power provided to the heating element 34 depending on the resistance of the sensor strip 36. Thus, the controller, power supply, heating element and temperature sensor form a closed circuit temperature control system for controlling the temperature of chamber 26. While a closed circuit system is presently preferred, in alternative embodiments the temperature sensor may be eliminated and the device may operate in an open circuit fashion. Further, processing electronics having, for example, one or more microprocessors, a multiplexer (multi-channel), power control circuit components, and sensor circuit components may be included in the device, or located outside the device body. May be connected to the apparatus main body.

The microfluidic device of the present invention is preferably used in combination with a cartridge or similar device for processing and analyzing a fluid sample. A suitable cartridge in which a microfluidic device can be used is disclosed in US patent application Ser. No. 08 / 998,188, filed Dec. 24, 1997. This disclosure is incorporated herein by reference.

FIG. 12 shows an example of the cartridge 101, and the microfluidic device of the present invention is used together with the cartridge. In this example, the cartridge 101 is designed to process a biological fluid sample and expand the nucleic acid, for example, by the polymerase chain reaction (PCR). The cartridge 101 includes a sample port 103 for conducting a fluid sample to the cartridge, a storage site 109 for storing a lysing reagent, and a storage site 125 for storing a washing reagent. The cartridge also includes a lysis site 107 for lysing the fluid sample, a filter site 119 for the fluid sample to contact the filter to remove organic deposits and cells from the sample, and a storage site 127 for storing the eluted fluid. Includes

The cartridge is preferably manufactured with a recess or recess or dent that houses the microfluidic device 20. The device 20 is inserted into the cartridge 101 such that the inlet port of the device is in fluid communication with one or more pretreatment sites, such as the lysis site 107 and the filter site 119. The outlet port of the device 20 is in fluid communication with the waste site 139 and the reagent site 141 containing the PCR reagent. The reagent site 141
It is fluidly connected to a PCR reaction site 143 for PCR amplification and detection.

The cartridge 101 preferably includes a flow controller 123 such as a fluid diode or fluid valve to control the flow of fluid through the cartridge. Cartridge 101 also preferably includes resistive sensors 115 that detect the presence of fluid in various grooves and sites. The cartridge 101 also includes or is connected to an external fluid power source that forces fluid through the cartridge. Generally, the fluid power source comprises a suitable mechanism for forcing fluid through the cartridge. The mechanism includes a pump, a pneumatic source and a suction device. In the embodiment of FIG. 12, the fluid power source comprises a plurality of electrolytic pumps or fluid-filled pouches located at sites 103, 109, 125, 127, the fluid-filled pouch being emptied by pneumatic, mechanical, or hydraulic pressure. Is done.

The cartridge 101 includes, for example, one or more microprocessors, a multiplexer, a power control circuit, and processing electronics 151 such as a sensor circuit, and controls the operation of the cartridge and the microfluidic device 20.
Processing electronics 151 are connected by electrical leads 147 to various sites and storage areas, pumps, sensors and channels in the cartridge.
In particular, the leads 147 connect the processing electronics 151 to the bonding pads of the microfluidic device 20 so that the temperature of the extraction chamber in the device is precisely controlled.

In FIG. 12, the processing electronics 151 is physically located on the cartridge 101, but the processing electronics may be located outside the cartridge, for example, in a large processing instrument into which the cartridge 101 is inserted. It must be understood. In this embodiment, the cartridge 101 preferably includes a thin card-like portion to electrically connect the cartridge to a mating connector in the instrument. Mating connectors are similar to standard edge connectors used on printed circuit boards. Instead,
The data link to the instrument of the cartridge may be other, such as a radio frequency link or an infrared link.

In operation, a fluid sample containing nucleic acids is applied to the sample port 103 of the cartridge and forced (continuously, eg, using an electrolytic pump or a mechanical pump) to flow through the channel 105, The dissolution site 107 is reached. At the same time, the lysis reagent is released from the storage site 109 and is forced and continuously flowed into the groove 111 to cause the
Reaches 7. Suitable lysis reagents include, for example, solutions with disturbing salts such as guanidine hydrogen chloride, guanidine thiocyanate, guanidine isothiocyanate, sodium iodide, urea, sodium perchlorate, potassium bromide.

The fluid sample and the lysing reagent moving in the grooves 105 and 111, respectively, are detected by the resistance type sensor 115. When the lysing reagent contacts the fluid sample flowing through the lysis site 107, the cells present in the fluid sample lyse. The fluid sample and the lysing reagent subsequently flow into the filter site 119, where the sample contacts the filter and debris is removed from the fluid sample. The fluid sample and the lysing reagent go from the filter site 119 to the groove 121 and are forcibly and continuously passed through the microfluidic device 20.

Referring again to FIG. 9, as the fluid sample and the lysing reagent flow through the extraction chamber 26, nucleic acids in the fluid sample adhere to the chamber walls and the surface of the column 32, while excess material exits the outlet port 30. And flows out of the chamber 26. The flow rate of the fluid sample through the chamber 26 is preferably in the range of 0.1 to 50 μL / sec. Referring again to FIG. 12, the fluid sample and the lysing reagent present in the device 20 flow through the channel 135, through the flow controller 41A, through the channel 136 to the waste site 139. In other embodiments, the fluid sample flows through the chamber and then is redirected and recirculated through the chamber an additional number of times.

After the fluid sample has been forced into the device 20, the cleaning reagent in the storage site 125 is flushed into the channel 129 and flows into the device 20. A preferred wash flow rate is about 0.5-50 μL / sec. Flow controller 123 in grooves 121, 129, 131
Prevents fluid from flowing upstream in the cartridge. The cleaning reagent is supplied to the device 2
Wash off residual contaminants, such as disturbing salts, from the zero internal adsorption surface. Changing p
Various suitable cleaning solutions of H, solvent components and ionic forces have been used for this purpose,
These cleaning solutions are well known in the art. For example, one suitable washing reagent is 80 mM potassium acetate, 8.3 mM trihydrochloride, and 40 uM pH 7.5.
It is a solution of ethylenediaminetetraacetic acid and 55% ethanol. The washing reagent then flows through flow controller 41A to waste site 139.

After cleaning the device 20, the elution fluid from the storage site 127 is forced to flow into the groove 131 and pass through the device 20. The nucleic acids are then released from the inner surface of the extraction chamber into the elution fluid. At this point, the flow controllers 41A, 41B have been modified to prevent elution fluid from flowing through the flow controller 41A and allow the elution fluid to flow into the reagent site 141 through the flow controller 41B. The flow rate of the elution fluid through the device 20 is preferably in the range of 0.1-1 μ / sec. The flow rate of the elution fluid is relatively slow compared to the fluid sample velocity, allowing for more nucleic acid to be released from the chamber.

Generally, a suitable elution fluid is used to elute nucleic acids from device 20. Such elution fluids are well-known in the art. For example, the elution fluid comprises molecular grade pure water or a buffer solution. The buffer solution may be, but is not limited to, TRIS / EDTA or, for example, TRIS / acetate / E of 4 mM Tris acetate (pH 7.8), 0.1 mM EDTA and 50 mM sodium chloride.
DTA, TRIS / borate, TRIS / borate / EDTA, potassium phosphate / DMSO / glycerol, NaCL / TRIS / EDTA, NaCL / TRIS / EDTA / TWEEN, TRIS / NaCL / TW
Includes solutions of EEN, phosphate buffer, TRIS buffer, HEPES buffer, nucleic acid expansion buffer, nucleic acid hybridization buffer.

Prior to forcing the elution fluid through device 20, an intermediate air gap stage is optionally performed. The gas, preferably air, is forced through the device 20. This is done after the washing solution has flowed into the device and before the eluent fluid has flowed into the device.
The air gap stage clearly separates the liquid phase and also helps to at least substantially dry the chamber of the remaining washing solution before elution.

The extraction chamber of device 20 is preferably heated as the elution fluid is forced through the device to increase elution efficiency. As previously described, this heating is preferably performed by powering the resistive heating element of device 20 in a closed circuit feedback system under the control of processing electronics 151.

In a preferred embodiment, the inner surface of the chamber is heated to a temperature in the range of 60 ° C. to 95 ° C. as the eluting fluid flows into chamber 1.

The elution fluid containing nucleic acids exits the device 20 and travels into the groove 135 where the reagent site 141
Leads to. The elution fluid and the nucleic acid come into contact with the dried PCR reagent contained in site 141 and are reconstituted. Then, the elution fluid, the nucleic acid and the PCR reagent subsequently flow into the reaction site 143 for PCR expansion and detection. In an alternative embodiment, it is not necessary to dry the reagent at site 141 because the elution fluid already contains the PCR reagent. The vent 145 is connected to the waste site 139, and the reaction site 143 can release gas during processing.

An advantage of the flow-through device of the preferred embodiment is that nucleic acids from a relatively large volume of fluid sample, eg, a few milliliters or more, can be concentrated into a much smaller volume of eluent fluid, eg, 25 μL or less. Whereas conventional microfluidic devices limit the volume of a fluid sample to microliter round masses, in contrast to this conventional microfluidic device, the device of the present invention efficiently converts nucleic acids from milliliter volumes of fluid sample. Extracted,
Exceptional enrichment rates are possible by eluting the nucleic acids into microliter volumes of eluent.

In particular, the ratio of the volume of the fluid sample forced through the device to the maximum capacity of the extraction chamber is preferably at least 2: 1 and more preferably at least 10: 1.
: 1. In a preferred embodiment, the extraction chamber has a maximum holding volume in the range of 0.1 to 25 μL. The volume of fluid sample forced through the device is in the range of 0.5 to 500 mL, allowing a concentration of 100 or more. For example, in this device, nucleic acids are captured from a 1 mL fluid sample and concentrated in 10 μL or less of the eluted fluid. These parameters are specific examples of preferred embodiments and are not intended to limit the scope of the present invention. In an alternative embodiment, the maximum holding volume of the extraction chamber and the volume of the fluid sample to be processed can be varied to tailor the device for a particular application.

Another advantage of the microfluidic device of the preferred embodiment is that rapid direct heating of the interior adsorption surface of the chamber is considered. The integrity and high thermal conductivity of the chamber wall and column structure cause a direct and rapid heat transfer from the heating element to the adsorption surface without requiring heating of the fluid in the chamber. This improvement in efficiency is important not only in reducing the power required for heating, but also in terms of heating rate and heating accuracy or accuracy. In particular, the rapid and direct heating of the interior surface to which the nucleic acids bind binds significantly increases the elution and elution efficiency of the nucleic acids and provides significant improvements over prior art methods and devices.

An advantage of the microfluidic device of the preferred embodiment is that the microfluidic device includes an array of integrally formed microstructures, preferably including a high aspect ratio column, to separate nucleic acids from a fluid sample. In that case, it provides high efficiency and control. In addition to the direct and rapid heating of the adsorption surface, this microstructure significantly increases the effective surface area of the chamber for capturing and eluting nucleic acids. In addition, the shape and arrangement of the microstructures, compared to beads, filters, and membranes, increase the likelihood that fluid samples or nucleic acids will be trapped in the device during fluid processing.

Further, with regularly spaced columns, the diffusion distance between the columns is constant and a uniform fluid flow exists. Thus, nucleic acids are exposed to the same "microenvironment", as opposed to beads or fibers with irregular properties. This uniformity allows for prediction of extraction parameters including the time, flow rate, heating volume, fluid volume, etc. required for each processing step. In addition, the increased efficiency obtained by using an array of internal structures, and the increased efficiency obtained by rapid and direct heating of the adsorption surface,
The relatively high flow rate of the fluid through the chamber allows efficient nucleic acid extraction and elution. This reduces the overall time required for extraction and elution.

Further, a slight variation of device 20 can be used to selectively extract nucleic acids from a solution or emulsion. Thin insulators based on silicon-glass with conductors underneath serve essentially as separators. The thickness of the insulator is 1 to 1,000 μm. A DC voltage (0.1 to 100 V) is applied to the conductor. The nucleic acids in the fluid sample are selectively attracted to the glass surface, and subsequently the nucleic acids are separated from the glass surface.

In one embodiment, the apparatus includes an extraction chamber, wherein the extraction chamber has vertical columns made from a single substrate, wherein the vertical columns are electrically and physically uniform. . The column is covered with a thin layer of silicon dioxide. The substrate has patterned bond pads thereon to provide ohmic contact to the substrate. The ohmic contact functions as a first electrode, and the device further includes a second electrode, preferably a wire disposed in the chamber, for making electrical contact with the fluid sample. The electrodes are
Used to charge the internal sorbent surface of the chamber to the fluid sample at a voltage that attracts and / or elutes nucleic acids.

As the sample flows through the device, a voltage is applied between the electrodes and a controlled density of surface charge is formed to uniformly cover the interior surface of the chamber. The components of the buffer and carrier solutions can be varied to control the charge distribution on the column surface, as well as the net charge of the target macromolecule. For example, the depth and density of charge on the surface of a microstructure is significantly affected by the pH and ionic force of the fluid. For example, by using amphoteric electrolytes and zwitterions and large amounts of macromolecules dissolved with the target, or by pulsing multiple applied voltages, as in double electrophoresis, the adsorption of the target component can be achieved. Increase. The latter method is used to separate weakly bound non-target mixtures from more strongly bound target components while the sample or wash solution is flowing.

Instead, the alternating voltage is tuned to a frequency that promotes the attraction and retention of DNA (but not other molecules) to the inner surface of the chamber. After the entire volume of the fluid sample has passed through the device, various cleaning solutions are introduced into the device to remove loosely bound unwanted material. Changes in pH or salt concentration, or the use of additives such as detergents and disruptors,
Used to increase such removal. A small amount of carrier solution is introduced into the device for elution. The polarity of the voltage is switched from positive (which holds the nucleic acid) to negative. The nucleic acids are then released from the inner surface and flow out of the device as highly concentrated lumps. By using double frequency electrophoresis with an alternating frequency selected to transfer nucleic acids from the internal surface, the efficiency of release is increased.

The adsorption of bioanalyte to the structure or the release of bioanalyte from the structure is increased by providing the device with ultrasonic energy transfer means. The structure is induced to oscillate, the frequency of contact between the individual molecules and the column increases during the extraction process, and the molecules are stripped from the structure during the release or elution stage. In addition, a piezoelectric ceramic disc is bonded to the outer surface of the device. By applying an AC voltage to this disk, bending occurs, and the arrangement of the microstructures is bent. At resonance, the movement of the structure is maximized. This objective can be achieved by incorporating a small ultrasonic horn into the device.

In another embodiment, another set of reactive structures of conductive material is included in the device. These reaction structures serve as electrodes for inducing electrophoretic pulses and increase the probability that macromolecules will encounter non-conductive but charged adsorption surfaces. The reaction structures are also used to facilitate the removal of bound target molecules from non-conductive structures, ie, columns. The latter is done with or without the reversal of the voltage polarity of the non-conductive capacitance electrode, and
Done with or without chemically mediated desorption.

In another aspect of the invention, a ligand binding method is used for the structure of the device. Ligand binding moieties, such as nucleic acids and proteins, are actively or passively attached to the surface of the structure to form a particular analyte capture surface. Ligand binding chemistries such as silane-based chemistries are used. A dual function linker is used, with one function binding to the internal surface and the other function binding to a target in the fluid sample. Next, the sample containing the test analyte is passed through the device and the analyte binds to the ligand-covered surface. After subsequent washing with one or more washing solutions, the ligand-analyte complex can be eluted. Alternatively, a secondary anti-analyte molecule conjugated to the reporting molecule is passed through the device and the conjugate is captured by the analyte. This complex is also eluted.

The devices of the present invention are useful for linking compounds of biopolymers such as oligonucleotides and polypeptides. Ligation compounds allow a large number of sequences to be synthesized in the device. This is accomplished by transporting, concentrating, and reacting the monomers in a separately addressable reaction / extraction microstructure to bind and deblock reagents and catalysts. By using in this manner, the capabilities of the device are utilized to isolate selected microstructures from each other and from nearby reagents.

The microfluidic device 20 of the preferred embodiment is fabricated using various techniques, including photolithography and / or micromachining. Fabrication is preferably
It is performed on silicon or on other suitable substrate materials such as glass, silicon dioxide, plastics and ceramics. A preferred method of fabricating a microfluidic device using deep reactive ion etching (DRIE) is described herein.

As the starting material of the base substrate 22, a 100 mm n-type (100) 0.1-0.2 ohm cm double-side polished silicon wafer is used. The thickness of the wafer is preferably in the range from 350 to 600 μm, depending on the desired structure. In one embodiment of making the device, the ohmic connection is preferably made by implanting phosphorous ions in the back region to a depth of 0.2 to 5 μm. Alternatively, a p-type silicon wafer may be used, and the ohmic contact is made using boron ion implantation. Subsequent to the implantation, the substrate is heated to activate the dopant.

Next, the wafer is spun, using photoresist on the front side (eg, available from Shipley) to obtain a photoresist thickness sufficient to mask the DRIE process. This thickness depends on the final desired thickness of the etching. The ratio of silicon etch rate to photoresist erosion rate is typically greater than 50: 1. For etching 200 μm structures, 4 μm photoresist is usually sufficient. The photoresist is soft baked at 90 ° C. for about 30 minutes, then exposed, developed, and hard baked with the desired mask pattern using processes well known in the silicon wafer processing art.

FIG. 11 shows a sample mask pattern on the front side of the wafer. The etch mask defines a chamber pattern 44 to form an extraction chamber in the substrate 22 and defines an array of column patterns 46 to form a corresponding array of columns in the substrate. Due to the spatial limitations of the drawing size, the etch mask is shown with only a few hundred columns 46.
However, the arrangement includes 1,000 to 10,000 column patterns, forming a corresponding number of columns in substrate 22.

Next, the patterned wafer is etched using a DRIE process to form an integral column with the extraction chamber. Equipment useful for etching is available from Surface Technology Systems of Redwood, California. The DRIE process alternates between dielectrically coupled plasma etching and deposition using a fluorine-based chemical. 20 in the etched structure
An aspect ratio of 1: 1 is easily achieved. Etch rate is typically 2 μm
/ Min or more.

After etching, the remaining photoresist is removed from the wafer by, for example, oxygen plasma etching or wet chemical stripping in sulfuric acid. next,
The substrate is oxidized so as to cover the inner surface of the chamber, that is, the chamber wall and the column surface with an oxide layer. The oxide layer is preferably 1-100 nm thick and is formed using well-known techniques such as, for example, thermal or chemical or electrochemical growth or deposition.

Next, an electrically conductive material, such as, for example, aluminum or gold or copper, is deposited and patterned on the back side of the substrate to form resistive heating elements, temperature sensors, and bonding pads. Other materials may be used to form the heating elements and sensors. Specific techniques for patterning a metal on a substrate are well known in the art. The substrate is then anodically bonded to a thin Pyrex glass cover, for example, 500 μm. The glass cover has holes formed therein, for example, by ultrasonic milling. The hole forms a fluid port to the chamber. After bonding, the substrate pair is cut into dices using a diamond saw. The resulting structure is shown schematically in FIG.

[0100] The exact size and structure of the microfluidic device will be varied to adapt the device to a particular application. Specific examples of possible devices according to the invention include: This device is 4.0 mm square and 0.9 mm thick. The extraction chamber has a depth of 200 μm and a length and width of 2.8 mm. The fluid ports each have a width of 0.4 mm. The apparatus has an array of honey columns occupying an area of 2.0 mm x 2.8 mm in the chamber. The columns have a height of 200 μm, a cross-sectional length of 50 μm, a cross-sectional width of 7 μm, a gap distance of 8 μm between adjacent columns in a row,
It has a center-to-center spacing of 15 μm. There are about 7,000 columns in the sequence. Of course, these dimensions are only representative examples, and do not limit the scope of the present invention. In alternative embodiments, the specific dimensions of each material of the device may preferably vary within the general guidelines set forth at the beginning of this document.

A specific technique for making a cartridge suitable for use with the microfluidic device of the present invention is disclosed in US patent application Ser. No. 08 / 998,188 filed Dec. 24, 1997. I have. For example, the cartridge is made from at least one injection molded or press molded or machined polymer part. The polymer part has depressions or depressions or dents formed in its surface to form several groove walls, reaction sites and storage sites. Examples of polymers suitable for injection molding or machining include, for example, polycarbonate, polystyrene, polypropylene, polyethylene, acrylic, and commercially available polymeric compounds. A second portion, interpolated in shape with respect to the first portion, fits over the first surface to form the remaining portion of the cartridge. The second part or the third part may be a printed circuit board for making electrical contact with the cartridge. Using these techniques, the microfluidic device is incorporated into one of the recesses of the cartridge. The device is applied to the cartridge with a flexible polymer such as silicone adhesive. Alternatively, a gasket is made with the holes aligned with the fluid ports of the device, and a sealed fluid assembly is formed between the microfluidic device and the cartridge body. The device is pressed tightly against the gasket material and sealed by bonding another plastic article onto the device so that the device is completely encapsulated in the cartridge.

Alternatively, the device is melted or welded directly to the cartridge without using a gasket. In a particularly advantageous embodiment, rather than using a separate substrate of eg Pyrex glass to form the cover, part of the cartridge becomes the cover of the device. In this embodiment, the substrate 22 is inserted into the cartridge,
Sealed to the cartridge wall. This wall has a hole therein and forms a fluid port to the extraction chamber.

FIG. 13 shows an alternative embodiment of the present invention, in which the microfluidic device has fluid ports 28, 30 formed in the base substrate 22 instead of the top substrate 24. The apparatus includes electrodes 48A and 48A for heating the interior surface of chamber 26.
B. These electrodes are preferably arranged on opposite sides on the bottom wall 23 of the extraction chamber. The base substrate 22 is made of thermally conductive, preferably silicon. Accordingly, the bottom wall 23 and the integrally formed column are heated by applying an appropriate voltage to the electrodes 48A and 48B. In a preferred embodiment, the device is preferably used in combination with a cartridge having a pre-treatment site and / or a post-treatment site for a fluid sample. The cartridge includes appropriate processing electrodes for powering electrodes 48A and 48B. The operation of the apparatus is similar to that of the preferred embodiment described above, except that the interior surface of chamber 26 is heated by applying a voltage to electrodes 48A and 48B. The bottom wall 23 functions as a resistive heating element for heating the chamber 26.

The microfluidic device of FIG. 13 is manufactured using various techniques, including photolithography and / or micromachining. A preferred method for manufacturing the device is described herein.

A 100 mm n-type (100) silicon wafer is used as a starting material for the berth substrate 22. The wafer preferably has a resistivity between 1 and 100 ohm-cm, depending on the desired final resistance between the electrodes 48A, 48B. The thickness of the wafer is preferably in the range from 350 to 600 μm, depending on the desired structure. The ohmic connection is made on the back side, preferably using implantation of phosphorous ions to a depth of 0.2 to 5 μm. Alternatively, a p-type silicon wafer may be used, and the ohmic connection may be made using boron ion implantation. After implantation, the substrate is heated to activate the dopant.

Next, on the back side of the wafer, a suitable masking material such as silicon nitride is deposited, patterned, and the silicon is anisotropically etched using this mask, thereby forming the fluid port 28 30 are formed. Next, the wafer is patterned with photoresist on the front side to obtain an etch mask for the DRIE process. As shown in FIG. 13, the etch mask defines a chamber pattern 44 for forming an extraction chamber on the substrate 22 and a column pattern 46 for forming an array of columns on the substrate. The patterned wafer is etched using a DRIE process to form an integral column with the extraction chamber. The wafer is etched to a sufficient depth so that the extraction chamber 26 has fluid ports 28 and 3
Meet zero.

After etching, the remaining photoresist is removed from the wafer, then the substrate is oxidized and the interior surface of chamber 26 is coated with an oxide layer, preferably 1 μm to 100 μm thick. Next, an electrically conductive material such as, for example, aluminum, gold, or copper is deposited on the implanted region on the back side of the substrate and patterned to form electrodes 48A and 48B. Next, the substrate 22 is anodically bonded to the cover 24, preferably to thin Pyrex glass. After bonding, the substrate pair is diced to form the final structure shown in FIG.

FIG. 14 shows a flow-through device 21 according to another embodiment of the present invention, wherein an internal adsorption surface for capturing and eluting nucleic acids is provided by one or more of the chambers contained in a chamber 26. Formed by a solid support of As the fluid sample flows through the chamber 26, the nucleic acids contact and adhere to the solid support. Chamber 26 is heated while the elution fluid is flowed into the chamber to elute the nucleic acids, thus releasing the nucleic acids from the solid support into the elution fluid. A suitable solid support for capturing nucleic acids is
Examples include beads, fibers, membranes, glass wool, filter paper, gels, etc., with substances that have a binding affinity for nucleic acids such as silica, diethylaminoether (DEAE) and polymers.

In the embodiment of FIG. 14, the solid support is a glass bead 50 packed into the chamber 26. In embodiments using beads, fibers, wool or gel as the solid support, preferably the device includes a barrier 52 located in the chamber 26 adjacent to the outlet port 30 so that the solid support is removed from the chamber. Prevent from spill. The barrier 52 may be any suitable retaining membrane or filter, such as a comb filter, for retaining the solid support material in the chamber 26. Alternatively, the barrier 52 may include a plurality of internals in the chamber, such as columns, that hold the solid support material with sufficiently small spacing. FIG. 1D shows a row of columns forming a filter, the columns being suitable for holding beads in a chamber.

In a preferred embodiment, preferably the device 21 is used in combination with a cartridge, said cartridge having a pretreatment site for fluid samples and / or a post-treatment site for eluted nucleic acids. The cartridge may also include appropriate processing electronics for powering the resistive heating element 34. Except that the internal adsorption surface within chamber 26 is provided by a solid support, such as bead 50, rather than by an array of integrally formed microstructures, the operation of device 21 is similar to that of the above embodiment. Similar in action.

The device 21 is manufactured using the same techniques as described in the previous examples, including photolithography and micromachining. A preferred method for making the device is described herein.

A 100 mm n-type (100) 0.1 to 0.2 ohm cm silicon wafer is preferably used as the starting material for the base substrate 22.
The wafer is patterned on the front side with photoresist to obtain an etch mask for the DRIE process. The etch mask defines a chamber pattern for forming the chamber 26 in the substrate 22, defines a barrier pattern for forming the internal barrier structure, and preferably forms closely spaced columns within the chamber 26. A barrier pattern to do this. Next, the patterned wafer is
Etched using E processing to form chamber 26 and internal barrier structure. Of course, the structures are spaced less than the diameter of the bead 50 and hold the bead 26 within the chamber 26.

After the etching, the remaining photoresist is removed from the wafer. Next, one or more electrically conductive materials are deposited and patterned on the back side of the substrate to form a resistive heating element, a temperature sensor, and a bond pad. The substrate is anodically bonded to a glass cover with holes forming fluid ports 28,30. Beads 50 are packed into the chamber before or after installing the cover, preferably after the cover has been installed. Bead 50 is inserted through inlet port 28. Of course, the barrier 52 can be put in place before the beads 50 are packed to prevent the beads from flowing out of the chamber.

FIG. 15 shows a flow-through device 31 according to another embodiment of the present invention, wherein the solid support housed in the chamber comprises a membrane or filter 60 for capturing nucleic acids. The device 31 includes a base substrate 58, a top substrate 54, and a middle substrate 56 sandwiched between the top substrate and the base substrate. The extraction chamber 26 is formed on the top substrate 54 and the base substrate 58. Filter 60 is in thermal contact with heater 34. Alternatively, the filter 60 may be located on the base substrate 58 adjacent to the outlet port 30.

The resistive heating element 34 is preferably located on a middle substrate 56 for heating the chamber 26. The heating element 34 includes a layer 62 of an insulating material such as, for example, silicon dioxide, silicon carbonate, silicon nitride, plastic, glue or other polymer, resist, or ceramic to protect the heating element 34 from the fluid flowing through the chamber 26. Covered by The middle substrate 56 includes holes (not shown in the cross-sectional view of FIG. 15) positioned around the heating element 34 so that fluid flows continuously from the inlet port 28 to the outlet port 30 through the chamber.

[0116] The heating element 34 may be a thin film of metal or polysilicon patterned on a substrate. Alternatively, substrate 56 may be a thin plastic flex circuit with heating element 34. In other embodiments, heating element 34 comprises a stacked heating source, such as an etched foil heating element attached to substrate 56. In embodiments where the heater is part of a laminated structure, the substrate 56 is a support for the heater. In yet another embodiment, the substrates 56 and 58, together with the heating element 34 and the insulator layer 62, can be fabricated from a single substrate using techniques known to those skilled in the art, such as, for example, thin film processing. The device 31 is preferably used in combination with a cartridge, which cartridge has a pretreatment site for fluid samples and / or
Or it has a post-processing site for eluted nucleic acids. The cartridge may also include appropriate processing electronics for powering the resistive heating element 34. In operation, the fluid sample is forced to flow through the device. As the fluid sample flows through the chamber 26, the nucleic acids contact and adhere to the filter 60.
The chamber is optionally cleaned to remove unwanted particulates. As the elution fluid flows into the chamber to elute the nucleic acids, the chamber 26 is heated by the heating element 34, releasing the nucleic acids from the filter 60 to the elution fluid.

The top substrate 54 and the base substrate 58 are preferably low-cost molded plastic parts, and the middle substrate 56 is preferably a plastic flex circuit. The filter 60 is pre-cut to predetermined dimensions and then the filter 60 and the substrates 54, 56, 58
Are assembled, and the device 31 is manufactured.

Overview, Subdivision-Related Issues, Scope The foregoing description contains many specificities, which should not be construed as limiting the scope of the invention, but rather merely limit the present Is merely an example of a preferred embodiment. Many other embodiments of the present invention are possible. For example, in one alternative embodiment, the interior adsorption surface of the chamber is charged with an AC or DC voltage to attract and / or elute nucleic acids. The present invention involves a combination of chemiphoresis, thermophoresis and electrophoresis to reduce the elution volume required to fully elute nucleic acids.
Combinations of more than one elution technique are possible. In another embodiment, the nucleic acids are treated after being bound to the adsorption surface. For example, a secondary binding member with a reporting molecule passes through the extraction chamber and binds to the already bound nucleic acid. Next, the complex is eluted. Further, the chamber is functionalized with specific capture components that bind specific targets such as cells, bacteria, viruses or specific nucleic acids.

For direct and rapid heating of the adsorption surface, it is preferable to heat the chamber using a resistor coupled to the chamber wall. However, the invention is not limited to this embodiment. Many different mechanisms or methods for heating the chamber
It will be clear to those skilled in the art. These include optical heating, ultrasonic heating, inductively coupled coils, microwaves or other suitable mechanisms for heating the chamber. Additionally, when the device is used in combination with a cartridge, the cartridge or the equipment used to process the cartridge includes a heater to overheat the device.

It is presently preferred to use the flow-through device of the invention in combination with a cartridge, but the scope of the invention is not limited to this embodiment. Given the above description that this device can be connected to a chemical analysis system, instead,
It will be apparent to those skilled in the art that the device can be used as a stand-alone device for extracting a desired substance from a fluid sample. Further, although nucleic acid extraction is presented as an exemplary embodiment of the present invention, many other desired samples from fluid samples such as organisms, cells, proteins, carbohydrates, virus particles, bacteria, chemicals, biochemicals, etc. It will be clear to those skilled in the art that the device of the present invention can be used to separate the following substances.

The scope of the invention should, therefore, be determined by the appended claims and their legal equivalents.

[Brief description of the drawings]

1A-1H are scanning electron micrographs of the device of the present invention, illustrating different arrangements of microstructures etched in silicon.

FIG. 1 shows an array of microcolumns located in a chamber having an inlet port and an outlet port.

FIG. 2 is an enlarged view of a portion of the array of microcolumns of FIG. 1A.

FIG. 3 shows different arrangements of microcolumns having pointed ends.

FIG. 4 shows a single row of microstructures forming a filter at one end of a chamber.

FIG. 5 is an electron micrograph showing six grooves converging on one common groove.

FIG. 6 shows two grooves merging into one common groove with a series of pillars arranged therein.

FIG. 7 shows another grooved device suitable for liquid phase separation.

FIG. 8 illustrates a hydrodynamic focusing device useful for flow cytometry.

FIG. 9 is a three-dimensional view of an apparatus where two deep grooves merge into a common groove for diffusion mixing of two fluids.

FIG. 10 is a three-dimensional view of another apparatus where two deep grooves merge into a common groove for diffusion mixing of two fluids.

FIG. 11 is a schematic plan view of an apparatus having a network of confluent grooves providing multiple interdiffusion regions for a fluid.

FIG. 12 is a three-dimensional view of an alternative device having two grooves that merge into one common groove with a series of pillars disposed therein.

FIG. 13 is a schematic cross-sectional view of a flow-through device for extracting a desired substance from a fluid sample according to a preferred embodiment of the present invention.

FIG. 14 is a bottom view of the device of FIG.

FIG. 15 is a three-dimensional view of a microcolumn formed in the extraction chamber of the device of FIG.

FIG. 16 is a schematic plan view of a micro column in the device of FIG.

FIG. 17 is a plan view of two adjacent microcolumns in the device of FIG.

FIG. 18 is a schematic view of an etch mask for forming a chamber pattern and a column pattern used in manufacturing the apparatus of FIG.

FIG. 19 is a schematic plan view of a cartridge including the device of FIG. 6 according to a preferred embodiment of the present invention.

FIG. 20 is a schematic cross-sectional view of a flow-through device for extracting a desired substance from a fluid sample according to an alternative embodiment of the present invention.

FIG. 21 is a schematic cross-sectional view of a flow-through device for extracting a desired substance from a fluid sample according to another embodiment of the present invention.

FIG. 22 is a schematic cross-sectional view of a flow-through device for extracting a desired substance from a fluid sample according to yet another embodiment of the present invention.

[Explanation of symbols]

 Reference Signs List 20: Flow-through device 26: Extraction chamber, 28: Inlet port, 30: Outlet port, 32: Column, 34: Heater, 48: Electrode, 101: Cartridge, 119: Filter site, 109, 125, 127: Storage site, 139 ... waste site, 141 ... reagent site, 143 ... reaction site, 151 ... processing electronics.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] February 14, 2000 (2000.2.14)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction contents]

[0007] As an alternative to traditional bench-top measurement techniques, attempts have been made to develop automated microfluidic devices, which provide some of the sample preparation and / or sample processing functions. Do Microfluidic systems have the potential for greater convenience, improved reproducibility, reduced contamination, reduced reagent consumption, reduced operator intervention, and lower measurement costs when compared to benchtop measurements. The technology of micro machining is
For example, semiconductor substrates can be used to enable the manufacture of microfluidic devices to manipulate, react, and detect microliter to picoliter volumes of sample. For example, Northrop et al., U.S. Pat. No. 5,639,423 and Winding U.S. Pat. No. 5,587,128, describe microfabricated devices for performing biochemical reactions, particularly those such as the polymerase chain reaction. A reactor (PCR) for performing a DNA-based reaction is disclosed. Another analytical device for determining the presence or amount of an analyte in a test sample is described in International Publication No. WO 96/10747 issued April 11, 1996 by Hansman.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction contents]

However, despite these advances in microfluidic technology, microfluidics can effectively extract nucleic acids from relatively large volumes of fluid samples and can concentrate nucleic acids into smaller volumes for subsequent analytical processing Equipment needs are still there. Particularly in diagnostic applications, it is frequently necessary to extract and concentrate low concentrations of nucleic acids from relatively large volumes of samples, for example several milliliters, such as pathogenic organs or cancer cells. Such treatments include, for example, toxic E. coli 0157 in food (toxic at the level of one organism per 10 grams of food), HIV in blood (less than 200 viral organisms per milliliter of blood), anthrax in bacterial warfare scenarios Bacteria (less than 100 organisms per liter of air), cancer cells in blood or urine (10
It is necessary for the detection of pathogens (up to 100 organisms per milliliter of fluid) in urine of sexually transmitted diseases (eg gonorrhea and chlamydia).

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0027

[Correction method] Change

[Correction contents]

Fluid mixing is a common and significant event in most biochemical analytical protocols. On the conventional macroscopic scale, there are a variety of effective means for mixing two or more fluids, including pipettes, vortexers and suction / dispensing robotics. However, it is well known that mixing fluids is difficult in microfluidic systems. This problem stems from the fact that in most microfluidic systems, the Reynolds number is generally very small (less than 100) and the flow is virtually always laminar. The Reynolds number is density × speed × groove width / viscosity. In microfluidic systems, the Reynolds number is small because the groove width is very small. In a macroscopic system, before the onset of turbulence, the Reynolds number must be about 2300 or more. In microfluidic systems, mixing occurs almost completely by diffusion, since the fluid flow is virtually never turbulent. Holmes et al., In WO 96/12541 published May 2, 1996, describe a method and apparatus for diffusion transfer between immiscible fluids. Unfortunately, mixing by diffusion can be very slow. As an example, a 300 μm deep and 600 μm wide “
There are "zigzag" grooves. This groove allows the fluid to mix completely with a flow length of only 100 mm.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

[Correction contents]

The present invention is useful for quickly and efficiently diffusing and mixing fluids and for making artificial emulsions that provide a means for performing the same protein extraction function as conventional stirring-based liquid phase separation methods. A microdevice suitable for producing a very large microfluidic interface is provided. New process technologies are becoming available to create ultra-micromachined microfluidic grooves to provide a structure that allows for efficient diffusion and mixing of fluids. With a method known as deep reactive ion etching (DRIE), it is possible to form very deep, but surprisingly narrow, grooves. Rapid diffusion mixing is achieved using this process by flowing two fluids through a deep vertical groove and joining them together. This allows two thin vertical sheaths (
Sheaths), which mix by diffusion over a much shorter distance than conventional grooves. A microgroove structure utilizing the DRIE process is designed to support the liquid phase separation process. As shown in FIGS. 1-5, the fluid channels are formed in various external forms on the solid substrate. FIG. 2 shows a device in which two deep grooves 70, 72 merge into one common groove 74. The groove is formed by etching the silicon substrate using DRIE. Each groove has a depth 73 greater than its width 75 and provides a surface area ratio greater than 3: 1. The fluid flowing in the separated grooves 70, 72 merges at the common groove 74 to form two co-current thin fluid sheaths. Grooves that are deeper in width provide a larger interfacial area during flow convection for diffusion mixing of the two fluids. FIG. 1E is a scanning electron micrograph of a similar device where six deep grooves merge into a single common groove. Fluids passing through the separate grooves merge at the common groove to form six co-current fluid sheaths.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07H 1/00 C07H 1/00 21/00 21/00 G01N 33/53 G01N 33/53 M 37/00 101 37/00 101 (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 , AZ (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, N, CU, CZ, DE, DK, 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, UZ, VN, YU, ZW (72) Inventor William A. McMillan United States 95014 Presidio Drive No. 8051, Cu Pertino, California, USA (72) Inventor M. Allen Northrop No. 923, Creston Road, Berkeley, CA 94708, United States Inventor Kurt E. Petersen 95148, United States San Jose, CA Valley Ridge Ridge Inn No. 3655 (72) Inventor Fazad Polar Madi Pajaro Drive 41013, Fremont, CA 94439 United States F-term (reference) 2G042 CB03 CB04 GA01 HA02 4C057 AA10 BB02 DD01 MM02 MM04 4D017 AA11 BA07 DA01 DB02 DB03 DB04 EA05 4G0 AB AB38 AD01 AD11 4G075 AA13 BB01 BB04 BD07 BD15 BD16 CA02 CA54 DA01 EB01 EE31 FA02 FB04

Claims (42)

[Claims] 1. A flow-through device for separating a desired substance in a fluid sample from other substances, comprising: a) an inlet port for introducing the fluid sample into the body; b) a fluid sample from the body. C) an extraction chamber which fluidly communicates the inlet port and the outlet port for extracting the desired substance from the fluid sample as the sample flows through the body. The inlet port and the outlet port are arranged such that the fluid can flow continuously through the extraction chamber, the chamber being formed by an inner surface of the body, At least one of the internal surfaces has a surface area large enough to capture the desired material as the fluid sample flows through the chamber and a surface area for the desired material. Apparatus characterized by and a sufficiently high binding affinity. 2. The apparatus of claim 1, wherein the interior surface comprises a surface of an interior microstructure integrally formed with at least one wall of the extraction chamber and extending into the chamber. An apparatus characterized in that: 3. The apparatus of claim 2, wherein said internal structure comprises an array of high aspect ratio columns. 4. The apparatus according to claim 2, wherein the rows are such that the internal structures are arranged in rows, and the microstructures in each row are adjacent to the substructures in the row. A device that is arranged so as not to be aligned on a straight line. 5. The apparatus of claim 4, wherein the rows are such that the microstructures in each row are not aligned with at least two of the substructures in the previous or next row. An apparatus characterized by being located. 6. The apparatus according to claim 2, wherein each of said microstructures comprises:
An apparatus having a cross-sectional length to width ratio of at least 4: 1. 7. The device according to claim 1, wherein the desired substance is selected from the group consisting of organisms, cells, proteins, nucleic acids, carbohydrates, virus particles, bacteria, chemicals and biochemicals. An apparatus characterized in that: 8. The device according to claim 1, wherein at least one of the inner surfaces is coated with a material having a binding affinity for the desired substance to facilitate capture of the desired substance. Characteristic device. 9. The device according to claim 8, wherein said material is selected from the group consisting of silicon, silicon dioxide, polymer, nucleic acid, metal, polypeptide, protein and polysaccharide. apparatus. 10. The apparatus according to claim 1, further comprising a heater for heating the extraction chamber. 11. The apparatus according to claim 10, wherein the heater comprises a resistive heating element coupled to at least one wall of the chamber. 12. The apparatus according to claim 10, wherein the heater is provided with an electrode for applying a potential difference to at least a part of the body forming the chamber. 13. The apparatus according to claim 10, further comprising processing electronics for controlling operation of the heater. 14. The apparatus of claim 1, wherein said apparatus is coupled to a cartridge having a pre-treatment site for said fluid sample, and said apparatus is inserted into said cartridge and said inlet port is connected to said front port. An apparatus characterized in that it is adapted to be in fluid communication with a processing site. 15. The apparatus of claim 1, wherein the apparatus is coupled to a cartridge having a post-processing site for eluent, and wherein the apparatus is inserted into the cartridge and the outlet port is connected to the post-processing port. A device characterized in that it is made in fluid communication with a site. 16. A method for separating a desired substance in a fluid sample from other substances, comprising: a) an inlet port, an outlet port, and an extraction chamber in fluid communication with the inlet port and the outlet port. Comprising providing a flow-through device comprising a body having an extraction chamber formed by an interior surface of the body, the interior surface having a sufficiently large surface area and a sufficiently high binding affinity for the desired substance. C) capturing the substance as the fluid sample flows through the chamber; and b) continuously forcing the fluid sample into the chamber so that the desired fluid sample flows through the chamber. C) dissolving said desired substance from said device by forcing an elution fluid through said chamber. Releasing the substance from the interior surface into the eluent fluid. 17. The method according to claim 16, wherein the desired substance is selected from the group consisting of organisms, cells, proteins, nucleic acids, carbohydrates, virus particles, bacteria, chemicals, and biochemicals. A method comprising: 18. The method of claim 16, wherein the volume of the fluid sample forced into the chamber is greater than a maximum capacity of the chamber. 19. The method of claim 16, wherein the step of eluting the substance further comprises heating the internal surface while forcing the eluting fluid through the chamber. Method. 20. The method of claim 16, further comprising forcing a gas into the chamber prior to forcing the eluting fluid into the chamber. 21. The method of claim 16, further comprising the step of contacting the fluid sample with a lysing reagent as the fluid sample flows into the chamber. 22. The method of claim 16, wherein the interior surface comprises an interior microstructure surface integrally formed with at least one wall of the extraction chamber. 23. The method according to claim 22, wherein the microstructure comprises a column extending into the chamber, and forcing the fluid sample into the chamber comprises displacing the fluid sample into the column. A method comprising the step of forcing flush between. 24. The method of claim 16, wherein a potential difference is applied between the body and the fluid sample while forcing the fluid sample into the chamber to facilitate capture of the desired substance. A method, further comprising a step. 25. A flow-through device for separating a desired substance in a fluid sample from another substance, comprising: a) a microfabricated chip, i) for introducing the fluid sample into the chip. Ii) an outlet port for removing the fluid sample from the chip; and iii) an extraction chamber for extracting the desired substance from the fluid sample when the fluid sample flows through the chip. The extraction chamber is in fluid communication with the inlet port and the outlet port, and the inlet port and the outlet port are arranged such that the fluid can flow continuously through the extraction chamber. B) a heater for heating the extraction chamber; and c) capturing the desired substance as the fluid sample continuously flows into the chamber. Apparatus characterized in that it comprises at least one solid support was placed in the capture chamber of the eye. 26. The apparatus according to claim 25, wherein said solid support comprises a support selected from the group consisting of beads, fibers, membranes, glass wool, filter paper and gel. Equipment to do. 27. The device according to claim 25, wherein the desired substance is selected from the group consisting of organisms, cells, proteins, nucleic acids, carbohydrates, virus particles, bacteria, chemicals, and biochemicals. An apparatus characterized in that: 28. The apparatus according to claim 25, wherein said heater comprises a resistive heating element coupled to at least one wall of said chamber. 29. The apparatus according to claim 25, wherein the heater comprises an electrode for applying a potential difference to at least a part of the chip forming the chamber. 30. The apparatus of claim 25, further comprising processing electronics for controlling operation of the heater. 31. The apparatus according to claim 25, wherein said apparatus is coupled to a cartridge having a pretreatment site for said fluid sample, and said apparatus is inserted into said cartridge and said inlet port is connected to said front port. An apparatus characterized in that it is adapted to be in fluid communication with a processing site. 32. The apparatus according to claim 25, wherein said apparatus is coupled to a cartridge having a post-processing site for eluent, and said apparatus is inserted into said cartridge and said outlet port is connected to said post-processing. A device characterized in that it is made in fluid communication with a site. 33. A method for separating a desired substance from another substance in a fluid sample, comprising: a) an inlet port and an outlet port and an extraction chamber in fluid communication with the inlet port and the outlet port. Providing a chip formed therein, wherein the inlet port and the outlet port are arranged to allow the fluid sample to flow continuously into the chamber, and wherein the extraction chamber comprises a fluid passage through the chamber. Including at least one solid support for capturing the desired material as it flows, b) continuously forcing the fluid sample into the chamber so that when the fluid sample flows through the chamber, Bonding said desired substance to said internal surface; c) transferring said desired substance to said chip by heating said chamber. Eluting from the solid support and releasing the substance from the solid support into the elution fluid. 34. The method according to claim 33, wherein the desired substance is selected from the group consisting of an organism, a cell, a protein, a nucleic acid, a carbohydrate, a virus particle, a bacterium, a chemical, and a biochemical. A method comprising: 35. The method of claim 33, wherein a volume of the fluid sample forced into the chamber is greater than a maximum capacity of the chamber. 36. The method of claim 33, further comprising forcing a gas into the extraction chamber prior to forcing the elution fluid into the chamber. 37. The method of claim 33, further comprising contacting the fluid sample with a lysing reagent as the fluid sample flows into the chamber. 38. An apparatus for manipulating a fluid, comprising: a substrate having at least a first groove and a second groove formed therein for passing at least a first fluid and a second fluid, respectively. The second groove merges with a common groove that provides a contact area for these fluid streams, the common groove having an internal surface area of at least three times its outer surface area to provide a large interfacial area between the fluid streams. An apparatus characterized in that: 39. The apparatus of claim 38, further comprising pillars located in said common groove to increase the stability of said fluid flow. 40. The apparatus according to claim 38, wherein said first groove and said second groove are provided.
An apparatus wherein the grooves merge into a plurality of common grooves to provide a corresponding plurality of contact areas for the fluid flow. 41. The apparatus according to claim 38, further comprising a third groove and a fourth groove that are in fluid communication with the common groove apart from the contact area,
Apparatus characterized by passing a fluid stream and said second fluid stream respectively. 42. An apparatus for manipulating a fluid, comprising: a) a first groove for passing a first fluid flow; b) a fluid communication with the first groove and the first fluid flow. A substrate having therein a second groove and a third groove branched from the first groove for separating the second and third fluid flows into a second fluid flow and a third fluid flow, each of the grooves having an outer surface area thereof. A device having an internal surface area greater than or equal to three times that of said fluid stream to provide a large interfacial area between said fluid streams.