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BACKGROUND OF THE INVENTION
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 .mu.m to about 500 .mu.m. 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 of microfluidic systems are myriad. For example, International
Patent Appln. WO 96/04547, published Feb. 15, 1996, describes the use of
microfluidic systems for capillary electrophoresis, liquid chromatography,
flow injection analysis, and chemical reaction and synthesis. U.S. patent
appln. Ser. No. 08/671,987, filed Jun. 28, 1996, 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
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, signalling and
other reactions. Biochemical systems of particular interest include, e.g.,
receptor-ligand interactions, enzyme-substrate interactions, cellular
signalling pathways, transport reactions involving model barrier systems
(e.g., cells or membrane fractions) for bioavailability screening, and a
variety of other general 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 mechanical micropumps and valves within the device. See, Published U.K.
Patent Application No. 2 248 891 (Oct. 18, 1990), Published European
Patent Application No. 568 902 (May 2, 1992), U.S. Pat. Nos. 5,271,724
(Aug. 21, 1991) and 5,277,556 (Jul. 3, 1991). See also, U.S. Pat. No.
5,171,132 (Dec. 21, 1990) to Miyazaki et 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. Pat. 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 (Dec. 30, 1988) to Kovacs, Harrison et al.,
Anal. Chem. (1992) 64:1926-1932 and Manz et al. J. Chromatog. (1992)
593:253-258, U.S. Pat. No. 5,126,022 to Soane. Electrokinetic forces have
the advantages of direct control, fast response and simplicity. However,
there are still some 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 in 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 in 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 present invention solves or substantially mitigates these problems of
electrokinetic transport. With the present invention, microfluidic systems
can move materials efficiently and without undesired change in the
transported materials. The present invention presents a high throughput
microfluidic system having direct, fast and straightforward control over
the movement of materials through the channels of the microfluidic system
with a wide range of applications, such as in the fields of chemistry,
biochemistry, biotechnology, molecular biology and numerous other fields.
SUMMARY OF THE INVENTION
The present invention provides for a microfluidic system which
electroosmotically moves subject material along channels in fluid slugs,
also termed "subject material regions," from a first point to a second
point in the microfluidic system. A first spacer region of high ionic
concentration contacts each subject material region on at least one side
and second spacer regions of low ionic concentration are arranged with the
subject material regions of subject material and first or high ionic
concentration spacer regions so that at least one low ionic concentration
region is always between the first and second points to ensure that most
of the voltage drop and resulting electric field between the two points is
across the low ionic concentration region.
The present invention also provides for a electropipettor which is
compatible with a microfluidic system which moves subject materials with
electroosmotic forces. The electropipettor has a capillary having a
channel. An electrode is attached along the outside length of the
capillary and terminates in a electrode ring at the end of the capillary.
By manipulating the voltages on the electrode and the electrode at a
target reservoir to which the channel is fluidly connected when the end of
the capillary is placed into a material source, materials are
electrokinetically introduced into the channel. A train of subject
material regions, high and low ionic concentration buffer or spacer
regions can be created in the channel for easy introduction into the
microfluidic system.
The present invention further compensates for electrophoretic bias as the
subject materials are electrokinetically transported along the channels of
a microfluidic system. In one embodiment a channel between two points of
the microfluidic system has two portions with sidewalls of opposite
surface charges. An electrode is placed between the two portions. With the
voltages at the two points substantially equal and the middle electrode
between the two portions set differently, electrophoretic forces are in
opposite directions in the two portions, while electroosmotic forces are
in the same direction. As subject material is transported from one point
to the other, electrophoretic bias is compensated for, while
electroosmotic forces move the fluid materials through the channel.
In another embodiment a chamber is formed at the intersection of channels
of a microfluidic system. The chamber has sidewalls connecting the
sidewalls of the intersecting channels. When a subject material region is
diverted from one channel into another channel at the intersection, the
chamber sidewalls funnel the subject material region into the second
channel. The width of the second channel is such that diffusion mixes any
subject material which had been electrophoretically biased in the subject
material region as it travelled along the first channel.
In still a further embodiment, the present invention provides a
microfluidic system and method of using that system for controllably
delivering a fluid stream within a microfluidic device having at least two
intersecting channels. The system includes a substrate having the at least
two intersecting channels disposed therein. In this aspect, the one of the
channels is deeper than the other channel. The system also includes an
electroosmotic fluid direction system. The system is particularly useful
where the fluid stream comprises at least two fluid regions having
different ionic strengths.
The present invention also provides a sampling system using the
electropipettor of the invention. The sampling system includes a sample
substrate, which has a plurality of different samples immobilized thereon.
Also included is a translation system for moving the electropipettor
relative to said sample substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic illustration of one embodiment of a microfluidic
system;
FIG. 2A illustrates an arrangement of fluid regions travelling in a channel
of the microfluidic system of FIG. 1, according to one embodiment of the
present invention; FIG. 2B is a scaled drawing of another arrangement of
different fluid regions travelling in a channel of the microfluidic system
according to the present invention;
FIG. 3A is another arrangement with high ionic concentration spacer regions
before a subject material region travelling within a channel of the
microfluidic system; FIG. 3B shows an arrangement with high ionic
concentration spacer regions after a subject material region travelling in
a channel of the microfluidic system;
FIG. 4A is a schematic diagram of one embodiment of a electropipettor
according to the present invention; FIG. 4B is a schematic diagram of
another electropipettor according to the present invention;
FIG. 5 is a schematic diagram of a channel having portions with oppositely
charged sidewalls in a microfluidic system, according to the present
invention; and
FIGS. 6A-6D illustrate the mixing action of funneling sidewalls at the
intersection of channels in a microfluidic system, according to the
present invention.
FIG. 7A shows the results of three injections of a sample fluid made up of
two oppositely charged chemical species in a low salt buffer, into a
capillary filled with low salt buffer. FIG. 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 run in a low salt buffer
filled capillary. FIG. 7C shows the results of three sample injections
similar to that of FIG. 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, without
allowing the sample elements to compromise subsequent or previous samples.
FIG. 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.
DETAILED DESCRIPTION OF THE INVENTION
I. General Organization of a Microfluidic System
FIG. 1 discloses a representative diagram of an exemplary microfluidic
system 100 according to the present invention. As shown, the overall
device 100 is fabricated in a planar substrate 102. Suitable substrate
materials are 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 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 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 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 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
incorporated into the device for these types of detection 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), polyurethane, polyvinylchloride (PVC),
polystyrene, polysulfone, polycarbonate and the like.
The system shown in FIG. 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 "microfluidic," these channels typically have very small
cross sectional dimensions, preferably in the range of from about 0.1
.mu.m to about 100 .mu.m. For the particular applications discussed below,
channels with depths of about 10 .mu.m and widths of about 60 .mu.m work
effectively, though deviations from 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 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. 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 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 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 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 access ports and/or reservoirs for introducing the various fluid
elements needed for a particular screen.
The system 100 shown in FIG. 1 also includes reservoirs 104, 106 and 108,
which are disposed and fluidly connected at the ends of the channels 114,
116 and 110 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 to a source of large numbers of
separate subject materials which are individually introduced into the
sample channel 112 and 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 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 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 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 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 96/04547 to Ramsey, which is
incorporated herein by reference in its entirety for all purposes.
II. Electrokinetic Transport
A. Generally
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 in the system of the present
invention 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 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 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 charged
species in the fluid pulls the solvent with them. The steady state
velocity of this fluid movement is generally given by the equation:
##EQU1##
where v is the solvent velocity, .epsilon. is the dielectric constant of
the fluid, .xi. is the zeta potential of the surface, .EPSILON. is the
electric field strength, and .eta. is the solvent viscosity. Thus, as can
be easily seen from this equation, the solvent 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 of subject materials from one point to another point
in the 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.
In accordance with the present invention, 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 | | |
