NZ504697A - Microfluidic transport with subject material slugs separated in channels by spacer slugs of different ionic strengths - Google Patents

Abstract

A microfluidic system for moving a number of materials from one location to another along a channel is disclosed. The system comprises a source for creating a voltage difference between the locations, a number of materials in the channel separated by spacers of high ionic strength and at least one spacer of low ionic strength.

Description

NEW ZEALAND PATENTS ACT, 1953 No: Divided out of Application No. 333345 Date: Dated 25 June 1997 COMPLETE SPECIFICATION IMPROVEMENTS RELATING TO MICROFLUIDIC SYSTEMS We, CALIPER TECHNOLOGIES CORPORATION, of 1275 California Avenue, Palo Alto, California 94304, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (Followed by page la) la This specification describes more than one invention. Claims to the inventions can be found in the present specification, divisional specification NZ and NZ 333345.

BACKGROUND OF THE INVENTIONS There has been a growing interest in the manufacture and use of microfluidic systems for the acquisition of chemical and biochemical information. Techniques commonly associated with the semiconductor electronics industry, such as photolithography, wet chemical etching, etc., are being used in the fabrication of these microfluidic systems. The term, "microfluidic", refers to a system or device having channels and chambers which are generally fabricated at the micron or submicron scale, e.g., having at least one cross-sectional dimension in the range of from about 0.1 /^m to about 500 /im. Early discussions of the use of planar chip technology for the fabrication of microfluidic systems are provided in Manz et al., Trends in Anal. Chem. (1990) 10 (5):144-149 and Manz et al., Avd. in Chromatog. (1993) 33:1-66, which describe the fabrication of such fluidic devices and particularly microcapillary devices, in silicon and glass substrates.

Applications in microfluidic systems are myriad. For example, International Patent Appln. WO 96/04547, published February 15, 1996, describes the use of microfluidic systems for capillary electrophoresis, liquid chromatography, flow injection analysis, and chemical reaction and synthesis. US 5,942,443 WO 98/00705 PCT/US97/10963 2 and incorporated herein by reference, discloses wide ranging applications of microfluidic systems in rapidly assaying large number of compounds for their effects on chemical, and preferably, biochemical systems. The phrase, "biochemical 5 system," generally refers to a chemical interaction which involves molecules of the type generally found within living organisms. Such interactions include the full range of catabolic and anabolic reactions which occur in living systems including enzymatic, binding, signaling and other reactions. 10 Biochemical systems of particular interest include, e.g., receptor-ligand interactions, enzyme-substrate interactions, cellular signaling pathways, transport reactions involving model barrier systems (e.g., cells or membrane fractions) for bioavailability screening, and a variety of other general 15 systems.

Many methods have been described for the transport and direction of fluids, e.g., samples, analytes, buffers and reagents, within these microfluidic systems or devices. One method moves fluids within microfabricated devices by 20 mechanical micropumps and valves within the device. See, Published U.K. Patent Application No. 2 248 891 (10/18/90), Published European Patent Application No. 568 902 (5/2/92), U.S. Patent Nos. 5,271,724 (8/21/91) and 5,277,556 (7/3/91). See also, U.S. Patent No. 5,171,132 (12/21/90) to Miyazaki et 25 al. Another method uses acoustic energy to move fluid samples within devices by the effects of acoustic streaming. See, Published PCT Application No. 94/05414 to Northrup and White. A straightforward method applies external pressure to move fluids within the device. See, e.g., the discussion in U.S. 30 Patent No. 5,304,487 to Wilding et al.

Still another method uses electric fields to move fluid materials through the channels of the microfluidic system. See, e.g., Published European Patent Application No. 376 611 (12/30/88) to Kovacs, Harrison et al., Anal. Chem. 35 (1992) 64:1926-1932 and Manz et al. J. Chromatoa. (1992) 593:253-258, U.S. Patent No. 5,126,022 to Soane.

Electrokinetic forces have the advantages of direct control, fast response and simplicity. However, there are still some WO 98/00705 PCT/US97/10963 3 disadvantages. For maximum efficiency, it is desirable that the subject materials be transported as closely together as possible. Nonetheless, the materials should be transported without cross-contamination from other transported materials. Further, the materials m one state at one location in a microfluidic system should remain in the same state after being moved to another location in the microfluidic system. These conditions permit the testing, analysis and reaction of the compound materials to be controlled, when and where as desired.

In a microfluidic system m which the materials are moved by electrokinetic forces, the charged molecules and ions in the subject material regions and in the regions separating these subject material regions are subjected to various electric fields to effect fluid flow.

Upon application of these electric fields, however; differently charged species within the subject material will exhibit different electrophoretic mobilities, i.e., positively charged species will move at a different rate than negatively charged species. In the past, the separation of different species within a sample that was subjected to an electric field was not considered a problem, but was, in fact, the desired result, e.g., in capillary electrophoresis. However, where simple fluid transport is desired, these varied mobilities can result in an undesirable alteration or "electrophoretic bias" in the subject material.

Without consideration and measures to avoid cross-contamination, the microfluidic system must either widely separate the subject materials, or, in the worst case, move the materials one at a time through the system. In either case, efficiency of the microfluidic system is markedly reduced. Furthermore, if the state of the transported materials cannot be maintained in transport, then many applications which require the materials to arrive at a location unchanged must be avoided.

The inventions solve or substantially mitigate these problems of electrokinetic transport. With the inventions, microfluidic systems can move materials 4 efficiently and without undesired change in the transported materials. The inventions present high throughput microfluidic systems having direct, fast and straightforward control over the movement of materials through the channels of the microfluidic systems with a wide range of applications, such as in the fields of chemistry, biochemistry, biotechnology, molecular biology and numerous other fields.

SUMMARY OF THE INVENTIONS In one aspect the present invention may be said to consist in a microfluidic system for moving a plurality of subject materials from a first location to a second location along a channel, said microfluidic system comprising: a source for creating a voltage difference across said first location and said second location; a plurality of subject material regions in said channel, said subject material regions separated by first spacer regions of high ionic strength; and at least one second spacer region of low ionic concentration.

In another aspect the present invention may be said to consist in a microfluidic system for transporting a plurality of subject material regions from a first point to a second point on a channel, said microfluidic system comprising: a pair of first spacer regions on either side of each subject material region, said first spacer regions having high ionic concentrations; at least one second spacer region, said second spacer region having a low ionic concentration.

In another aspect the present invention may be said to consist in a method of transporting fluid samples within a microfluidic channel, comprising: introducing at least a plug of a first fluid material having a first ionic strength into said channel; introducing at least a first sample fluid plug into said channel; introducing at least a second fluid material plug having said first ionic strength into said channel; introducing at least a third fluid material plug having a second ionic strength, said second ionic strength being lower than said first ionic strength; and applying a voltage across said channel.

In another aspect the present invention may be said to consist in a microfluidic system comprising a substrate having a channel, in which at least a first subject material is transported from at least a first location to a second location along "said channel, utilizing at least one region of low ionic concentration which is transported along said channel due to an applied voltage. 6 Embodiments of the inventions described may be put into a plurality of different uses, which are themselves inventive, for example, as follows: The use of a substrate having a channel, in transporting at least a first subject material from at least a first location to a second location along the channel, utilizing* at least one region of low ionic concentration which is transported along the channel due to an applied voltage. ;A use of an embodiment m which the ionic concentration of the one region is substantially lower than that of the subject material. ;A use of an embodiment wherein a plurality of subject materials are transported, separated by high ionic concentration spacer regions. ;The use of a substrate having a channel along which at least a first subject material may be transported, in electrophoretic bias compensation, the channel being divided into a first and a second portion, in which the wall or walls of the channel are oppositely cnarged, such that electrophoretic bias on the at least first subject material due to transportation in the first portion is substantially compensated for by electrophoretic bias due to transport in the second portion. ;A use of an embodiment in which a first electrode is located at a remote end of the first portion, a second electrode is located at the intersection between the portions and a third electrode is located at a remote end of the second portion. ;A use of an embodiment m which the suDStrate is a microfluidic system. ;A use of an embodiment m which the substrate is an electropipettor. ;WO 98/00705 PCT/US97/10963 ;7 ;A use of an embodiment in which the electropipettor has a main channel for transportation of the subject material and at least one further channel fluidly connected to the main channel from which a 5 further material to be transported along the main channel is obtained. ;A use of an embodiment in which the further material is drawn into the main channel as a buffer region between each of a plurality of separate ;10 subject materials. ;The use of a taicrofluidic system having at least a first and a second fluid channel which intersect, in optimizing flow conditions, the channels having different depths. ;15 A use of an embodiment in which one channel is between 2 to 10 times deeper than the other channel. ;The use of a microfluidic system having a first channel and a second channel intersecting the first ;20 channel, in electrophoretic compensation, the intersection between the channels being shaped such that a fluid being transported along the first channel towards the second channel is mixed at the intersection and any electrophoretic bias m the fluid is dissipated. ;25 ;) BRIEF DESCRIPTION OF THE DRAWINGS ;Figure 1 shows a schematic illustration of one embodiment of a microfluidic system; ;Figure 2A illustrates an arrangement of fluid ;30 regions traveling in a channel of the microfluidic system of Figure 1, according to one embodiment; ;Figure B is a scaled drawing of another arrangement of different fluid regions traveling in a channel of the microfluidic system according to an embodiment; ;35 Figure 3A is another arrangement with high ionic concentration spacer regions before a subject material region traveling within a channel of the microfluidic system; Figure 3B shows an arrangement with high ionic concentration spacer ;WO 98/00705 PCT/US97/10963 ;8 ;regions after a subject material region traveling in a channel of the microfluidic system; ;Figure 4A is a schematic diagram of one embodiment of an electropipettor; ;5 Figure 4B is a schematic diagram of another electropipettor embodiment; ;Figure 5 is a schematic diagram of a channel having portions with oppositely charged sidewalls in a microfluidic system; and ;10 Figures 6A-6D illustrate the mixing action of funneling sidewalls at>the intersection of channels in a microfluidic system. ;Figure 7A shows the results of three injections of a sample fluid made up of two oppositely charged chemical 15 species in a low salt buffer, into a capillary filled with low salt buffer. Figure 7B shows the results of three sample injections where the sample is in high salt buffer, high salt buffer fluids were injected at either end of the sample region to function as guard bands, and the sample/guard bands were 20 run in a low salt buffer filled capillary. Figure 7C shows the results of three sample injections similar to that of Figure 7B except that the size of the low salt spacer region between the sample/high salt spacers (guard bands) is reduced, allowing partial resolution of the species within the sample, 25 without allowing the sample elements to compromise subsequent or previous samples. ;Figure 8 shows a schematic illustration of an electropipettor for use with a sampling system using samples immobilized, e.g., dried, on a substrate sheet or matrix. 30 Figure 9A is a plot of fluorescence versus time which illustrates the movement of a sample fluid made up of test chemical species which is periodically injected into, and moved through, an electropipettor. ;Figure 9B is another plot which shows the movement 35 of the sample fluid with the chemical species through a microfluidic substrate which is connected to the electropipettor, under different parameters. Figure 9C is a plot which illustrates the movement of the sample fluid and ;WO 98/00705 PCT/US97/10963 ;9 ;chemical species through an electropipettor formed from an air abraded substrate. ;Figure 10 is a plot which again illustrates the movement of a chemical species in a sample fluid which has 5 been periodically injected into an electropipettor, accordi-ng to one embodiment. In this experiment, the species is a small molecule compound. ;DETAILED DESCRIPTION OF THE INVENTION 10 I. General Organization of a Microfluidic System ;Fig. 1 disclcses a representative diagram of an exemplary microfluidic system 100 according to one embodiment. As shown, the overall device 100 is fabricated m a planar substrate 102. Suitable substrate materials are 15 generally selected based upon their compatibility with the conditions present in the particular operation to be performed by the device. Such conditions can include extremes of pH, temperature, ionic concentration, and application of electrical fields. Additionally, substrate materials are also 20 selected for their inertness to critical components of an analysis or synthesis to be carried out by the system. ;Useful substrate materials include, e.g., glass, quartz and silicon, as well as polymeric substrates, e.g., plastics. In the case of conductive or semiconductive 25 substrates, there should be an insulating layer on the ^ substrate. This is particularly important where the device incorporates electrical elements, e.g., electrical fluid direction systems, sensors and the like, or uses electroosmotic forces to move materials about the system, as 30 discussed below. In the case of polymeric substrates, the substrate materials may be rigid, semi-rigid, or non-rigid, opaque, semi-opaque or transparent, depending upon the use for which they are intended. For example, devices which include an optical or visual detection element, will generally be 35 fabricated, at least in part, from transparent materials to allow, or at least, facilitate that detection. Alternatively, transparent windows of, e.g., glass or quartz, may be incorDorated into the device for these types of detection ;WO 98/00705 PCT/US97/10963 ;10 ;elements. Additionally, the polymeric materials may have linear or branched backbones, and may be crosslinked or non-crosslinked. Examples of particularly preferred polymeric materials include, e.g., polydimethylsiloxanes (PDMS), 5 polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate and the like. ;The system shown in Figure 1 includes a series of channels 110, 112, 114 and 116 fabricated into the surface of the substrate 102. As discussed in the definition of 10 "microfluidic," these channels typically have very small cross sectional dimensions, preferably in the range of from about 0.1 /im to about 100 jum. For the particular applications, discussed below, channels with depths of about 10 £im and widths of about 60 nm work effectively, though deviations from 15 these dimensions are also possible. ;Manufacturing of these channels and other microscale elements into the surface of the substrate 102 may be carried out by any number of microfabrication techniques that are well known in the art. For example, lithographic techniques may be 20 employed in fabricating glass, quartz or silicon substrates, for example, with methods well known in the semiconductor manufacturing industries. Photolithographic masking, plasma or wet etching and other semiconductor processing technologies define microscale elements in and on substrate surfaces. 25 Alternatively, micromachining methods, such as laser drilling, micromilling and the like, may be employed. Similarly, for polymeric substrates, well known manufacturing techniques may also be used. These techniques include injection molding techniques or stamp molding methods where large numbers of 30 substrates may be produced using, e.g., rolling stamps to produce large sheets of microscale substrates, or polymer microcasting techniques where the substrate is polymerized within a microfabricated mold. ;Besides the substrate 102, the microfluidic system 35 includes an additional planar element (not shown) which overlays the channeled substrate 102 to enclose and fluidly seal the various channels to form conduits. The planar cover element may be attached to the substrate by a variety of ;WO 98/00705 PCT/US97/10963 ;11 ;means, including, e.g., thermal bonding, adhesives or, in the case of glass, or semi-rigid and non-rigid polymeric substrates, a natural adhesion between the two components. The planar cover element may additionally be provided with 5 access ports and/or reservoirs for introducing the various fluid elements needed for a particular screen. ;The system 100 shown in Figure 1 also includes reservoirs 104, 106 and 108, which are disposed and fluidly connected at the ends of the channels 114, 116 and 110 10 respectively. As shown, sample channel 112, is used to introduce a plurality of different subject materials into the device. As such, the channel 112 is fluidly connected tQ a source of large numbers of separate subject materials which are individually introduced into the sample channel 112 and 15 subsequently into another channel 110. As shown, the channel 110 is used for analyzing the subject materials by electrophoresis. It should be noted that the term, "subject materials," simply refers to the material, such as a chemical or biological compound, of interest. Subject compounds may 20 include a wide variety of different compounds, including chemical compounds, mixtures of chemical compounds, e.g., polysaccharides, small organic or inorganic molecules, biological macromolecules, e.g., peptides, proteins, nucleic acids, or extracts made from biological materials, such as 25 bacteria, plants, fungi, or animal cells or tissues, naturally occurring or synthetic compositions. ;The system 100 moves materials through the channels 110, 112, 114 and 116 by electrokinetic forces which are provided by a voltage controller that is capable of applying 3 0 selectable voltage levels, simultaneously, to each of the reservoirs, including ground. Such a voltage controller can be implemented using multiple voltage dividers and multiple relays to obtain the selectable voltage levels. ;Alternatively, multiple independent voltage sources may be 35 used. The voltage controller is electrically connected to each of the reservoirs via an electrode positioned or fabricated within each of the plurality of reservoirs. See, for example, published International Patent Application No. wo ;WO 98/00705 PCT/US97/10963 ;12 ;96/04547 to Ramsey, which is incorporated herein by reference in its entirety for all purposes. ;II. Electrokinetic Transport 5 A. General 1y ;The electrokinetic forces on the fluid materials in the channels of the system 100 may be separated into electroosmotic forces and electrophoretic forces. The fluid control systems used m the system 10 employ electroosmotic force to move, direct and mix fluids in the various channels and reaction chambers present on the surface of the substrate 102. In brief, when an appropriate fluid is placed in a channel or other fluid conduit having functional groups present at the surface, those groups can 15 ionize. Where the surface of the channel includes hydroxyl functional groups at the surface, for example, protons can leave the surface of the channel and enter the fluid. Under such conditions, the surface possesses a net negative charge, whereas the fluid possesses an excess of protons or positive 20 charge, particularly localized near the interface between the channel surface and the fluid. ;By applying an electric field across the length of the channel, cations flow toward the negative electrode. Movement of the positively cnarged species m the fluid pulls 25 the solvent with them. The steady state velocity of this fluid movement is generally given by the equation: ;30 ;v = e£E 4nri where v is the solvent velocity, s is the dielectric constant of the fluid, £ is the zeta potential of the surface, S is the electric field strength, and n is the solvent viscosity. ;Thus, as can be easily seen from this equation, the solvent 35 velocity is directly proportional to the zeta potential and the applied field. ;Besides electroosmotic forces, there are also electrophoretic forces which affect charged molecules as they move through the channels of the system 100. In the transport 40 of subject materials from one point to another point in the ;WO 98/00705 PCT/US97/10963 ;13 ;system 100, it is often desirable for the composition of the subject materials to remain unaffected in the transport, i.e., that the subject materials are not electrophoretically differentiated in the transport. ;The subject materials are moved about the channels as slugs of fluid (hereafter termed "subject material regions"), which have a high ionic concentration to minimize electrophoretic forces on the subject materials within these particular regions. To minimize the effect of electrophoretic forces within the subject material regions, regions of spacer fluids ("first spacer regions") are placed on either side of a slug. These first spacer regions have a high ionic concentration to minimize the electric fields in these regions, as explained below, so that subject materials are essentially unaffected by the transport from one location to another location in a microfluidic system. The subject materials are transported through the representative channels 110, 112, 114, 116 of the system 100 in regions of certain ionic strengths, together with other regions of ionic strengths varying from those regions bearing the subject materials. ;A specific arrangement is illustrated in Figure 2A, which illustrates subject material regions 200 being transported from point A to point B along a,channel of the microfluidic system 100. In either side of the subject material regions 200 are first spacer regions 201 of high ionic strength fluid. Additionally, second spacer regions 202 of low ionic concentration fluid periodically separate arrangements of subject material regions 200 and first spacer regions 201. Being of low ionic concentration, most of the voltage drop between points A and B occurs across these second spacer regions 202. The second or low concentration spacer regions 202 are interspersed between the arrangements of subject material region 200 and first spacer region 201 such that, as the subject material regions 200 and the first spacer regions 201 are electroosnotically pumped through the channel, there is always at least one second or low ionic concentration spacer region 202 between the points A and B. This ensures ;WO 98/00705 PCT/US97/10963 ;14 ;that most of the voltage drop occurs in the second spacer region 202, rather than across the subject material region 200 and first spacer regions 201. Stated differently, the electric field between points A and B is concentrated in the 5 second spacer region 202 and the subject material regions 200 and first spacer regions 201 experience low electric fields (and low electrophoretic forces). Thus, depending upon the relative ionic concentrations in the subject material regions ;200, first spacer regions 201 and second or low ionic ;10 concentration spacer regions 202, other arrangements of these subject material regiohs-200, and first and second spacer-regions 201 and 202 can be made. ;For example, Figure 2B illustrates an arrangement in which a second or low ionic concentration spacer region 202 is 15 regularly spaced between each combination of first spacer region 201/subject material region 200/first spacer region ;201. Such an arrangement ensures that there is always at least one second or low concentration spacer region 202 between points A and B. Furthermore, the drawings are drawn ;20 to scale to illustrate the relative lengths of a possible combination of subject material region 200, first or high concentration spacer region 201 and second or low concentration spacer region 202. In the example of Figure 2B, the subject material region 200 holds the subject material in 25 a high ionic concentration of 150 mM of NaCl. The subject material region 200 is 1 mm long in the channel. The two first spacer regions 201 have ionic concentrations of 150 mM of NaCl. Each first spacer region 201 is 1 mm long. The second spacer region 202 is 2 mm and has an ionic 30 concentration of 5 mM of borate buffer. This particular configuration is designed to maintain a rapidly electrophoresing compound in the subject material region 200 and buffer regions 201 while the compound travels through the channels of the microfluidic system. For example, using these 35 methods, a subject material region containing, e.g., benzoic acid, can be flowed through a microfluidic system for upwards of 72 seconds without suffering excessive electrophoretic bias. ;WO 98/00705 PCT/US97/10963 ;15 ;Stated more generally, the velocity of fluid flow, veof/ through the channels of the microfluidic system can be determined and, by measurement, it is possible to determine the total distance, 1T, which a subject matter molecule is to 5 travel through the channels. Thus the transit time, tTr, for the subject matter molecule to travel the total distance is: ;tTr = 1t/VEof ;To contain a subject matter molecule x within the first spacer region 201 next to the subject material region 200, the length 10 of the first spacer region 201, lq, should be greater than the electrophoretic velocity of the subject matter molecule x in the first spacer region 201, vgx, multiplied by the transit time: ;It > (vqx) (tTr)" ;15 Since the electrophoretic velocity is proportional to the electric field in the first spacer region 201, the present invention allows control over vgx so that the subject materials can be contained in transport through the microfluidic system channels. ;20 In the arrangements in Figures 2A and 2B, the first or high ionic concentration spacer regions 201 help maintain the position of the subject materials m the vicinity of its subject material region 200. No matter what the polarity of the charges of the subject material, the first spacer regions 25 201 on either side of the subject material region 200 ensures ^ that any subject material leaving the subject material region ;200 is only subject to a low electric field due to the relative high ionic concentrations in the first spacer regions 201. If the polarity of the subject material is known, then ;3 0 the direction of the electrophoretic force on the molecules of the subject material is also known. ;Figure 3A illustrates an example where charges of the subject material in all the subject material regions 200 are such that the electrophoretic force on the subject 35 material molecules are in the same direction as the direction of the electroosmotic flow. Hence the first spacer regions ;201 precede the subject material regions 200 in the direction of flow. There are no first spacer regions 201 following the ;WO 98/00705 PCT/US97/10963 ;16 ;subject material regions 200 because the electrophoretic force keeps the subject material from escaping the subject material region 200 in that direction. By eliminating one-half of the first spacer regions 201, more subject material regions 200 5 with their subject material can be carried per channel length. This enhances the transportation efficiency of the microfluidic system. The second or low ionic concentration spacer regions 2 02 are arranged with respect to the subject material regions 200 and the first or high ionic concentration 10 spacer regions 201 so that high electric fields fall in the second spacer regions 202 and the electric fields (and _ electrophoretic forces) in the subject material regions 200 and first spacer regions 201 are kept low. ;In Figure 3B the first spacer regions 201 follow 15 the subject material regions 200 in the direction of the electroosmotic flow. In this example, the charges of the subject material in all the subject material regions 200 are such that the electrophoretic force on the subject matter molecules are in the opposite direction as the direction of 20 the electroosmotic flow. Hence the subject material may escape the confines of its subject material region, in effect, being left behind by its subject material region 200. The first spacer regions 201 following the subject material regions 200 keep the subject material from migrating too far 25 from its subject material region 200. Likewise, the second or low ionic concentration spacer regions 202 are arranged with the subject material regions 200 and the first or high ionic concentration spacer regions 201 so that high electric fields fall in the second spacer regions 202 and the electric fields 30 in the subject material regions 200 and first spacer regions 2 01 are kept low. ;Various high and low ionic strength solutions are selected to produce a solution having a desired electrical conductivity for the first and second spacer regions 2 01 and 35 202. The specific ions that impart electrical conductivity to the solution maybe derived from inorganic salts (such as NaCl, KI, CaCl2, FeF3/ (NH4)2S04 and so forth), organic salts (such as pyridimum benzoate, benzalkonium laurate) , or mixed ;WO 98/00705 PCT/US97/10963 ;17 ;inorganic/organic salts (such as sodium benzoate, sodium deoxylsulfate, benzylaminehydrochloride). These ions are also selected to be compatible with the chemical reactions, separations, etc. to be carried out in the microfluidic 5 system. In addition to aqueous solvents, mixtures of aqueous/organic solvents, such as low concentrations of DMSO in water, may be used to assist in the solubilization of the subject matter molecules. Mixtures of organic solvents, such as CHCl3:MeOH, may be also used for the purpose of 10 accelerating assays for phospholipase activity, for example. ;Generally, when aqueous solvents are used, solution conductivity is adjusted using inorganic ions. When les^ ;polar solvents are used, organic or mixed inorganic/organic ions are typically used. In cases where two immiscible 15 solvents may be simultaneously present (e.g., water and a hydrocarbon such as decane) such that electrical current must flow from one into the other, ionophores (e.g., valinomycin, nonactin, various crown ethers, etc.) and their appropriate ions may be used to conduct current through the non-polar 20 solvent. ;B. Electrokinetic Control of Pressure Based Flow ;In the electrokinetic flow systems described herein, the presence of differentially mobile fluids (e.g., having a different electrokinetic mobility in the particular system) in 25 a channel may result in multiple different pressures being present along the length of a channel in the system. For example, these electrokinetic flow systems typically employ a series of regions of low and high ionic concentration fluids (e.g., first and second spacer regions and subject material 30 regions of subject material) in a given channel to effect electroosmotic flow, while at the same time, preventing effects of electrophoretic bias within a subject material containing subject material region. As the low ionic concentration regions within the channel tend to drop the most 35 applied voltage across their length, they will tend to push the fluids through a channel. Conversely, high ionic concentration fluid regions within the channel provide ;WO 98/00705 PCT/US97/10963 ;18 ;relatively little voltage drop across their lengths, and tend to slow down fluid flow due to viscous drag. ;As a result of these pushing and dragging effects, pressure variations can generally be produced along the length of a fluid filled channel. The highest pressure is typically found at the front or leading edge of the low ionic concentration regions (e.g., the second spacer regions), while the lowest pressure is typically found at the trailing or back edge of these low ionic strength fluid regions. ;While these pressure differentials are largely irrelevant in straight "channel systems, their effects can result in reduced control over fluid direction and manipulation in microfluidic devices that employ intersecting channel arrangements, i.e., the systems described m US 5,942,443, previously incorporated by reference. For example, where a second channel is configured to intersect a first channel which contains fluid regions of varying ionic strength, the pressure fluctuations described above can cause fluid to flow in and out of the intersecting second channel as these different fluid regions move past the intersection. This fluctuating flow could potentially, significantly disturb the quantitative electroosmotically driven flow of fluids from the second channel, and/or perturb the various fluid regions within the channel. ;By reducing the depth of the intersecting channel, e.g., the second channel, relative to the first or main channel, the fluctuations in fluid flow can be substantially eliminated. In particular, in electroosmotic fluid propulsion or direction, for a given voltage gradient, the rate of flow (volume/time) generally varies as the reciprocal of the depth of the channel for channels having an aspect ratio of >10 (width:depth). With some minor, inconsequential error for the calculation, this general ratio also holds true for lower aspect ratios, e.g., aspect ratios >5. Conversely, the pressure induced flow for the same channel will vary as the third power of the reciprocal of the channel depth. Thus, the pressure build-up in a channel due to the simultaneous ;WO 98/00705 PCT/US97/10963 ;19 ;presence of fluid regions of differing ionic strength will vary as the square of the reciprocal of the channel depth. ;Accordingly, by decreasing the depth of the intersecting second channel relative to the depth of the first 5 or main channel by a factor of X, one can significantly reduce the pressure induced flow, e.g., by a factor of X3, while only slightly reducing the electroosmotically induced flow, e.g., by a factor of X. For example, where the second channel is reduced in depth relative to the first channel by one order of 10 magnitude, the pressure induced flow will be reduced 1000 times while the electroosmotically induced flow will be -reduced by only a factor of ten. Accordingly, in some aspects, the present invention provides microfluidic devices as generally described herein, e.g., having at least first and 15 second intersecting channels disposed therein, but where the first channel is deeper than the second channel. Generally, the depths of the channels may be varied to obtain optimal flow conditions for a desired application. As such, depending upon the application, the first channel may be greater than ;2 0 about two times as deep as the second channel, greater than about 5 times as deep as the second channel, and even greater than about ten times as deep as the second channel. ;In addition to their use in mitigating pressure effects, varied channel depths may also be used to 25 differentially flow fluids within different channels of the ^ same device, e.g., to mix different proportions of fluids from different sources, and the like. ;Ill. Electropipettor ;3 0 As described above, any subject material can be transported efficiently through the microfluidic system 100 in or near the subject material regions 200. With the first and second spacer regions 201 and 202, the subject materials are localized as they travel through the channels of the system. 35 For efficient introduction of subject matter into a microfluidic system, the present invention also provides an electropipettor which introduces subject material into a microfluidic system in the same serial stream of combinations ;WO 98/00705 PCT/US97/10963 ;20 ;of subject material region 200, first and second spacer regions 201 and 202. ;A. Structure and Operation ;As illustrated in Figure 4A, an electropipettor 250 is formed by a hollow capillary tube 251. The capillary tube ;251 has a channel 254 with the dimensions of the channels of the microfluidic system 100 to which the channel 254 is fluidly connected. As shown in Figure 4A, the channel 254 is a cylinder having a cross-sectional diameter in the range of 1-100 ixm, with a diameter of approximately 3 0 jum being preferable. An electrode 252 runs down the outside wall of the capillary tube 251 and terminates in a ring electrode 253 around the end of the tube 251. To draw the subject materials in the subject material regions 200 with the buffer regions 201 and 202 into the electropipettor channel 254, the electrode 252 is driven to a voltage with respect to the voltage of a target reservoir (not shown) which is fluidly connected to the channel 254. The target reservoir is in the microfluidic system 100 so that the subject material regions 200 and the buffer regions 201 and 202 already m the channel 254 are transported serially from the electropipettor into the system 100. ;Procedurally, the capillary channel end of the electropipettor 250 is placed into a source of subject material. A voltage is applied to the electrode 252 with respect to an electrode in the target reservoir. The ring electrode 253, being placed in contact with the subject material source, electrically biases the source to create a voltage drop between the subject material source and the target reservoir. In effect, the subject material source and the target reservoir become Point A and B in a microfluidic system, i.e., as shown in Figure 2A. The subject material is elecrrokinetically introduced into the capillary channel 254 to create a subject material region 200. The voltage on the electrode 252 is then turned off and the capillary channel end is placed into a source of buffer material of high ionic concentration. A voltage is again applied to the electrode ;252 with respect to the target reservoir electrode such that ;WO 98/00705 PCT/US97/10963 ;21 ;the first spacer region 201 is electrokinetically introduced into the capillary channel 2 54 next to the subject material region 200. If a second or low ionic concentration spacer region 202 is then desirable in the electropipettor channel 5 254, the end of the capillary channel 254 is inserted into a source of low ionic concentration buffer material and a voltage applied to the electrode 252. The electropipettor 250 can then move to another source of subject material to create another subject material region 200 in the channel 254. 10 By repeating the steps above, a plurality of subject material regions 200 with different subject materials, which are separated by first and second spacer regions 201 and„202, are electrokinetically introduced into the capillary channel 254 and into the microfluidic system 100. ;15 Note that if the sources of the subject material and the buffer materials (of low and high ionic concentration) ;have their own electrode, the electrode 252 is not required. Voltages between the target reservoir and the source electrodes operate the electropipettor. Alternatively, the 20 electrode 252 might be in fixed relationship with, but separated from, the capillary tube 251 so that when the end of the tube 251 contacts a reservoir, the electrode 252 also contacts the reservoir. Operation is the same as that described for the Fig. 4A electropipettor. 25 Figure 4B illustrates a variation of the electropipettor 250 of Figure 4A. In this variation the electropipettor 27 0 is not required to travel between a subject material source and buffer material sources to create the first and second spacer regions 201 and 202 within the 30 pipettor. The electropipettor 270 has a body 271 with three capillary channels 274, 275 and 276. The main channel 274 operates identically to the channel 254 of the previously described electropipettor 250. However, the two auxiliary capillary channels 275 and 276 at one end are fluidly 3 5 connected to buffer source reservoirs (not shown) and the other end of the channels 275 and 276 is fluidly connected to the main channel 27 4. One reservoir (i.e., connected to auxiliary channel 275) holds buffer material of high ionic ;WO 98/00705 PCT/US97/10963 ;22 ;concentration, and the other reservoir (i.e., connected to the channel 276) holds buffer material of low ionic concentration. ;All of the reservoirs are connected to electrodes for electrically biasing these reservoirs for operation of the 5 electropipettor 270. The electropipettor 270 may also have an electrode 272 along the walls of its body 271 which terminates in a ring electrode 273 at the end of the main channel 274. By applying voltages on the electrode 272 (and ring electrode 273) to create voltage drops along the channels 274, 275, 276, 10 not only can subject material be drawn into the main channel ;274 from subject material sources, but buffer material of high and low ionic concentrations can also be drawn from the „ auxiliary channels 275 and 276 into the main channel 274. ;To operate the electropipettor 270 with the 15 electrode 272, the end of the main capillary channel 274 is placed into a source 280 of subject material. A voltage is applied to the electrode 272 with respect to an electrode in the target reservoir to create a voltage drop between the subject material source 280 and the target reservoir. The 20 subject material is electrokmetically drawn into the capillary channel 274. The capillary channel end is then removed from the subject material source 28 0 and a voltage drop is created between the target reservoir connected to the channel 274 and the reservoir connected to the channel 275. A 25 first or high ionic strength spacer region 201 is formed in the channel 274. Capillary action inhibits the introduction of air into the channel 274 as the buffer material is drawn from the auxiliary channel 275. If a second or low ionic concentration spacer region 202 is then desired in the main 30 channel 274, a voltage is applied to the electrodes in the target reservoir and in the reservoir of low ionic concentration buffer material. A second spacer region 202 is electrokinetically introduced into the capillary channel 274 from the second auxiliary channel 276. The electropipettor 3 5 27 0 can then move to another source of subject material to create another subject material region 200 in the channel 274. ;By repeating the steps above, a plurality of subject material regions 200 with different subject materials, which ;WO 98/00705 PCT/US97/10963 ;23 ;are separated by first and second spacer regions 201 and 202, are electrokinetically introduced into the capillary channel 274 and into the microfluidic system 100. ;If it is undesirable to expose the subject material 5 source to oxidation/reduction reactions from the ring electrode 273, the electropipettor may be operated without the electrode 272. Because electroosmotic flow is slower in solutions of higher ionic strength, the application of a potential (- to +) from the reservoir connecting the channel 10 274 to the reservoir connecting the channel 275 results in the formation of a vacuum kt the point where the channels 27A and 27 5 intersect. This vacuum draws samples from the subject material source into the channel 274. When operated in this mode, the subject material is somewhat diluted with the 15 solutions in the channels 275 and 276. This dilution can be mitigated by reducing the relative dimensions of the channels 276 and 275 with respect to the channel 274. ;To introduce first and second spacer regions 201 and 202 into the capillary channel 274, the electropipettor 270 is 2 0 operated as described above. The capillary channel end is removed from the subject material source 280 and a voltage drop is created between the target reservoir for the channel 274 and the reservoir connected to the selected channels 275 or 276. ;2 5 Although generally described in terms of having two auxiliary channels and a main channel, it will be appreciated that additional auxiliary channels may also be provided to introduce additional fluids, buffers, diluents, reagents and the like, into the main channel. ;3 0 As described above for intersecting channels within a microfluidic device, e.g., chip, pressure differentials resulting from differentially mobile fluids within the different pipettor channels also can affect the control of fluid flow within the pipettor channel. Accordingly, as 3 5 described above, the various pipettor channels may also be provided having varied channel depths relative to each other, in order to optimize fluid control. ;B. Method of Electropipettnr Manufacture ;WO 98/00705 PCT/US97/10963 ;24 ;The electropipettor might be created from a hollow capillary tube, such as described with respect to Fig. 4A. For more complex structures, however, the electropipettor is optimally formed from the same substrate material as that of 5 the microchannel system discussed above. The electropipettor channels (and reservoirs) are formed in the substrate in the same manner as the microchannels for a microfluidic system and the channeled substrate is covered by a planar cover element, also described above. The edges of the substrate and cover 10 element may then be shaped to the proper horizontal dimensions of the pipettor, especially its end, as required. Techniques, such as etching, air abrasion (blasting a surface with particles and forced air), grinding and cutting, may be'used. Electrodes are then created on the surface of the substrate 15 and possibly cover, as required. Alternatively, the edges of the substrate and the cover element may be shaped prior to being attached together. This method of manufacture is particularly suited for multichannel electropipettors, such as described immediately above with respect to Fig. 4B and 20 described below with respect to Fig. 8. ;IV. Sampling Svstem ;As described above, the methods, systems and apparatuses described above will generally find widespread 25 applicability m a variety of disciplines. For example, as noted previously, these methods and systems may be particularly well suited to the task of high throughput chemical screening in, e.g., drug discovery applications, such as is described m copending US 5,942,443 and previously 30 incorporated by reference. ;A. Sample Matrices ;The pipetting and fluid transport systems of the invention are generally described in terms of sampling numbers 35 of liquid samples, i.e., from multi-well plates. In many instances, however, the number or nature of the liquid based samples to be sampled may generate sample handling problems. For example, in chemical screening or drug discovery ;WO 98/00705 PCT/US97/10963 ;25 ;applications, libraries of compounds for screening may number in the thousands or even the hundreds of thousands. As a result, such libraries would require extremely large numbers of sample plates, which, even with the aid of robotic systems, 5 would create myriad difficulties in sample storage, ;manipulation and identification. Further, in some cases, specific sample compounds may degrade, complex or otherwise possess relatively short active half-lives when stored in liquid form. This can potentially result in suspect results 10 where samples are stored in liquid form for long periods prior to screening. ;Accordingly, embodiments provide sampling systems which address these further problems, by providing the compounds to be sampled in an immobilized format. By 15 "immobilized format" is meant that the sample material is provided in a fixed position, either by incorporation within a fixed matrix, i.e., porous matrix, charged matrix, hydrophobic or hydrophilic matrix, which maintains the sample in a given location. Alternatively, such immobilized samples include 20 samples spotted and dried upon a given sample matrix. ;In preferred aspects, the compounds to be screened are provided on a sample matrix in dried form. Typically, such sample matrices will include any of a number of materials that can be used in the spotting or immobilization of materials, 25 including, e.g., membranes, such as cellulose, nitrocellulose, PVDF, nylon, polysulfone and the like. Typically, flexible sample matrices are preferred, to permit folding or rolling of the sample matrices which have large numbers of different sample compounds immobilized thereon, for easy storage and 3 0 handling. ;Generally, samples may be applied to the sample matrix by any of a number of well known methods. For example, sample libraries may be spotted on sheets of a sample matrix using robotic pipetting systems which allow for spotting of 35 large numbers of compounds. Alternatively, the sample matrix may be treated to provide predefined areas for sample localization, e.g., indented wells, or hydrophilic regions surrounded by hydrophobic barriers, or hydrophobic regions ;WO 98/00705 ;26 ;PCT/US97/10963 ;surrounded by hydrophilic barriers (e.g., where samples are originally in a hydrophobic solution), where spotted materials will be retained during the drying process. Such treatments then allow the use of more advanced sample application 5 methods, such as those described in U.S. Patent No. 5,474,796, wherein a piezoelectric pump and nozzle system is used to direct liquid samples to a surface. Generally, however, the methods described in the '796 patent are concerned with the application of liquid samples on a surface for subsequent 10 reaction with additional liquid samples. However, these methods could be readily modified to provide dry spotted samples on a substrate. ;Other immobilization or spotting methods may be similarly employed. For example, where samples are stable in 15 liquid form, sample matrices may include a porous layer, gel or other polymer material which retain a liquid sample without allowing excess diffusion, evaporation or the like, but permit withdrawal of at least a portion of the sample material, as desired. In order to draw a sample into the pipettor, the 20 pipettor will free a portion of the sample from the matrix, ;e.g., by dissolving the matrix, ion exchange, dilution of the sample, and the like. ;B. ResoTubilizina Pipettor ;As noted, the sampling and fluid transport methods 25 and systems of the present invention are readily applicable to screening, assaying or otherwise processing samples immobilized in these sample formats. For example, where sample materials are provided in a dried form on a sample matrix, the electropipetting system may be applied to the 30 surface of the matrix. The electropipettor is then operated to expel a small volume of liquid which solubilizes the previously dried sample on the matrix surface (dissolves a retaining matrix, or elutes a sample from an immobilizing support), e.g., by reversing the polarity of the field applied 35 to the pipettor, or by applying a potential from the low ionic concentration buffer reservoir to the high ionic concentration buffer reservoir, as described above. Once the sample is resolubilized, the pipettor is then operated in its typical ;WO 98/00705 PCT/US97/10963 ;27 ;forward format to draw the solubilized sample into the pipettor channel as previously described. ;A schematic illustration of one embodiment of an electropipettor useful in performing this function, and its 5 operation, is shown in Figure 8. Briefly, the top end 8 02 of the pipettor (as shown) 800 is generally connected to the assay system, e.g., a microfluidic chip, such that voltages can be supplied independently to the three channels of the pipettor 804, 806 and 808. Channels 804 and 808 are typically 10 fluidly connected to buffer reservoirs containing low and high ionic concentration fluids, respectively. In operation-, the tip of the pipettor 810 is contacted to the surface of a sample matrix 812 where an immobilized(e.g., dried) sample 814 is located. A voltage is applied from the low ionic 15 concentration buffer channel 804 to the high ionic concentration buffer channel 808, such that buffer is forced out of the end of the pipettor tip to contact and dissolve the sample. As shown, the pipettor tip 816 may include a recessed region or "sample cup" 818 in order to maintain the expelled 20 solution between the pipettor tip and the matrix surface. In some cases, e.g., where organic samples are being screened, in order to ensure dissolution of the sample, an appropriate concentration of an acceptable solvent, e.g., DMSO, may be included with the low ionic concentration buffer. Voltage is 25 then applied from the high ionic concentration buffer channel to the sample channel 8 06 to draw the sample into the pipettor in the form of a sample plug 82 0. Once the sample is completely withdrawn from the sample cup into the pipettor, the high surface tension resulting from air entering the 3 0 sample channel will terminate aspiration of the sample, and high ionic concentration buffer solution will begin flowing into the sample channel to form a first spacer region 8 22, following the sample. Low ionic concentration buffer solution may then be injected into the sample channel, i.e., as a 35 second spacer region 824, by applying the voltage from the low ionic concentration buffer channel 804 to the sample channel 806. Prior to or during presentation of the next sample position on the matrix, a first or high ionic concentration ;WO 98/00705 PCT/US97/10963 ;28 ;spacer region 822 may be introduced into the sample channel by applying the voltage between the high ionic concentration buffer channel and the sample channel. As noted previously, a roll, sheet, plate, or numerous rolls, sheets or plates, of 5 sample matrix having thousands or hundreds of thousands of different compounds to be screened, may be presented in this manner, allowing their serial screening in an appropriate apparatus or system. ;10 V. Elimination of Electrophoretic Bias ;As explained* above, electrokinetic forces are used to transport the subject material through the channels of the microfluidic system 100. If the subject material is charged in solution, then it is subject to not only electroosmotic 15 forces, but also to electrophoretic'forces. Thus the subject material is likely to have undergone electrophoresis in traveling from one point to another point along a channel of the microfluidic system. Hence the mixture of the subject material or the localization of differently charged species in 20 a subject material region 200 at the starting point is likely to be different than the mixture or localization at the arrival point. Furthermore, there is the possibility that the subject material might not even be in the subject material region 2 00 at the arrival point, despite the first spacer 25 regions 201.

Therefore, another embodiment compensates for electrophoretic bias as the subject materials are transported through the microfluidic system 100. One way to compensate for electrophoretic bias is illustrated in 30 Figure 5. In the microfluidic system 100 described above, each of the channels 110, 112, 114 and 116 was considered as a unitary structure along its length. In Figure 5, an exemplary channel 140 is divided into two portions 142 and 144. The sidewalls of each channel portion 142 and 144 have surface 3 5 charges which are of opposite polarity. The two channel portions 142 and 144 are physically connected together by a salt bridge 133, such as a glass frit or gel layer. While the salt bridge 133 separates the fluids in the channel 140 from WO 98/00705 PCT/US97/10963 29 an ionic fluid in a reservoir 135 which is partially defined by the salt bridge 13 3, the salt bridge 13 3 permits ions to pass through. Hence the reservoir 135 is in electrical, but not fluid, communication with the channel 140.

To impart electroosmotic and electrophoretic forces along the channel 140 between points A and B, electrodes 132 and 134 are arranged at points A and B respectively. Additionally, a third electrode 13 7 is placed in the reservoir 135 at the junction of the two portions 142 and 144. The 10 electrodes 13 2 and 134 are maintained at the same voltage and the electrode 137 at a'nother voltage. In the example -illustrated in Figure 5, the two electrodes 132 and 134'are at a negative voltage, while the electrode 13 7 and hence the junction of the two portions 142 and 144 are at zero voltage, 15 i.e., ground. Thus the voltage drops, and hence the electric fields, in the portions 142 and 144 are directed in opposite directions. Specifically, the electric fields point away from each other. Thus the electrophoretic force on a particularly charged molecule is in one direction in the channel portion 20 142 and in the opposite direction in the channel portion 144. Any electrophoretic bias on a subject material is compensated for after traveling through the two portions 142 and 144.

The electroosmotic force in both portions 142 and 144 are still in the same direction, however. For example, 25 assuming that the sidewalls of the channel portion 142 have positive surface charges, which attract negative ions in solution, and the sidewalls of the channel portion 144 have negative surface charges, which attract positive ions in solution, as shown in Figure 5, the electroosmotic force in 30 both portions 142 and 144 is to the right of the drawing.

Thus the subject material is transported from point A to point B under electroosmotic force, while the electrophoretic force is in one direction in one portion 142 and in the opposite direction in the other portion 144.

To create a channel with sidewalls having positive or negative surface charges, one or both portions of the channel is coated with insulating film materials with surface charges, such as a polymer. For example, in the microfluidic WO 98/00705 PCT/US97/10963 system 100 the substrate 102 and the channels may be formed of glass. A portion of each channel is coated with a polymer of opposite surface charge, such as polylysine, for example, or is chemically modified with a silanizing agent containing an 5 amino function, such as aminopropyltrichlorosilane, for example. Furthermore, the surface charge densities and volumes of both channel portions should be approximately the same to compensate for electrophoretic bias.

Rather than being formed in a solid planar 10 substrate, the channel can also be formed by two capillary tubes which are butted together with a salt bridge which separates an ionic" fluid reservoir from fluids in the capillary tubes. An electrode is also placed in the ionic fluid reservoir. One capillary tube has a negative surface 15 charge and the other capillary tube a positive surface charge. The resulting capillary channel operates as described above.

Figures 6A-6D illustrate another embodiment for which the effects of electrophoretic bias induced upon the subject material is moving from point A 20 to point B are compensated. In this embodiment the subject material is mixed at point B, an intersection between two channels, such as illustrated in Figure 1.

Figures 6A-6D show a chamber 160 is formed at the intersection of the channels 150, 152, 154 and 156. The 25 chamber 160 has four sidewalls 162, 164, 166 and 168. The sidewall 162 connects a sidewall of the channel 152 to a sidewall of the channel 150; the sidewall 164 connects a sidewall of the channel 154 to the other sidewall of the channel 152; the sidewall 166 connects a sidewall of the 30 channel 156 to the other sidewall of the channel 154; and the sidewall 168 connects the opposite sidewall of the channel 156 to the opposite sidewall of the channel 150. Assuming a flow of materials through the channel 152 toward the channel 156, the sidewalls 162 and 168 form as a funnel if the materials 3 5 are diverted into the channel 150.

The dimensions of the sidewalls 162 and 168 accommodate the length of a subject material plug 2 00 traveling along the channel 152. The sidewalls 162 and 168 WO 98/00705 PCT/US97/10963 31 funnel down the plug 2 00 into the width of the channel 150. The width of the channel 150 is such that diffusion of the subject material occurs across the width of the channel 150, i.e., mixing occurs and any electrophoretic bias of the 5 subject material created in the subject material region 200 traveling along the channel 162 is eliminated. For example, if the channel 150 is 50 wide, diffusion across the channel occurs in approximately one second for a molecule having a diffusion constant of 1 x 10"5 cm2/sec.

In Figure 6A a plug 200 of cationic subject material is moving along the channel 152 towards the channel 156, By the time the plug 200 reaches the chamber 160, the subject material has undergone electrophoresis so that the material is more concentrated at the forward end of the subject material 15 region 200. This is illustrated by Figure 6B. Then the voltage drop impressed along the channels 152 and 156 is terminated and a voltage drop is created along the channels 154 and 150 to draw the subject material region 2 00 into the channel 150. The sidewalls 162 and 168 of the chamber 160 2 0 funnel the subject material region 2 00 with its electrophoretically biased subject material. This is illustrated by Figure 6C. By diffusion the subject material is spread across the width of the channel 150 before the subject material travels any significant distance along the 25 channel 150; the subject material in the subject material region 200 is mixed and ready for the next step of operation in the microfluidic system 100.

In addition to its use in correcting electrophoretic bias within a single sample, it will be appreciated that the 30 structure shown in Fig. 6 will be useful in mixing fluid elements within these microfluidic devices, e.g., two distinct subject materials, buffers, reagents, etc.

EXAMPLES Example l- Forced Co-Migration of Differentially Charged Species, in Electropjpettor-Tvpe Format In order to demonstrate the efficacy of methods used to eliminate or reduce electrophoretic bias, two oppositely WO 98/00705 PCT/US97/10963 32 charged species were electrokinetically pumped in a capillary channel, and comigrated in a single sample plug. A Beckman Capillary Electrophoresis system was used to model the electrophoretic forces in a capillary channel.

In brief, a sample containing benzylamine and benzoic acid in either low ionic concentration (or "low salt") (5 mM borate), or high ionic concentration ("high salt") (500 mM borate) buffer, pH 8.6, was used in this experiment. The benzoic acid was present at approximately 2X the concentration 10 of the benzylamine. All injections were timed for 0.17 minutes. Injection plug length was determined by the -injection voltage," 8 or 30 kV. The low salt and high salt buffers were as described above.

In a first experiment, three successive injections 15 of sample in low salt buffer were introduced into a capillary filled with low salt buffer. Injections were performed at 8 kV and they were spaced by low salt injections at 3 0 kV. Data from these injections is shown in Figure 7A. These data show that benzylamine (identifiable as short peaks, resulting from 20 its lesser concentration) from the first and second injections precede the benzoic acid peak (tall peak) from the first injection. Further, the benzylamine peak from the third injection is nearly coincident with the first benzoic acid peak. Thus, this experiment illustrates the effects of 25 electrophoretic bias, wherein sample peaks may not exit a capillary channel in the same order they entered the channel. As can be clearly seen, such separations can substantially interfere with characterization of a single sample, or worse, compromise previously or subsequently introduced samples. 30 In a second experiment, the capillary was filled with low salt buffer. Samples were injected by first introducing/injecting high salt buffer into the capillary at 8 kV (first spacer region 1). This was followed by injection of the sample in high salt buffer at 8 kV, which was followed by 3 5 a second injection of high salt buffer at 8 kV (first spacer region 2) . Three samples were injected in this manner, and spaced apart by injecting a low salt buffer at 30 kV. As can be seen in Figure 7B, both compounds contained in the sample WO 98/00705 PCT/US97/10963 33 were forced to co-migrate through the capillary channel in the same sample plug, and are represented by a single peak from each injection. This demonstrates sample registration, irrespective of electrophoretic mobility.

By reducing the size of the low salt spacer plug between the samples, relative to the size of the samples, partial resolution of the components of each sample injection can be accomplished. This may be useful where some separation of a sample is desired during electrokinetic pumping, but 10 without compromising subsequently or previously injected samples. This was carried out by injecting the low-sal-t spacer plug at 8 kV rather than 3 0 kV. The data from this example is shown in Figure 7C.

Example 2 - Migration of Subject Materials through 15 Electropipettor into Microfluidic System Substrate Figs. 9A-9C illustrate the experimental test results of the introduction of a subject material, i.e., a sample, into a microfluidic system substrate through an electropipettor as described above. The sample is rhodamine B 20 in a phosphate buffered saline solution, pH 7.4. A high ionic concentration ("high salt") buffer was also formed from the phosphate buffered saline solution, pH 7.4. A low ionic concentration ("low salt") buffer was formed from 5 mM Na borate, pH 8.6, solution.

In the tests subject material regions containing the fluorescent rhodamine B were periodically injected into the capillary channel of an electropipettor which is joined to a microfluidic system substrate. High salt and low salt buffers were also injected between the subject material regions, as 3 0 described previously. Fig. 9A is a plot of the fluorescence intensity of the rhodamine B monitored at a point along the capillary channel near its junction with a channel of the substrate versus time. (In passing, it should be observed that the numbers on fluorescent intensity axis of the Figs. 3 5 9A-9C and 10 plots are for purposes of comparative reference, rather than absolute values.) The injection cycle time was 7 seconds and the electric field to move the subject material regions through the electropipettor was 1000 volts/cm. The WO 98/00705 PCT/US97/10963 34 integration time for the photodiode monitoring light from the capillary channel was set at 10 msec. From the Fig. 9A plot, it should be readily evident that light intensity spikes appear at 7 second intervals, matching the injection cycle 5 time of the fluorescent rhodamine B.

In another experiment, the same buffers were used with the rhodamine B samples. The monitoring point was in the channel of the substrate connected to the electropipettor. The injection cycle time was set at 13.1 seconds and the 10 voltage between the source reservoir containing the rhodamine B and destination reservoir in the substrate set at -3000 volts. The monitoring photodiode integration time was 400 msec. As shown in Fig. 9B, the fluorescent intensity spikes closely match the rhodamine B injection cycle time. 15 The results of a third experimental test are illustrated in Fig. 9C. In this experiment the electropipettor was formed from a substrate and shaped by air abrasion. The monitoring point is along the capillary channel formed in the substrate (and a planar cover). Here the sample 20 material is 100 nM of rhodamine B in a buffer of PBS, pH 7.4. The high salt buffer solution of PBS, pH 7.4, and a low salt buffer solution of 5 mM Na borate, pH 8.6, are also used.

Again periodic spikes of fluorescent intensity match the cyclical injection of rhodamine B into the electropipettor. 25 Fig. 10 illustrates the results of the cyclical injection of another subject material into an electropipettor, according to one embodiment. In this experiment the sample was a small molecule compound of 100 /uM with 1% DMSO in a phosphate buffered saline solution, pH 7.4. A high salt 30 buffer of the same phosphate buffered saline solution, pH 7.4, and a low salt buffer of 5 mM Na borate, pH 8.6, was also used. The applied voltage to move the subject material regions through the electropipettor was -4000 volts, and the integration time for the photodiode monitoring light from the 3 5 capillary channel was set at 400 msec. The samples were periodically injected into the electropipettor as described previously. As in the previous results, the Fig. 10 plot illustrates that for small molecule compounds, the WO 98/00705 PCT/US97/10963 electropipettor moves the samples at evenly spaced time (and space) intervals.

While the foregoing inventions have been "described "in some detail for purposes of clarity and understanding, it will 5 be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the present invention. For example, all the techniques described above may be used in various combinations. All publications and 0 patent documents cited m this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were so individually denoted. 36

Claims (13)

WHAT WE CLAIM IS:

1. A microfluidic system for moving a plurality of subject materials from a first location to a second location along a channel, said microfluidic system comprising: a source for creating a voltage difference across said first location and said second location; a plurality of subject material regions in said channel, said subject material regions separated by first spacer regions of high ionic strength; and at least one second spacer region of low ionic concentration.

2. A microfluidic system for transporting a plurality of subject material regions from a first point to a second point on a channel, said microfluidic system comprising: a pair of first spacer regions on either side of each subject material region, said first spacer regions having high ionic concentrations; at least one second spacer region, said second spacer region having a low ionic concentration.

3. The microfluidic system of claim 2, wherein said second spacer region has an ionic concentration at least two orders of magnitude less than that said first spacer region.

4. The microfluidic system of claim 3 wherein said second spacer region has an ionic concentration m the range of 1 to 10 mM, and said first spacer regions have ionic concentrations in the range of 100 to 1000 mM.

5. A method of transporting fluid samples within a microfluidic channel, comprising: 37 introducing at least a plug of a first fluid material having a first ionic strength into said channel; introducing at least a first sample fluid plug into said channel; introducing at least a second fluid material plug having said first ionic strength into said channel; introducing at least a third fluid material plug having a second ionic strength, said second ionic strength being lower than said first ionic strength; and applying a voltage across said channel.

6. The method of claim 5, wherein said steps of introducing said at least first fluid material plug, said at least sample fluid plug, said at least second fluid material plug and said at least third fluid material plug comprise: placing an end of said channel in contact with a source of said at least first fluid material, and applying a voltage from said source of said at least first fluid material to said channel, whereby said first fluid material is introduced into said channel; placing an end of said channel in contact with a source of said at least first sample fluid and applying a voltage from said source of said at least first sample fluid to said channel, whereby said first sample fluid is introduced into said channel; placing an end of said channel in contact with a source of said at least second fluid material and applying a voltage from said source of said at least second fluid material to said channel, whereby said second fluid material is introduced into said channel; and placing an end of said channel m contact with a source of said at least third fluid material and applying a voltage from said source of said at least third fluid 38 material to said channel, whereby said third fluid material is introduced into said channel.

7. A microfluidic system comprising a substrate having a channel, in which at least a first subject material is transported from at least a first location to a second location along said channel, utilizing at least one region of low ionic concentration which is transported along said channel due to an applied voltage.

8. The system of claim 7, in which the ionic concentration of said one region is substantially lower than that of said subject material.

9. A system of claim 7 or claim 8, wherein a plurality of subject materials are transported, separated by high ionic concentration spacer regions.

10. A microfluidic system for moving a plurality of subject materials from a first location to a second location along a channel substantially as hereinbefore described with reference to the accompanying drawings.

11. A microfluidic system for transporting a plurality of subject material regions from a first point to a second point on a channel substantially as hereinbefore described with reference to the accompanying drawings.

12. A method of transporting fluid samples within a microfluid channel substantially as hereinbefore described with reference to the accompanying drawings.

13. A microfluidic system according to any one of claims 7-9 substantially as hereinbefore described with reference to the accompanying drawings.