JP2005049357A - Electronic pipetter and compensation means for electrophoretic bias - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To introduce a substance into a micro fluid system. <P>SOLUTION: The micro fluid system has a capillary channel constituted to introduce the substances into the micro fluid system from a plurality of sources, and having an end part, and a voltage source for impressing a voltage potential to an electrode of the micro fluid system. In the system, the end part of the capillary channel is brought into contact with the objective substance source, the voltage is impressed to the objective substance source, as to the electrode, the source of the spacer substance having a prescribed concentration is selected, and a voltage is impressed to the spacer substance source, as to the electrode. The system includes a process for repeating the process hereinbefore, using the different substance sources, to introduce the plurality of different substances separated by the spacer substance electrokinetically into the capillary channel, without being mixed between the different substances, and to be transported toward the micro fluid system. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Cross-reference to related applications This application is a US patent filed on December 6, 1996, which is a continuation-in-part of US Patent Application Serial No. 08 / 671,986, filed June 28, 1996. This is a continuation-in-part of application serial No. 08 / 760,446. All of these applications are hereby incorporated by reference for all purposes.

  There is an increasing interest in the manufacture and use of microfluidic systems for obtaining chemical and biochemical information. Techniques such as photolithography and wet chemical etching, usually associated with the semiconductor electronics industry, are also used to manufacture these microfluidic systems. The term “microfluidic” refers to a system or device having channels and chambers, generally on a micron or submicron scale (eg, such that at least one cross-sectional dimension has a range from about 0.1 μm to about 500 μm). . An early consideration of the use of planar chip technology for the manufacture of microfluidic systems is given in Manz et al. Trends in Anal. Chem. (1990) 10 (5): 144-149 and Manz et al., Avd. In Chromatog. (1993) 33: 1-66. They describe the production of fluidic devices as described above, in particular microcapillary devices, on silicon and glass substrates.

  There are countless applications for microfluidic systems. For example, international patent application WO 96/04547, published February 15, 1996, describes the use of microfluidic systems for capillary electrophoresis, liquid chromatography, flow injection analysis, and chemical reactions and synthesis. ing. US Patent Application Serial No. 08 / 671,987, filed June 28, 1996 (incorporated herein by reference), describes the effects of numerous compounds in chemical and preferably biochemical systems. Discloses a wide range of applications for microfluidic systems in rapidly assaying The phrase “biochemical system” generally refers to a chemical interaction involving a type of molecule commonly found in living organisms. Such interactions include a variety of catabolic and anabolic reactions that occur in living systems, including enzymatic reactions, binding reactions, signaling reactions, and other reactions. Biochemical systems of particular interest include, for example, receptor-ligand interactions, enzyme-substrate interactions, cell signaling pathways, model barrier systems for bioavailability screening (eg, cell or membrane fractions, etc.) Transport reactions, and various other common systems.

  There are many methods described for transporting and directing fluids such as samples, analytes, buffers and reagents within these microfluidic systems or devices. In one method, fluid in a microfabricated device is moved by mechanical micropumps and valves in the device. Published UK patent application No. 2 248 891 (10/18/90), published European patent application No. 568902 (5/2/92), and U.S. Pat.No. 5,271,724 (8/21/91) ) And 5,277,556 (7/3/91). See also Miyazaki et al. US Pat. No. 5,171,132 (12/21/90). In another method, acoustic energy is used to move a fluid sample within the device due to an acoustic streaming effect. See published Northrup and White PCT Application No. 94/05414. As a straightforward method, the fluid in the device is moved by applying external pressure. See, for example, the description in Wilding et al., US Pat. No. 5,304,487.

In yet another method, fluid material is moved through the channels of the microfluidic system by using an electric field. See, for example, published Kovacs European Patent Application No. 376 611 (12/30/88), Harrison et al., Anal. See Chem. (1992) 64: 1926-1932 and Manz et al., J. Chromatog. (1992) 593: 253-258, and Soane, US Pat. No. 5,126,022. Dynamic power has the advantages of direct control, fast response and simplicity. However, it still has some drawbacks. In order to maximize efficiency, it is desirable to transport the target substances together as much as possible. However, the material should be transported without cross-contamination from other transported materials. Further, a substance in one state that exists at a location in a microfluidic system should remain the same after it has been moved to another location in the microfluidic system. These conditions make it possible to control the testing, analysis and reaction of compound materials when and when desired.
In microfluidic systems where substances are moved by electrokinetic forces, charged molecules and ions in target substance regions and regions that separate these target substance regions are exposed to various electric fields, thereby causing fluid flow. Wake up.

However, when these electric fields are applied, differently charged species in the target material exhibit different electrophoretic mobilities. That is, positively charged species move at a different speed than negatively charged species. In the past, the separation of different species in samples exposed to an electric field has not been considered a problem and in fact has been a desired result, for example in capillary electrophoresis. However, if simple fluid transport is desired, these varying mobilities can lead to undesirable changes or “electrophoretic bias” in the material of interest.
Without consideration and means to avoid cross-contamination, microfluidic systems must broadly separate target materials or, in the worst case, materials must move through the system one at a time. In either case, the efficiency of the microfluidic system is greatly reduced. Furthermore, if the state of the material being transported is not maintained during transport, many applications must be avoided that require the material to remain in place.
International patent application WO 96/04547 US Patent Application Serial No. 08 / 671,987 UK Patent Application No. 2 248 891 European Patent Application No. 568902 U.S. Pat.No. 5,271,724 U.S. Pat.No. 5,277,556 U.S. Pat.No. 5,171,132 PCT Application No. 94/05414 U.S. Pat.No. 5,304,487 European Patent Application No. 376 611 U.S. Pat.No. 5,126,022 Manz et al. Trends in Anal. Chem. (1990) 10 (5): 144-149 Manz et al. Avd. In Chromatog. (1993) 33: 1-66 Harrison et al. Chem. (1992) 64: 1926-1932 Manz et al., J. Chromatog. (1992) 593: 253-258

  The present invention solves or substantially alleviates these problems with electrokinetic transport. With the present invention, microfluidic systems can move material efficiently and without causing undesirable changes in the material being transported. The present invention provides direct, fast and straightforward control over the movement of materials through channels of microfluidic systems in a wide range of applications such as chemistry, biochemistry, biotechnology, molecular biology, and many other fields. A high-throughput microfluidic system is provided.

The present invention provides a microfluidic system that moves the target material by electroosmosis from a first point to a second point of the microfluidic system along a channel in the fluid slug, also referred to as a “target material region”. The high ion concentration first spacer region is in contact with each target material region on at least one side, and the low ion concentration second spacer region is the target material region of the target material and the first or high ion concentration Arranged with the spacer region, the at least one low ion concentration region is always between the first point and the second point, and most of the voltage drop and the electric field obtained between the two points is low. Make sure you cross the territory.
The present invention also provides an electronic pipetter that is compatible with a microfluidic system that moves an object of interest with electroosmotic force. The electronic pipettor includes a capillary having a channel. The electrodes are attached along the outside length of the capillary and terminate in the form of an electrode ring at the end of the capillary. By manipulating the voltage and electrode on the electrode at the target reservoir where the channel is in fluid communication when the end of the capillary is placed in the substance source, the substance is electrokinetically induced into the channel. A row of target material regions, high and low ionic concentration buffer or spacer regions can be formed in the channel for easy introduction into the microfluidic system.

The present invention further compensates for electrophoretic bias as the material of interest is electrokinetically transported along the channels of the microfluidic system. In one embodiment, the channel between two points of the microfluidic system has two parts with opposite surface charge sidewalls. The electrode is arranged between the two parts. When the voltages at the two points are substantially equal and the intermediate electrode between the two parts is set differently, the electrophoretic force is in the opposite direction in the two parts and the electroosmotic force is in the same direction . As the material of interest is transported from one point to another, the electrophoretic bias is compensated while the electroosmotic force moves the fluid material through the channel.
In other embodiments, the chamber is formed at the intersection of the channels of the microfluidic system. The chamber has side walls connecting the side walls of the intersecting channels. When the target material region is directed from one channel to the other at the intersection, the chamber sidewalls funnel the target material region toward the second channel. The second channel has such a width that all the target substances energized by electrophoresis in the target substance region are mixed when the target substance moves along the first channel by diffusion.

In yet another embodiment, the present invention provides a microfluidic system and method of using the system for controllably delivering a fluid stream in a microfluidic device having at least two intersecting channels. The system has a substrate with at least two crossing channels provided therein. In this respect, one of the channels is deeper than the other channel. The system also has an electroosmotic fluid direction system. The system is particularly useful when the fluid stream includes at least two fluid regions having different ionic strengths.
The present invention also provides a sample extraction system using the electronic pipettor of the present invention. The sample extraction system has a sample substrate, and a plurality of different samples are fixed to the sample substrate. A transition system for moving the electronic pipettor relative to the sample substrate is also included.
As described above, the present invention is used for a plurality of different applications, and the applications themselves are progressive. For example, the present invention is as follows.

A channel when transporting at least a first target substance from at least a first position to a second position along a channel and using at least one region of low ion concentration transported along the channel by an applied voltage; Use of a substrate having.
Use of the above invention, wherein the ion concentration in one region is substantially lower than the ion concentration of the target substance.
Use of the above invention, wherein a plurality of target substances are transported and separated by a high ion concentration spacer region.
Use in electrophoretic bias compensation of a substrate having a channel through which at least a first target substance can be transported, wherein the channel is divided into first and second portions, and one or more walls of the channel are opposite Use wherein the electrophoretic bias on the at least one target substance due to transport in the first part is substantially compensated by the electrophoretic bias due to transport in the second part.
The first electrode is disposed at the distal end of the first portion, the second electrode is disposed at the intersection between the first portion and the second portion, and the third electrode is disposed in the second portion. Use of the above invention, located at the distal end of
Use of the above invention, wherein the substrate is a microfluidic system.
Use of the above invention, wherein the substrate is an electronic pipettor.
The electronic pipettor has a main channel for transporting the target substance and at least one other channel in fluid communication with the main channel, from which the other channel is transported along the main channel Use of the above invention, wherein other substances are obtained.
The use of the invention described above, wherein the other substance is introduced into the main channel as a buffer region between each of a plurality of individual target substances.
Use of a microfluidic system having at least first and second fluidic channels that intersect with channels having different depths in optimizing flow conditions.
Use of the above invention wherein one channel is between 2 and 10 times deeper than the other channel.
Use of a microfluidic system having a first channel and a second channel intersecting the first channel in electrophoretic compensation, wherein the intersection between the channels is a second channel along the first channel Use, where the fluid being transported to is mixed at the intersection and shaped to diffuse the electrophoretic bias within the fluid.

BEST MODE FOR CARRYING OUT THE INVENTION

I. General Configuration of Microfluidic System FIG. 1 is a representative diagram of an example of a microfluidic system 100 according to the present invention. As shown, the entire device 100 is fabricated in a planar substrate 102. Appropriate substrate materials are generally selected based on suitability for the conditions present during the particular operation to be performed by the device. Such conditions may include application of extreme values of pH, temperature, ion concentration, and electric field. The substrate material is also selected to be inert to the critical components of analysis or synthesis performed by the system.
Useful substrate materials include, for example, glass, quartz and silicon, and polymeric substrates such as plastic. In the case of a conductive or semiconductive substrate, an insulating layer is required on the substrate. This is particularly important when the device is equipped with an electrical element such as an electrofluidic system or sensor, as described below, or when a substance is moved through the system using electroosmotic forces. For polymeric substrates, the substrate material can be rigid, quasi-rigid or non-rigid, opaque, translucent or transparent, depending on the intended use. For example, devices having optical or visual detection elements are generally manufactured at least in part from a transparent material to allow or at least facilitate detection. Alternatively, for these types of detection elements, the device may be provided with a transparent window, for example made of glass or quartz. Furthermore, the polymer material may have a linear or branched skeleton, and may or may not be crosslinked. Examples of particularly suitable polymer materials include, for example, polydimethylsiloxane (PDMS), polyurethane, polyvinyl chloride (PVC), polystyrene, polysulfone, and polycarbonate.

The system shown in FIG. 1 has a series of channels 110, 112, 114 and 116 fabricated in the surface of the substrate 102. As stated in the definition of “microfluidic”, these channels typically have very small cross-sectional dimensions, preferably in the range of about 0.1 μm to about 100 μm. In certain applications described below, channels having a depth of about 10 μm and a width of about 60 μm work effectively, but it is possible to deviate from these dimensions.
Fabricating these channels and other microscale elements in the surface of the substrate 102 can be done using any number of microfabrication techniques well known in the art. For example, a glass, quartz or silicon substrate may be manufactured using lithography techniques with methods well known in the semiconductor manufacturing industry. Photolithographic masking, plasma etching or wet etching, and other semiconductor processing techniques define microscale elements in or on the substrate. Alternatively, micromachining methods such as laser drilling and micromilling may be used. Similarly, well-known manufacturing techniques can be used for the polymer substrate. These techniques include injection molding techniques or stamping techniques that can produce a large number of substrates, for example by creating large sheets of microscale substrates using rolling stamps, or advanced techniques for polymerizing substrates in microfabrication molds. Includes molecular microcasting technology.

  In addition to the substrate 102, the microfluidic system has additional planar elements (not shown) that overlap the channeled substrate 102, thereby enclosing and fluidly sealing the various channels. Form. The planar cover element is attached to the substrate by various means including thermal bonding, adhesive, or in the case of glass or semi-rigid and non-rigid polymer substrates, natural adhesion between the two components, etc. obtain. The planar cover element may further be provided with access ports and / or reservoirs for introducing various fluid elements necessary for a particular screening.

  The system 100 shown in FIG. 1 also has reservoirs 104, 106 and 108 located and in fluid communication with the ends of the channels 114, 116 and 110, respectively. As shown, the sample channel 112 is used to introduce a plurality of different substances of interest into the device. In this way, the channel 112 is in fluid communication with a number of separate target material sources, and a number of separate target materials are individually introduced into the sample channel 112 and then into another channel 110. As shown, the channel 110 is used for analyzing the target substance by electrophoresis. Note that the term “substance” simply refers to a substance such as the compound or biological compound in question. The compound of interest is a compound, a mixture of compounds (eg, polysaccharides), small organic or inorganic molecules, biological macromolecules (eg, peptides, proteins, nucleic acids, or organisms such as bacteria, plants, fungi, or animal cells or tissues. A wide range of different compounds, including extracts made from biological materials), naturally occurring or synthetic compositions, and the like.

  System 100 moves material through channels 110, 112, 114, and 116 with dynamic power supplied from a voltage controller capable of simultaneously applying a selected voltage level (including ground) to each reservoir. Such a voltage controller can be realized by using a plurality of voltage dividers and a plurality of relays to obtain a selective voltage level. Alternatively, a plurality of independent voltage sources may be used. The voltage controller is electrically connected to each reservoir via electrodes located within or manufactured from each of the plurality of reservoirs. See, for example, published Ramsey international patent application WO 96/04547. This entire document is incorporated herein by reference for all purposes.

II. Electrokinetic transport A. Overview The dynamic power to the fluid material in the channels of the system 100 can be divided into electroosmotic and electrophoretic forces. The fluid control system used in the system of the present invention uses electroosmotic forces to move, orient, and mix fluids in various channels and reaction chambers on the surface of the substrate 102. That is, when an appropriate fluid is placed in a channel or other fluid conduit with functional groups on the surface, these groups can ionize. For example, when the surface of the channel contains a hydroxyl functional group on the surface, protons can leave the surface of the channel and enter the fluid. Under such conditions, particularly near the channel surface-fluid interface, the surface has a substantially negative charge and the fluid has an excess of protons or positive charges.

By applying an electric field across the length of the channel, cations flow toward the negative electrode. As positively charged species in the fluid move, the solvent moves with these species. The steady state velocity of such fluid motion is approximately given by the following equation:
v = εξE
4πη
Where v is the velocity of the solvent, ε is the dielectric constant of the fluid, ξ is the zeta potential of the surface, E is the electric field strength, and η is the viscosity of the solvent. Thus, as can be readily seen from this equation, the speed of the solvent is directly proportional to the zeta potential and the applied electric field.
In addition to electroosmotic forces, there are also electrophoretic forces that affect charged molecules as they travel through the channels of system 100. When the target substance is transported from one point in the system 100 to another point, the composition of the target substance remains unaffected during transport, that is, the target substance is separated electrically during transport. Often it is desirable not to.

  According to the present invention, the target substance moves through the channel as a plurality of fluid slugs (hereinafter referred to as “target substance regions”). These target substance regions have a high ion concentration, thereby minimizing the electrophoretic force applied to the target substance in a specific region. In order to minimize the influence of the electrophoretic force in the target substance region, a spacer fluid region (“first spacer region”) is disposed on either side of the slag. Such first spacer regions have a high ion concentration, as will be described below, thereby minimizing the electric field in these regions so that the target material can be moved from one location in the microfluidic system to another. It is virtually unaffected by being transported to the location. The target substance is transported through the representative channels 110, 112, 114, 116 of the system 100 in a region with a certain ionic strength, along with other regions having a different ionic strength than the region containing the target material. .

  A particular configuration is shown in FIG. 2A. FIG. 2A shows the target material region 200 being transported from point A to point B along the channel of the microfluidic system 100. There is a first spacer region 201 of high ionic strength fluid on either side of the target material region 200. Further, the second spacer region 202 of the low ion concentration fluid separates the arrangement of the target substance region 200 and the first spacer region 201 at a constant period. Due to the low ion concentration, most of the voltage drop between point A and point B occurs in the second spacer region 202. When the target material region 200 and the first spacer region 201 are electroosmotically pumped through the channel, there is always at least one second low concentration spacer region 202 between points A and B. The second low-concentration spacer region 202 is interspersed between the target material region 200 and the first spacer region 201. This ensures that most of the voltage drop occurs in the second spacer region 202 and not in the target material region 200 and the first spacer region 201. In other words, the electric field between the points A and B is concentrated in the second spacer region 202, and a low electric field (and low electrophoretic force) is applied to the target material region 200 and the first spacer region 201. Therefore, depending on the relative ion concentration in the target material region 200, the first spacer region 201 and the second spacer region, that is, the low ion concentration spacer region 202, the target material region 200, the first and second spacer regions 201, and Other arrangements of 202 can be formed.

  For example, FIG. 2B shows an arrangement in which the second spacer region, ie, the low ion concentration spacer region 202 is spaced regularly between each combination of the first spacer region 201 / target material region 200 / first spacer region 201. Indicates. Such an arrangement ensures that there is always at least one second or low concentration spacer region 202 between points A and B. Further, the drawings are drawn to scale to show the relative lengths of possible combinations of target material region 200, first spacer region or high concentration spacer region 201 and second spacer region or low concentration spacer region 202. It is. In the embodiment of FIG. 2B, the target substance region 200 holds the target substance in NaCl having a high ion concentration of 150 mM. The target substance region 200 is 1 mm long in the channel. The two first spacer regions 201 have NaCl with an ion concentration of 150 mM. Each first spacer region 201 is 1 mm long. The second spacer region 202 is 2 mm and has a borate buffer with an ionic concentration of 5 mM. This particular configuration is designed to maintain the rapidly electrophoretic compound in the buffer region 201 and the target material region 200 and to move the compound through multiple channels of the microfluidic system. For example, using these methods, a target substance region containing, for example, benzoic acid, can flow upwards in a microfluidic system for 72 seconds without suffering an excessive electrophoretic bias.

More generally, it is possible to determine the velocity V _EoF of the fluid flow in the channel of the microfluidic system and to determine the total distance l _t that the object molecule travels in the channel by measurement. is there. Therefore, the movement time t _Tr for the target molecule to move the total distance is
t _Tr = l _T / V _EoF
It is. The object molecule x, the order including the first spacer region 201 adjacent to the subject material region 200, the length l _g of the first spacer region 201, electric objects molecule x within the first spacer region 201 It should be greater than the migration speed V _gx multiplied by the transport time.
l _g > (V _gx ) (t _Tr )
Since the electrophoretic velocity is proportional to the electric field of the first spacer region 201, the present invention makes it possible to control V _gx so that it can contain the target substance during transport through the microfluidic system channel. .

  2A and 2B, the first spacer region, that is, the high ion concentration spacer region 201, helps maintain the position of the target substance in the vicinity of the target substance area 200. Regardless of the charge polarity of the target material, the first spacer region 201 on either side of the target material region 200 causes the target material that leaves the target material region 200 to have a relatively high ion concentration in the first spacer region 201. Only exposed to low electric fields. When the polarity of the target substance is known, the direction of the electrophoretic force applied to the molecules of the target substance is also known.

  FIG. 3A shows an embodiment in which the charges of all target substances in the target substance region 200 are such that the electrophoretic force applied to the target molecule is in the same direction as the electroosmotic flow direction. Therefore, the first spacer region 201 is in front of the target substance region in the flow direction. There is no first spacer region 201 after the target material region 200. This is because the electrophoretic force prevents the target substance from escaping from the target substance region 200 in that direction. By removing half of the first spacer region 201, it becomes possible to carry more target material regions 200 having the target material for each channel length. This increases the transport efficiency of the microfluidic system. The second spacer region, that is, the low ion concentration spacer region 202 has a high electric field applied to the second spacer region, and the electric field (and electrophoretic force) of the target material region 200 and the first spacer region 201 is kept low. The material region 200 and the first spacer region, ie, the high ion concentration spacer region 201 are configured.

  In FIG. 3B, the first spacer region 201 is after the target material region 200 in the direction of electroosmotic flow. In this embodiment, the charges of all target substances in the target substance region 200 are such that the electrophoretic force applied to the target molecule is in the direction opposite to the electroosmotic flow direction. Thus, the target substance can escape from the boundary of the target substance region and effectively remain behind the target substance region 200. The first spacer region 201 after the target material region 200 prevents the target material from moving too far from the target material region 200. Similarly, the second spacer region, that is, the low ion concentration spacer region 202 has a target material region in which a high electric field is applied to the second spacer region 202 and the electric fields of the target material region 200 and the first spacer region 201 are kept low. 200 and a first spacer region, that is, a high ion concentration spacer region 201.

For the first and second spacer regions 201 and 202, various high and low ionic strength solutions are selected to produce a solution having the desired electrical conductivity. Specific ions that give conductivity to aqueous solutions include inorganic salts (eg, NaCl, KI, CaCl ₂ , FeF ₃ , (NH ₄ ) ₂ SO ₄ ), organic salts (eg, pyridinium benzoate, benzalkonium laurate) Alternatively, it may be derived from a mixed inorganic / organic salt (for example, sodium benzoate, sodium deoxyl sulfate, benzylamine hydrochloride) and the like. These ions are selected to be compatible with chemical reactions and separations that occur within the microfluidic system. In addition to aqueous solvents, aqueous / organic solvent mixtures, such as low concentration aqueous solutions of DMSO, can be used to assist in solubilization of target molecules. Mixtures of organic solvents such as CHCl ₃ : MeOH can also be used, for example, to facilitate analysis of phospholipase activity.
In general, when an aqueous solvent is used, the conductivity of the aqueous solution is adjusted using inorganic ions. When less polar solvents are used, organic or mixed inorganic / organic ions are typically used. Two immiscible solvents (eg, water and hydrocarbons such as decane) can be present at the same time, and if the current always flows from one solvent to the other, ion-permeable pores (eg, valinomycin, nonactin, various Currents can be passed through non-polar solvents using crown ethers and the like and their appropriate ions.

B. Electrokinetic control of pressure based flow The electrokinetic flow system described herein is differentially mobile (eg, different for a particular system) When fluid (with electrokinetic mobility) is present in the channel, multiple different pressures can occur along the channel length in the system. For example, these kinetic current systems typically represent a series of regions of low and high ionic concentration fluid (eg, first and second spacer regions and a target material region of a target material) within a given channel. In this way, an electroosmotic flow is generated, and at the same time, the influence of the electrophoretic bias in the target substance including the target substance region is prevented. The low ion concentration region in the channel tends to push the fluid through the channel because it tends to cause the highest applied voltage to drop over the region length. Conversely, high ionic concentration fluid regions within the channel tend to provide a relatively small voltage drop over the region length and slow the fluid flow by viscous drag.

As a result of such pushing and dragging effects, pressure variations can generally be generated along the length of the channel filled with fluid. The highest pressure is typically found at the front or tip of the low ion concentration region (eg, the second spacer region), and the lowest pressure is typically found at the trailing or trailing edge of the low ionic strength fluid region.
While such pressure disparities are largely irrelevant in linear channel systems, these effects are related to microfluidic devices using crossed channel arrangements, ie, previously incorporated US patent application Ser. No. 08/671, 987 can result in reduced control of fluid direction and operation in the system described in 987. For example, if the second channel is configured to intersect a first channel that includes a plurality of fluid regions with different ionic strengths, the pressure fluctuations described above may occur when these different fluid regions move past the intersection. Can flow into and out of the second channel that intersects. This fluctuating flow potentially disturbs the flow of fluid driven by quantitative electroosmosis from the second channel and / or perturbs various fluid regions within the channel. obtain.

  By reducing the depth of the intersecting channel, eg, the second channel, relative to the first or main channel, fluid flow fluctuations can be substantially eliminated. In particular, in the propulsion or direction of electroosmotic fluid for a given voltage gradient, the flow rate (volume / time) generally varies as the reciprocal of the channel depth of a channel having an aspect ratio (width: depth) of> 10. To do. Even if there are some minor errors, such an approximate ratio holds even for low aspect ratios, such as> 5 aspect ratios. Conversely, the pressure-induced flow for the same channel varies as the third power of the inverse of the channel depth. Thus, the increase in pressure in the channel due to the simultaneous presence of fluid regions of different ionic strength varies as the square of the reciprocal of the channel depth.

Therefore, the depth of the second channel intersecting the depth of the first channel or the main channel, by reducing the 1 X minutes, the pressure induced flow, significantly reduced in example X ³ min 1 However, electroosmotically induced flow can be reduced, for example, only by a factor of X. For example, if the depth of the second channel is decreased by an order of magnitude relative to the first channel, the pressure-induced flow is reduced by a factor of 1000 while the electroosmotically induced flow is 10 minutes. It can only be reduced to 1. Accordingly, in some aspects, the present invention provides a microfluidic device as generally described herein. The device has at least first and second intersecting channels, for example disposed in the device, wherein the first channel is deeper than the second channel. In general, the channel depth can be varied to obtain optimal flow conditions for the desired application. Thus, depending on the application, the first channel can be about two times deeper than the second channel, about five times deeper, and much deeper than about ten times.
In addition to use to reduce the effects of pressure, a variety of different channel depths can be used to flow fluids differently in different channels of the same device, e.g., at different rates from different sources. It is possible to mix fluids.

III. As described above, any target substance is efficiently transported through the microfluidic system 100 in or near the target substance region 200. The first and second spacer regions 201 and 202 localize the material of interest as it moves through the channels of the system. In order to efficiently introduce the target substance into the microfluidic system, the present invention further provides that the target substance is the same serial stream of the target substance region 200 and the combination of the first and second spacer regions 201 and 202. An electronic pipetter for introduction into a microfluidic system is provided.

A. Structure and Operation As shown in FIG. 4A, the electronic pipetter 250 is formed by a hollow capillary tube 251. The capillary tube 251 has a channel 254 having the dimensions of the channel of the microfluidic system 100, and the channel 254 is in fluid communication with the microfluidic system 100. As shown in FIG. 4A, the channel 254 is a cylinder having a cross-sectional diameter in the range of 1-100 μm, preferably about 30 μm in diameter. The electrode 252 descends along the outer wall of the capillary tube 251 and becomes a ring electrode 253 around the end of the tube 251. In order to draw the target material region 200 and the target material in the buffer regions 201 and 202 to the electronic pipettor channel 254, the electrode 252 is up to a voltage relative to the voltage of the target reservoir (not shown) in fluid communication with the channel 254 Driven. The target reservoir is in the microfluidic system 100 such that the target substance region 200 and buffer regions 201 and 202 already in the channel 254 are transferred in series from the electronic pipetter to the system 100.

  As a procedure, the end of the capillary channel of the electronic pipetter 250 is put into the source of the target substance. A voltage is applied to the electrode 252 with respect to the electrode in the target reservoir. The ring electrode 253 is placed in contact with the target material source, and generates a voltage drop between the target material source and the target reservoir by applying an electrical bias to the source. In practice, the target material source and the target reservoir are point A and point B in the microfluidic system, ie, as shown in FIG. 2A. The target substance is electrokinetically introduced into the capillary channel 254 to form the target substance region 200. The voltage to electrode 252 is then turned off and the capillary channel end is placed in a source of buffer material having a high ion concentration. Again, a voltage is applied to the electrode 252 relative to the target reservoir electrode, and as a result, the first spacer region 201 is electrokinetically introduced into the capillary channel 254 adjacent to the target material region 200. In doing so, if a second spacer region, i.e., a low ion concentration spacer region 202 is desired in the electron pipettor channel 254, the end of the capillary channel 254 is inserted into the source of the low ion concentration buffer material and the electrode A voltage is applied to 252. The electronic pipetter 250 then moves to another source of the target material, thereby forming another target material region 200 in the channel 254.

By repeating the above process, a plurality of target substance regions 200 having different target substances separated by the first and second spacer regions 201 and 202 are electrokinetically transferred to the capillary channel 254 and the microfluidic system 100. Introduced.
If the source of the target substance and buffer substance (having low and high ionic concentrations) has its own electrode, the electrode 252 is not necessary. The voltage between the target reservoir and the source electrode operates the electronic pipettor. Alternatively, the electrode 252 may be in a fixed relationship with the capillary tube 251 but separated from the capillary tube 251 so that when the end of the tube 251 contacts the reservoir, the electrode 252 also contacts the reservoir To do. The operation is the same as described for the electronic pipettor of FIG. 4A.

FIG. 4B shows a modification of the electronic pipettor 250 of FIG. 4A. In this variation, the electronic pipettor 270 need not move between the target material source and the buffer material source in order to form the first and second spacer regions 201 and 202 in the pipettor. The electronic pipetter 270 has a body 271 having three capillary channels 274, 275 and 276. The main channel 274 operates in the same manner as the channel 254 of the electronic pipetter 250 already described. However, the two auxiliary capillary channels 275 and 276 are in fluid communication at one end with a buffer source reservoir (not shown) and the other ends of the channels 275 and 276 are in fluid communication with the main channel 274. One reservoir (ie, in communication with the auxiliary channel 275) holds a buffer material having a high ionic concentration, and the other reservoir (ie, in communication with the channel 276) has a low ionic concentration. Retains buffer material.
All of the reservoirs are connected to electrodes that electrically bias these reservoirs for operation of the electronic pipettor 270. The electronic pipetter 270 can further include an electrode 272 along the wall of the body 271. The electrode 272 becomes a ring electrode 273 at the end of the main channel 274. By applying a voltage to electrode 272 (and ring electrode 273) to create a voltage drop along channels 274, 275, and 276, the target material can be drawn from the target material source into main channel 274, as well as high And buffer material having a low ionic concentration may also be drawn from the auxiliary channels 275 and 276 into the main channel 274.

  In order to operate the electronic pipettor 270 with the electrode 272, the end of the main capillary channel 274 is placed in the source 280 of the target substance. A voltage is applied to the electrode 272 with respect to the electrode in the target reservoir, thereby causing a voltage drop between the target material source 280 and the target reservoir. The target substance is electrokinetically drawn into the capillary channel 274. Thereafter, the capillary channel end is removed from the target material source 280 and a voltage drop is generated between the target reservoir communicating with the channel 274 and the reservoir communicating with the channel 275. A first spacer region or high ionic strength spacer region 201 is formed in the channel 274. As the buffer material is drawn from the auxiliary channel 275, the capillary action prevents the introduction of air into the channel 274. In doing so, if a second spacer region or low ionic concentration spacer region 202 is desired in the main channel 274, a voltage is applied to the electrode in the target reservoir and the reservoir having the low ionic concentration buffer material. The second spacer region 202 is then electrokinetically introduced from the second auxiliary channel 276 into the capillary channel 274. Thereafter, the electronic pipettor 270 also moves to another source of the target material, thereby forming another target material region 200 in the channel 274.

By repeating the above steps, a plurality of target substance regions 200 having different target substances separated by the first and second spacer regions 201 and 202 are electrokinetically transferred to the capillary channel 274 and the microfluidic system 100. To be introduced.
If it is not desired to expose the source of interest to the oxidation / reduction reaction from the ring electrode 273, the electronic pipettor can operate without the electrode 272. Since electroosmotic flow is slower in solutions with higher ionic strength, applying a potential (-+) from a reservoir communicating with channel 274 to a reservoir communicating with channel 275 causes channels 274 and 275 to cross. As a result, a vacuum is formed at the point to be performed. This vacuum draws the sample into the channel 274 from the source of interest. When operating in this mode, the substance of interest is diluted to some extent by the solution in channels 275 and 276. This dilution can be mitigated by reducing the relative dimensions of channels 276 and 275 relative to channel 274.

In order to introduce the first and second spacer regions 201 and 202 into the capillary channel 274, the electronic pipettor 270 operates as described above. The capillary channel end is removed from the target material source 280 and a voltage drop is generated between the target reservoir for channel 274 and the reservoir in communication with the selected channel 275 or 276.
Although generally described in the context of having two auxiliary channels and a main channel, additional auxiliary channels can also be provided to introduce additional fluids, buffers, diluents, reagents, and the like into the main channel. Is understood.
As noted above with respect to microfluidic devices, eg, intersecting channels in a chip, the resulting pressure differential of different moving fluids in different pipetter channels also affects the control of fluid flow in the pipettor channel. . Thus, as described above, various pipettors can also be provided having channel depths that vary with one another to optimize fluid control.

B. Method for Manufacturing an Electronic Pipetter An electronic pipettor can be formed from a hollow capillary tube as described above with respect to FIG. 4A. However, for more complex structures, the electronic pipettor is optimally formed from the same substrate material as that of the microchannel system described above. The electronic pipettor channel (and reservoir) is formed in the substrate in the same manner as the microchannel for the microfluidic system, and the substrate having the channel is covered by the planar cover element also described above. The edges of the substrate and cover element can then be shaped into an appropriate horizontal dimension of the pipettor, especially at the end, if desired. Techniques such as etching, air ablation (blowing particles and forced air to the surface), grinding, and cutting can be used. Thereafter, an electrode is formed on the surface of the substrate, and possibly on the cover as needed. Alternatively, the edges of the substrate and cover element can be shaped before being attached. This manufacturing method is particularly suitable for the multi-channel electronic pipetter described immediately above with reference to FIG. 4B and below with reference to FIG.

IV. Sampling System As noted above, the methods, systems, and apparatus described above generally find wide applicability in various fields. For example, as already mentioned, these methods and systems are described, for example, in copending US patent application serial number 08 / 671,987, filed June 28, 1996, which is already incorporated by reference. Is particularly suitable for the task of high-throughput chemical screening applied to drug delivery as described in.
A. Sample Matrix Pipetting and fluid transport systems of the present invention are generally described in terms of the number of liquid samples, ie, samples from a multiwell plate. However, in many instances, the number and nature of liquid-based samples to be sampled can create sample handling problems. For example, the number of libraries of screening compounds applied to chemical screening or drug delivery can range from thousands to tens of thousands, or even hundreds of thousands to millions, tens of millions. As a result, such libraries require a very large number of sample plates, which create incomparable difficulties in sample storage, manipulation and identification with the help of robotic systems. . Further, in some cases, certain sample compounds may have degradation, complexation, or otherwise a relatively short active half-life when stored in liquid form. This can potentially cause suspicious results when samples are stored in liquid form for extended periods prior to screening.

  The present invention thus provides a sampling system that addresses these additional problems by providing the compound to be sampled in an immobilized form. “Immobilized form” means that a sample is incorporated by incorporation into a fixed matrix that maintains the sample in a given location, ie, a porous matrix, charged matrix, hydrophobic or hydrophilic matrix. It means that the material is supplied to a fixed position. Alternatively, such immobilized samples include samples that are spotted and dried on a given sample matrix. In a preferred aspect, the compounds to be screened are provided on the sample matrix in a dry form. Typically, such sample matrices include any of a number of materials that can be used in spotting or immobilizing materials, including membranes such as cellulose, nitrocellulose, PVDF, nylon, and polysulfone. Typically, a flexible sample matrix is suitable for easy storage and handling to allow folding or entrainment of a sample matrix having many different sample compounds in an immobilized state.

  In general, the sample can be applied to the sample matrix by any of a number of well-known methods. For example, a sample library can be spotted onto a sample matrix sheet using a robotic pipetting system that allows spotting of multiple compounds. Alternatively, the sample matrix may be a predetermined region for sample localization, such as a well having a recess, a hydrophilic region surrounded by a hydrophobic barrier, or a hydrophobic region surrounded by a hydrophilic barrier (e.g. (If originally in a hydrophobic solution). In such areas, the spotted material is retained during the drying process. Such processing then enables the use of more advanced sample application methods such as those described in US Pat. No. 5,474,796. In the above patent, a piezoelectric pump and a nozzle system are used to direct the liquid sample to the surface. However, in general, the method described in the '796 patent relates to applying a liquid sample to a surface for subsequent reaction with an additional liquid sample. However, these methods can be easily modified to provide a dried and spotted sample on the substrate.

  Other immobilization or spotting methods can be used as well. For example, if the sample is stable in liquid form, the sample matrix will hold the liquid sample without allowing excessive diffusion or evaporation, etc., but will allow at least a portion of the sample material to be removed if desired. , Porous layers, gels or other polymeric materials. In order to draw the sample into the pipettor, the pipetter liberates a portion of the sample from the matrix, such as by matrix degradation, ion exchange, and sample dilution.

B. As described above, the sampling and fluid transport methods and systems of the present invention are readily applicable to screening, assaying or otherwise processing samples immobilized in these sample formats. It is. For example, if the sample material is supplied in a dry form on the sample matrix, an electropipetting system can be applied to the surface of the matrix. The electronic pipetter then operates to solubilize a small amount of liquid that solubilizes the already dried sample on the matrix surface (disintegrates the retention matrix or elutes the sample from the immobilized support). This can be done, for example, by reversing the polarity of the electric field applied to the pipetter or by applying a potential from the low ion concentration buffer reservoir to the high ion concentration buffer reservoir as described above. Once the sample is re-solubilized, the pipetter then operates in a typical forward format to draw the solubilized sample into the pipettor channel as previously described.

  A schematic diagram of one embodiment of an electronic pipettor useful for performing the above functions and its operation is shown in FIG. Briefly, the upper end 802 of the pipettor 800 (as shown) is generally connected to an assay system, such as a microfluidic chip, so that voltage can be separately supplied to the three channels 804, 806 and 808 of the pipettor. Is done. Channels 804 and 808 are typically fluidly connected to a buffer reservoir containing fluids with low and high ionic concentrations, respectively. In operation, the pipettor tip 810 contacts the surface of the sample matrix 12 where the fixed (eg, dry) sample 814 is located. A voltage is applied from the low ionic concentration buffer channel 804 to the high ionic concentration buffer channel 808, whereby the buffer is pushed out of the pipettor tip and contacts and dissolves the sample. As shown, the pipettor tip 816 includes a recessed area or “sample cup” 818 that can maintain the solution released between the pipettor tip and the matrix surface. In some cases, for example, when screening organic samples, an appropriate concentration of an acceptable solvent, such as DMSO, may be included with a low ionic concentration buffer to ensure sample lysis. A voltage is then applied from the high ion concentration buffer channel to the sample channel 806 to draw the sample into the pipetter in the form of a sample plug 820. When the sample is completely aspirated from the sample cup into the pipettor, the sample channel is terminated because air enters the sample channel and the surface tension is high, and the buffer solution with a high ion concentration starts flowing into the sample channel. Then, a first spacer region 822 is formed following the sample. Next, a low ionic concentration buffer solution may be injected into the sample channel as a second spacer region 824 by applying a voltage from the low ionic concentration buffer channel 804 to the sample channel 806. By applying a voltage between the high ion concentration buffer channel and the sample channel before or during the next sample location on the matrix, the first or high ion concentration spacer region 822 is sampled. Can be introduced into the channel. As mentioned above, single or multiple rolls, sheets, plates of a sample matrix having thousands or hundreds of thousands of different compounds to be screened can be presented in this way, so that in an appropriate device or system Continuous screening is possible.

V. Removal of Electrophoretic Bias As described above, electromotive force is used to transport the substance of interest through the channels of the microfluidic system 100. When the target substance is charged in a solution, it receives not only electroosmotic force but also electrophoretic force. Thus, the target substance tends to undergo electrophoresis as it moves from one point to another along the channel of the microfluidic system. Thus, the arrangement of species with different mixing or charging of the target substance in the target substance region 200 at the start point tends to be different from the mixing or arrangement at the arrival point. Furthermore, there is a possibility that the target substance is not in the target substance area 200 at the arrival point even though the first spacer area 201 exists.

  Accordingly, another aspect of the present invention compensates for electrophoretic bias when the substance of interest is transported through the microfluidic system 100. One way to compensate for electrophoretic bias is shown in FIG. In the microfluidic system 100 described above, each of the channels 110, 112, 114, and 116 was considered to be a unitary structure along its length. In FIG. 5, the example channel 140 is divided into two portions 142 and 144. The side walls of each channel portion 142 and 144 have surface charges of opposite polarity. The two channel portions 142 and 144 are physically connected by a salt bridge 133 such as a glass frit or gel layer. The salt bridge 133 separates the fluid in the channel 140 from the ionic fluid in the reservoir 135 defined in part by the salt bridge 133 while allowing ions to pass through the salt bridge 133. Accordingly, reservoir 135 is in electrical communication with channel 140 but not in fluid communication.

Electrodes 132 and 134 are positioned at points A and B, respectively, to provide electroosmotic and electrophoretic forces along the channel 140 between points A and B. Further, a third electrode 137 is disposed in the reservoir 135 at the junction of the two portions 142 and 144. Electrodes 132 and 134 are maintained at the same voltage, and electrode 137 is maintained at a different voltage. In the example shown in FIG. 5, the two electrodes 132 and 134 are at a negative voltage, and the electrode 137, and thus the junction of the two portions 142 and 144, is at zero voltage, or ground voltage. Thus, the voltage drops so that the electric field in portions 142 and 144 is in the opposite direction. Specifically, the electric fields refer to directions away from each other. Therefore, the direction of the electrophoretic force acting on a specific charged molecule is opposite between the channel portion 142 and the channel portion 144. After passing through the two portions 142 and 144, any electrophoretic bias to the target substance is compensated.
However, the electroosmotic forces in both portions 142 and 144 are still in the same direction. For example, as shown in FIG. 5, the sidewall of the channel portion 142 has a positive surface charge and attracts negative ions in the solution, and the sidewall of the channel portion 144 has a negative surface charge and is positive in the solution. Assuming that the ions are attracted, the electroosmotic forces in both portions 142 and 144 are directed to the right side of the drawing. Therefore, while the electrophoretic force is in the opposite direction between the one portion 142 and the other portion 144, the target substance is transported from the point A to the point B under the electroosmotic force.

To form a channel with sidewalls having a positive or negative surface charge, one or both portions of the channel are coated with an insulating film material having a surface charge, such as a polymer. For example, in the microfluidic system 100, the substrate 102 and the channel can be formed of glass. A portion of each channel is coated with a polymer with an opposite surface charge, such as polylysine, or chemically modified with a silanizing agent containing an amino functional group, such as aminopropyltrichlorosilane. . Further, the surface charge density and volume of both channel portions should be approximately the same so that the electrophoretic bias is compensated.
Rather than being formed in a solid planar substrate, the channel can be formed by two capillary tubes. The capillary tube is joined by a salt bridge that separates the ionic fluid reservoir from the fluid in the capillary tube. Again, electrodes are provided in the ionic fluid reservoir. One capillary tube has a negative surface charge and the other capillary tube has a positive surface charge. The resulting capillary channel operates as described above.

6A-6D illustrate another embodiment of the present invention. Here, the effect of the electrophoretic bias induced when the target substance moves from the point A to the point B is compensated. In this embodiment, as shown in FIG. 1, the target substance is mixed at point B, which is the intersection between the two channels.
6A-6D show the chamber 160 being formed at the intersection of the channels 150, 152, 154 and 156. FIG. Chamber 160 has four sidewalls 162, 164, 166 and 168. Side wall 162 connects the side wall of channel 152 to the side wall of channel 150. Side wall 164 connects the side wall of channel 154 to the side wall of channel 152. Sidewall 166 connects the side wall of channel 156 to the side wall of channel 154. Side wall 168 then connects the opposite side wall of channel 156 to the opposite side wall of channel 150. Looking at the flow of material through channel 152 towards channel 156, sidewalls 162 and 168 form a funnel when the material is deflected toward channel 150.
The dimensions of the sidewalls 162 and 168 accommodate the length of the material plug 200 that travels along the channel 152. Side walls 162 and 168 allow plug 200 to flow into the width of channel 150 in a funnel shape. The width of the channel 150 is such that the target material diffuses across the width of the channel 150, ie, the electrophoresis of the target material generated in the target material region 200 that moves along the channel 162 when mixing occurs. The size is such that the bias is removed. For example, if the width of the channel 150 is 50 μm, diffusion across the channel occurs in about 1 second for molecules with a diffusion constant of 1 × 10 ⁻⁵ cm ² / sec.

In FIG. 6A, the plug 200 of the cationic target substance is moving along the channel 152 toward the channel 156. By the time the plug 200 arrives at the chamber 160, the target substance is subjected to electrophoresis, so that the substance is concentrated more on the front end of the target substance region 200. This is shown in FIG. 6B. Next, the applied voltage drop along channels 152 and 156 ends, creating a voltage drop along channels 154 and 150, thereby attracting material region 200 to channel 150. Side walls 162 and 168 of the chamber 160 guide the target material region 200 having target material biased by electrophoresis to the funnel. This is shown in FIG. 6C. Due to diffusion, the target material spreads across the width of the channel 150 before the target material travels a significant distance along the channel 150. The target substance in the target substance region 200 is mixed, and the preparation for the next operation step in the microfluidic system 100 is completed.
In addition to being used to correct electrophoretic bias in a single sample, the structure shown in FIG. 6 mixes fluid elements in a microfluidic device, such as two separate analytes, buffers, reagents, etc. Can be useful to do.

Example 1- Two oppositely charged species are capillaryized to demonstrate the effectiveness of the method used to remove or reduce the forced co-migration electrophoretic bias of differently charged species in an electronic pipetter type format Electrokinetically sucked into the channel and co-migrated within a single sample plug. The Beckman capillary tube electrophoresis system was used to model the electrophoresis force in the capillary channel.
Briefly, in this experiment, a low ionic concentration (or “low salt”) (5 mM borate) or a high ionic concentration (or “high salt”) (500 mM borate) buffer, pH 8.6. A sample containing benzylamine and benzoic acid was used. Benzoic acid was present at a concentration approximately twice that of benzylamine. All injections were for 0.17 minutes. The length of the injection plug was determined by the injection voltage 8 or 30 kV. The low salt and high salt buffer were as described above.

  In the first experiment, a sample in low salt buffer was injected three times in succession into a capillary tube filled with low salt buffer. The injection was performed at 8 kV with a low salt injection at 30 kV in between. Data obtained from these injections is shown in FIG. 7A. These data show that benzylamine from the first and second injections (which can be identified as a short peak as a result of the lower concentration) precedes the benzoic acid peak (high peak) from the first injection. I understand that. In addition, the benzylamine peak from the third injection occurs almost simultaneously with the first benzoic acid peak. This experiment thus shows that the effect of electrophoretic bias, that is, the sample peak may not exit in the same order as it entered the capillary channel. As clearly shown, such separation substantially interferes with the properties of a single sample or, in the worse case, at the expense of already introduced or subsequently introduced samples. obtain.

In the second experiment, the capillary tube was filled with a low salt buffer. First, a sample was injected by introducing / injecting a high salt buffer into the capillary tube at 8 kV (first spacer region 1). Following this, the sample in high salt buffer was injected at 8 kV, followed by a second injection of high salt buffer at 8 kV (first spacer region 2). Three samples were injected in this way and spaced by injecting low salt buffer at 30 kV. As can be seen in FIG. 7B, both compounds contained in the sample are forced to move together through the capillary channel within the same sample plug, which are represented by a single peak from each injection. . This indicates that the sample is aligned regardless of the mobility by electrophoresis.
By reducing the size of the low salt spacer plug between samples relative to the sample size, partial dissolution of the components of each sample injection can be achieved. This can be useful if some separation of the sample is desired during electrokinetic pumping without sacrificing subsequently injected or already injected sample. This was performed by injecting a low salt spacer plug at 8 kV instead of 30 kV. Data from this example is shown in FIG. 7C.

Example 2— Transfer of a Target Substance through an Electronic Pipetter to a Microfluidic System Substrate FIGS. 9A-9C illustrate experimental testing of introducing a target substance, i.e., a sample, through an electronic pipetter to a microfluidic system substrate , as described above. Results are shown. The sample is rhodamine B in phosphate buffered saline pH 7.4. A high ion concentration (“high salt”) buffer was also formed from phosphate buffered saline pH 7.4. Low ionic concentration (“low salt”) buffer was formed from a 5 mM Na borate pH 8.6 solution.
In the test, the target substance region containing fluorescent rhodamine B was periodically injected into the capillary channel of an electronic pipettor connected to the substrate of the microfluidic system. High salt and low salt buffer were also injected between the areas of interest as described above. FIG. 9A is a plot of rhodamine B fluorescence intensity versus time monitored at a point near the junction with the substrate channel along the capillary channel. (Note that the numbers on the fluorescence intensity axes in FIGS. 9A to 9C and FIG. 10 are not absolute values, but for comparison reference.) The injection cycle time is 7 seconds and the target substance region The electric field for moving the through the electron pipettor was 1000 volts / cm. The total time for the photodiode to monitor the light from the capillary channel was set to 10 milliseconds. From the plot of FIG. 9A, it will be apparent that light intensity spikes appear at 7 second intervals, which corresponds to the injection cycle time of fluorescent rhodamine B.

In another experiment, the same buffer was used with the Rhodamine B sample. The monitoring point was in the channel of the substrate connected to the electronic pipettor. The injection cycle time was set to 13.1 seconds and the voltage between the source reservoir containing rhodamine B and the target reservoir in the substrate was set to -3000 volts. The total monitor time with the photodiode was 400 milliseconds. As shown in FIG. 9B, the fluorescence intensity spike is very consistent with the rhodamine B injection cycle time.
The result of the third experimental test is shown in FIG. 9C. In this experiment, an electronic pipettor was formed from the substrate, and its shape was determined by air erosion. The monitoring point is a position along the capillary channel formed in the substrate (and the flat cover). In this case, the sample material is 100 μM rhodamine B in PBS buffer, pH 7.4. A high salt buffer solution in PBS, pH 7.4, and a 5 mM Na borate low salt buffer solution, pH 8.6 were also used. Again, periodic spikes in fluorescence intensity are consistent with periodic injection of rhodamine B into the electron pipettor.

  FIG. 10 shows the result of periodic injection of another target substance into the electronic pipettor of the present invention. In this experiment, the sample was 100 μM small molecule compound with 1% DMSO in phosphate buffered saline, pH 7.4. The same phosphate buffered saline high salt buffer, pH 7.4, and 5 mM Na borate low salt buffer, pH 8.6 were also used. The applied voltage for moving the target substance region through the electron pipettor was −4000 volts, and the total time for the photodiode to monitor the light from the capillary tube channel was set to 400 milliseconds. Samples were periodically injected into an electronic pipetter as described above. Similar to the previous result, it can be seen from the plot of FIG. 10 that for low molecular weight compounds, the electron pipettor moves the sample at evenly spaced time (and space) intervals.

  Although the present invention has been described in some detail for purposes of clarity and understanding, it is to be understood that various changes in form and detail may be made without departing from the spirit and scope of the invention. Can be obvious to those skilled in the art. For example, all of the techniques described above can be used in various combinations. All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as each publication or patent document is presented individually.

1 is a schematic diagram of one embodiment of a microfluidic system. FIG. 2A shows the configuration of a fluid region moving through one channel of the microfluidic system of FIG. 1 according to one embodiment of the present invention. FIG. 2B is a scaled illustration of another configuration of different fluid regions moving through one channel of a microfluidic system according to the present invention. FIG. 3A is another configuration having a high ion concentration spacer region in front of a region of interest moving through one channel of the microfluidic system. FIG. 3B shows a configuration having a high ionic concentration spacer region after the region of interest moving through one channel of the microfluidic system. FIG. 4A is a schematic diagram of one embodiment of an electronic pipettor according to the present invention. FIG. 4B is a schematic diagram of another electronic pipettor according to the present invention. FIG. 2 is a schematic view of a channel with a portion having oppositely charged sidewalls in a microfluidic system according to the present invention. 6A-6D illustrate the mixing action of funneling sidewalls at the intersection of channels in a microfluidic system according to the present invention. Shown is the result of three injections of a sample fluid consisting of two oppositely charged species in low salt buffer into a capillary filled with low salt buffer. The sample is in high salt buffer, high salt buffer fluid is injected to act as a guard band at both ends of the sample region, and the sample / guard band is run in a capillary filled with low salt buffer. The results of three sample injections are shown. FIG. 7B shows the results of three sample injections similar to FIG. 7B, except that the sample element can be a later or previous sample by reducing the size of the low salt spacer region between the sample / high salt spacer (guard band). Allows partial resolution of species in a sample without sacrificing 1 shows a schematic diagram of an electronic pipettor for use with a sample extraction system that uses a sample that has been solid-phased, eg, by drying, on a substrate sheet or matrix. FIG. 4 is a fluorescence versus time plot showing the movement of a sample fluid consisting of test species periodically injected into and moved through an electronic pipetter according to the present invention. FIG. 6 is another plot showing the movement of a sample fluid having chemical species through a microfluidic substrate connected to an electronic pipettor under different parameters. FIG. 5 is a plot showing the movement of sample fluid and species through an electronic pipettor formed from an air abraded substrate. 4 is a plot showing the migration of chemical species in a sample fluid periodically injected into an electronic pipettor according to the present invention. The species in this experiment is a small molecule compound.

Explanation of symbols

200 Target substance regions 201, 202 Spacer region (buffer agent region)
250 Electron pipetter 251 Capillary tube 252 Electrode 253 Ring electrode 254 Capillary channel 270 Electronic pipettor 271 Body 272 Electrode 273 Ring electrode 274 Main capillary channel 275 Auxiliary capillary channel 276 Auxiliary capillary channel 280 Target material source

Claims (31)

A method of introducing a substance from a plurality of sources into a microfluidic system, the microfluidic system having a capillary channel having an end and a voltage source for applying a voltage potential to an electrode of the microfluidic system. And the method
Bringing the end of the capillary channel into contact with a target material source;
Applying a voltage to the target material source with respect to the electrode such that the target material from the source is electrokinetically introduced into the capillary channel toward the microfluidic system;
Selecting a source of spacer material having a predetermined ion concentration;
Applying a voltage to the spacer material source with respect to the electrode such that the spacer material is electrokinetically introduced into the capillary channel next to the target material;
Different materials so that a plurality of different materials separated by the spacer material are introduced electrokinetically into the capillary channel and transported towards the microfluidic system without mixing between the different materials Repeating the above steps using a source. The method of claim 1, wherein the spacer material comprises a high ionic concentration solution. The method of claim 1, wherein the spacer material comprises a substantially non-mixable fluid. The method of claim 1, wherein the spacer material comprises ion permeable pores. The method of claim 1, wherein the capillary channel has a cross-sectional area of about 10 to 1000 (μm) ² . Bringing the end of the capillary channel into contact with the target material source and applying a voltage to the spacer material source with respect to the first electrode so that the spacer material is electrokinetically introduced into the capillary channel. The step of applying
Placing the end of the capillary channel in the source of the first spacer material;
Applying a voltage to the source of the first spacer material with respect to the electrode such that the first spacer material is electrokinetically introduced into the capillary channel;
Placing the end of the capillary channel in the source of a second spacer material;
Applying a voltage to the source of the second spacer material with respect to the electrode, such that the second spacer material is electrokinetically introduced into the capillary channel;
The method further comprises repeating the first two of the steps so that a plurality of different target materials are separated by the regions of the first, second and first spacer materials. the method of. The method of claim 6, wherein the first spacer material comprises a high ionic concentration solution and the second spacer material comprises a low ionic concentration solution. Disposing the second electrode from the capillary channel along the end of the capillary channel such that the second electrode contacts the source when the end of the capillary channel contacts the material or spacer source. The method of claim 1 further comprising the step of applying a voltage comprising creating a voltage difference between an electrode of the microfluidic system and the second electrode. The method of claim 1, wherein applying the voltage comprises applying a negative voltage to the target material or spacer source with respect to an electrode of the microfluidic system. A microfluidic system for moving a plurality of objects of interest along a channel from a first location to a second location, the microfluidic system comprising:
A source for creating a voltage difference between the first position and the second position;
A plurality of target material regions in the channel, wherein the target material regions are separated by a first spacer region having a high ion concentration and at least one second spacer region having a low ion concentration; system. A microfluidic system for transporting a plurality of regions of interest from a first point on a channel to a second point, the microfluidic system comprising:
A pair of first spacer regions on both sides of each target material region, the first spacer region having a high ion concentration;
At least one second spacer region, the second spacer region having a low ion concentration. The system of claim 11, wherein the second spacer region has an ion concentration that is at least two orders of magnitude less than the first spacer region. 13. The microfluidic system of claim 12, wherein the second spacer region has an ion concentration in the range of 1-10 mM, and the first spacer region has an ion concentration in the range of 100-1000 mM. The substrate having at least a first channel and at least a second channel disposed on the substrate, wherein the at least second channel intersects the first channel, and the first channel is A substrate deeper than the second channel;
An electroosmotic fluid directing system. 15. The microfluidic system of claim 14, wherein the at least first channel is at least twice as deep as the second channel. 15. The microfluidic system of claim 14, wherein the at least first channel is at least 5 times deeper than the second channel. 15. The microfluidic system of claim 14, wherein the at least first channel is at least about 10 times deeper than the second channel. In an electroosmotic fluid directing system, a method for controllably delivering a fluid stream along a first channel, wherein the first channel intersects at least a second channel, and the fluid streams are different. A method comprising at least two fluidic regions having an ionic concentration, the method comprising providing the first channel with a depth greater than the second channel. A method of transporting a fluid sample within a microfluidic channel, comprising:
Introducing a plug of at least a first fluid material having a first ion concentration into the channel;
Introducing at least a first sample fluid plug into the channel;
Introducing at least a second fluidic material plug having the first ion concentration into the channel;
Introducing at least a third fluid material plug having a second ion concentration, wherein the second ion concentration is lower than the first ion concentration;
A method further comprising applying a voltage to the channel. Introducing the at least first fluid material plug, the at least sample fluid plug, the at least second fluid material plug, and the at least third fluid material plug;
An end of the channel is brought into contact with the source of the at least first fluid material to apply a voltage to the channel from the source of the at least first fluid material, whereby the first fluid material is Introducing into the channel;
An end of the channel is brought into contact with the source of the at least first sample fluid to apply a voltage from the source of the at least first sample fluid to the channel so that the first sample fluid is Introducing into the channel;
An end of the channel is brought into contact with the source of the at least second fluid material to apply a voltage to the channel from the source of the at least second fluid material, whereby the second fluid material is Introducing into the channel;
An end of the channel is brought into contact with the source of the at least third fluid material to apply a voltage from the source of the at least third fluid material to the channel so that the third fluid material is And introducing into the channel. Use of a substrate having a channel, wherein at least a first target material is transported along the channel due to an applied voltage when transporting along the channel from at least a first location to a second location Use of at least one low ion concentration region. The use according to claim 21, wherein an ion concentration of the one region is lower than an ion concentration of the target substance. 23. Use according to claim 21 or 22, wherein a plurality of target substances separated by a high ion concentration spacer region are transported. 24. Use according to any of claims 21 to 23, wherein the substrate is a microfluidic system. Use of a microfluidic system in optimizing flow conditions, wherein the microfluidic system has at least first and second fluid channels, and the channels have different depths. 26. Use according to claim 25, wherein one channel is 2 to 10 times deeper than the other channel. A microfluidic system comprising a substrate having a channel, wherein at least a first target substance is at least along the channel using at least one low ion concentration region transported along the channel for an applied voltage A microfluidic system transported from a first location to a second location. 28. The system according to claim 27, wherein an ion concentration of the one region is substantially lower than an ion concentration of the target substance. 29. A system according to claim 27 or 28, wherein a plurality of target substances separated by a high ion concentration spacer region are transported. A microfluidic system having at least first and second fluid channels intersecting each other, the channels having different depths to optimize flow conditions. 32. The system of claim 30, wherein one channel is 2 to 10 times deeper than the other channel.