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BACKGROUND OF THE INVENTION
There has been a growing interest in the development and manufacturing of microscale fluid systems for the acquisition of chemical and biochemical information, in both preparative and analytical capacities. Adaptation of technologies from the
electronics industry, such as photolithography, wet chemical etching and the like, has helped to fuel this growing interest.
One of the first areas in which microscale fluid systems have been used for chemical or biochemical analysis was in the area of capillary electrophoresis (CE). CE systems generally employ fused silica capillaries, or more recently, etched
channels in planar silica substrates, filled with an appropriate separation matrix or medium. A sample fluid that is to be analyzed is injected at one end of the capillary or channel. Application of a voltage across the capillary then permits the
electrophoretic migration of the species within the sample. Differential electrophoretic mobilities of the constituent elements of a sample fluid, e.g., due to their differential net charge or size, permits their separation, identification and analysis. In order to optimize the separation aspect of the CE applications, researchers have sought to maximize the electrophoretic mobility of charged species relative to each other and relative to the flow of the fluid through the capillary resulting from,
e.g., electroosmosis. See, e.g., U.S. Pat. No. 5,015,350, to Wiktorowicz, and U.S. Pat. No. 5,192,405 to Petersen et al.
In comparison to these CE applications, the technologies of the electronics industry have also been focused on the production of small scale fluidic systems for the transportation of small volumes of fluids over relatively small areas, to perform
one or more preparative or analytical manipulations on that fluid. These non-CE fluidic systems differ from the CE systems in that their goal is not the electrophoretic separation of constituents of a sample or fluid, but is instead directed to the bulk
transport of fluids and the materials contained in those fluids. Typically, these non-CE fluidic systems have relied upon mechanical fluid direction and transport systems, e.g., miniature pumps and valves, to affect material transport from one location
to another. See, e.g., Published PCT Application No. 97/02357. Such mechanical systems, however, can be extremely difficult and expensive to produce, and still fail to provide accurate fluidic control over volumes that are substantially below the
microliter range.
Electroosmotic (E/O) flow systems have been described which provide a substantial improvement over these mechanical systems, see, e.g., Published PCT Application No. WO 96/04547 to Ramsey et al. Typically, such systems function by applying a
voltage across a fluid filled channel, the surface or walls of which have charged or ionizeable functional groups associated therewith, to produce electroosmotic flow of that fluid in the direction of the current. Despite the substantial improvements
offered by these electroosmotic fluid direction systems, there remains ample room for improvement in the application of these technologies. The present invention meets these and other needs.
SUMMARY OF THE INVENTION
The present invention generally provides methods, systems and devices which provide for enhanced transportation and direction of materials using electroosmotic flow of a fluid containing those materials. For example, in a first aspect, the
present invention provides methods of enhancing material direction and transport by electroosmotic flow of a fluid containing that material, which method comprises providing an effective concentration of at least one zwitterionic compound in the fluid
containing the material.
In a related aspect, the present invention also provides methods of reducing electrophoretic separation of differentially charged species in a microscale fluid column, where that fluid column has a voltage applied across it, which method
comprises providing an effective concentration of at least one zwitterionic compound in the fluid.
The present invention also provides microfluidic systems which incorporate these enhanced fluid direction and transport methods, i.e., provide for such enhanced fluid transport and direction within a microscale fluid channel structure. In
particular, these microfluidic systems typically include at least three ports disposed at the termini of at least two intersecting fluid channels capable of supporting electroosmotic flow. Typically, at least one of the intersecting channels has at
least one cross-sectional dimension of from about 0.1 .mu.m to about 500 .mu.m. Each of the ports may include an electrode placed in electrical contact with it, and the system also includes a fluid disposed in the channels, whereby the fluid is in
electrical contact with those electrodes, and wherein the fluid comprises an effective concentration of a zwitterionic compound.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1a-1c are a schematic illustration of the effects of electrophoretic mobility of charged species on the migration of those species in a coherent electroosmotic fluid flow. FIG. 1A illustrates an optimal scenario where differentially
charged chemical species contained in discrete fluid volumes have apparent mobilities that are substantially the same as the electroosmotic flow rate for the fluid. FIG. 1B illustrates the situation wherein the apparent mobility of positively charged
species is greater than the rate of electroosmotic flow and the apparent mobility of negatively charged species is less than or opposite to the rate of electroosmotic flow, resulting in the electrophoretic biasing of the charged species within the
discrete fluid volumes. FIG. 1C illustrates the situation where the apparent mobilities of charged species are substantially different from the rate of electroosmotic flow of the fluid, such that the charged species in the two discrete fluid volumes
overlap.
FIG. 2 is a graph showing the effect of the addition of sulfobetaine on electroosmotic flow and apparent mobility of charged species, under conditions of electroosmotic flow.
FIG. 3 illustrates a microfluidic device used to perform enzyme inhibitor assays.
FIG. 4 illustrates a graphical comparison of enzyme inhibition assays in the presence and absence of a zwitterionic compound, NDSB.
DETAILED DESCRIPTION OF THE INVENTION
I. General
The present invention generally provides methods and systems for the enhanced transportation and direction of materials within fluidic systems, which utilizes the electroosmotic flow of fluids containing those materials. By "enhanced
transportation and direction" is generally meant the electroosmotic flow and direction of fluids within fluidic systems, which shows: (1) a reduction in the electrophoretic mobility of a charged species relative to the electroosmotic flow of the fluid
containing that charged species; and/or (2) an increase in the overall electroosmotic flow of that fluid, relative to such systems not incorporating the present invention, as described herein.
A. Reduction of Electrophoretic Mobility of Charaed Species
As noted previously, in capillary electrophoresis applications, the general goal is to maximize the separation between different species contained in a sample of interest, in order to separately analyze those species, identify their presence
within the sample, or the like. This is accomplished by maximizing the differences in the electrophoretic mobilities of these species, which differences may result from differences in their size and/or net charge.
In the E/O fluid direction systems described herein, however, the goals are somewhat different from those of CE systems. In particular, the general object of these E/O fluid direction systems is the transport and/or direction of material of
interest contained in a volume of fluid or multiple discrete volumes of fluid, from one location in the system to another, using controlled E/O flow. Because these fluids are generally to be subjected to further manipulation or combination with other
fluids, it is generally desirable to affect the transportation of these fluids without substantially altering their make-up, i.e., electrophoretically separating or biasing differentially charged or sized materials contained within those fluids.
Similarly, where these systems are being used to serially transport small volumes of fluids or multiple discrete volumes of different fluids along the same channels, it is generally desirable to transport these fluid volumes as coherently as
possible, i.e., minimizing smearing of materials or diffusion of fluids. In particular, because these systems are preferably utilized in microfluidic applications, the improved coherency of a particular fluid volume within the E/O flow system permits
the transport of larger numbers of different fluid volumes per unit time. Specifically, maintaining higher fluid volume coherency allows separate volumes to be transported closer together through the channels of the system, without resulting in
excessive intermixing of these volumes. Further, maintenance of maximum fluid volume coherency during the transport and direction of the fluids permits more precise control of volumetric delivery of materials within these systems.
Despite the differing goals of the CE systems and the E/O flow systems used in the present invention, in each case, the application of an electrical field across a fluid of interest has the same basic result. Specifically, where the fluid of
interest comprises charged species, or is made up of a plurality of differentially charged chemical species, application of a voltage across that fluid, e.g., to obtain E/O flow, will result in those charged species electrophoresing within the fluid, and
the differentially charged species electrophoresing at different rates. As such, in a channel having a negative surface potential, negatively charged species will have an electrophoretic mobility opposite to the direction of E/O flow, whereas positively
charged species will have an electrophoretic mobility in the same direction of E/O flow. The greater the number of charges a particular species has, the greater its electrophoretic mobility in the same or opposite direction of E/O flow. In systems
employing electroosmotic fluid direction, this results in a net separation of differentially charged species that are contained within the fluid that is being transported.
Where one is transporting a particular volume of a given sample fluid, this separation can result in an electrophoretic biasing of the sample, where the positively charged species have a greater apparent mobility, than negatively charged species. "Apparent mobility" as used herein, generally refers to the overall mobility of a given species within the fluidic system. in the systems of the present invention, apparent mobility is typically defined as the rate of E/O mobility plus the
electrophoretic mobility. Where electrophoretic mobility is opposite to the direction of E/O flow, i.e., negative, this leads to an apparent mobility that is less than the E/O mobility.
In the case of species having high electrophoretic mobility, e.g., highly charged species, the effect can be magnified to the point that the apparent mobility of such species is substantially different from the E/O mobility of the fluid
containing them. For example, species possessing multiple negative charges may have an electrophoretic mobility substantially opposite the direction of E/O mobility, resulting in a substantial reduction in the apparent mobilitv of that species. Where
that reduction is sufficiently large, it can result in that species being effectively "left behind" by the particular volume of fluid that is being transported.
Conversely, a species bearing multiple positive charges may have an apparent mobility that is far greater than that of the fluid being transported and other species contained therein, such that the species is transported well ahead of the fluid
volume.
This problem is not as significant where one is transporting large volumes of fluid from one location to another. Specifically, one can reduce the effects of the electrophoretic separation of a fluid by collecting larger volumes, thereby
reducing the contribution that biased portions of the fluid have on the overall fluid delivered.
However, the problem is substantially magnified when one wishes to transport a relatively small volume, or multiple small volumes of the same or different fluids, without separating the materials contained in the individual fluid volumes or
intermixing the materials contained in separate volumes. Specifically, in transporting a one or a series of discrete volumes of a particular fluid or fluids, e.g., samples, test compounds, various elements of a screening system, species that have
apparent mobilities that are substantially different from the E/O mobility of the particular fluid volume will travel ahead of, and behind the fluid volume, effectively smearing the materials that are sought to be delivered. As described above, this is
a significant disadvantage where relatively precise fluid control is desired, or where smaller effective volumes are used. For example, where one is screening for compounds which affect a particular reaction mix, e.g., a biochemical system, it is
generally desirable to be able to mix the elements necessary for that screen, e.g., enzyme, substrate and test inhibitor, and allow those elements to incubate together while transporting them to the ultimate detection area. Where those elements separate
based upon their differential electrophoretic mobilites, this can have substantial adverse effects on the overall efficacy of the screening system.
More importantly, where a species in a first volume being transported has an apparent mobility that is substantially less than the E/O mobility of the fluid, while a species in a second or following volume has an apparent mobility that is
substantially greater than the E/O mobility of the fluid that is being delivered, those two species can overlap within the flow system.
The above described problems are schematically illustrated in FIG. 1. FIG. 1A shows an optimal situation where discrete volumes or regions of fluids in a channel (fluids AB and XY, shown underlined) contain differentially charged species, e.g.,
X+ and Y-, and A+ and B-. In this optimal situation, these differentially charged chemical species have an apparent mobility that is not substantially different from the E/O mobility of the fluid containing those species. As a result, the various
species are maintained substantially within their separate fluid regions. FIG. 1B illustrates the smearing effect which results when charged species, as a result of their greater electrophoretic mobilities, begin to migrate outside of their respective
fluid volumes or regions. This results in a smearing of the materials that are being transported and substantially reduces the precision with which these materials can be transported. Finally, FIG. 1C illustrates the situation where the apparent
mobility of the charged species is so substantially different from the E/O mobility of the fluid regions, that it results in the overlapping and intermixing of differentially charged species from different fluid regions. The intermixing of separate
fluid volumes creates substantial problems where the fluid system is being used in the serial transport of multiple different fluids, e.g., as described in U.S. patent application Ser. No. 08/761,575, filed Dec. 6, 1996, and incorporated herein by
reference in its entirety for all purposes.
Methods have been developed to prevent and/or correct for the excessive electrophoretic mobility of charged species, when those species are being transported in E/O fluid direction systems, by incorporating fluid barriers around the fluid being
transported, in which the electrophoretic mobility of these charged species is substantially reduced, see, e.g., commonly assigned U.S. patent application Ser. No. 08/760,446, filed Dec. 6, 1996 (now U.S. Pat. No. 5,880,071), and incorporated herein
by reference in its entirety for all purposes.
Generally, the enhanced E/O material transport and direction produced by the present invention is carried out by providing within the fluid component of the system, a compound or compounds that are capable of reducing the effects such an E/O
system has on charged species contained within the fluid. For example, incorporation of these compounds within the fluid component of the E/O flow system typically results in a reduction in the electrophoretic mobility of charged species, and thus,
reduces the differential electrophoretic mobility and apparent mobility of differentially charged species.
In preferred aspects, zwitterionic compounds or combinations thereof, are used to reduce the electrophoretic mobility of materials that are contained within the fluids that are sought to be transported using these E/O fluid direction systems,
thereby achieving or substantially achieving the optimal situation shown in FIG. 1A.
Without being bound to a particular theory of operation, it is believed that such zwitterionic compounds interact with the charged species in a layer-like complex. The "complex" has the same net charge as the charged species, but that charge is
spread over a much larger structure effectively reducing the charge:size ratio, and reducing the electrophoretic mobility of the complex. Because zwitterions are dipolar molecules, they can be effectively employed with respect to positively or
negatively charged species.
While other methods can be used to effectively reduce the charge:size ratio of compounds in an E/O fluid direction system, these methods have numerous associated problems. For example, raising or lowering pH of the fluid containing the species
can effectively reduce the level of charge of a chemical species by protonating or deprotonating functional groups present therein. While effective in reducing net charge of a given species, this method can have substantial adverse effects.
Specifically, where the fluidic system is being utilized in the analysis of biological systems, e.g., enzymatic reactions, receptor/ligand interactions, or in transporting other materials sensitive to extremes of pH, the substantial variation of pH,
e.g., from neutral or physiological conditions, can place the system well outside the optimal pH for subsequent manipulation or analysis. In some cases, the optimal pH for reducing the net charge of a particular species may denature or otherwise degrade
active components of the materials that are being transported.
The incorporation of zwitterionic compounds as described herein, on the other hand, is readily compatible with systems to be used for the transport of pH sensitive materials, e.g., systems used in analysis of biological systems. In particular,
different zwitterionic compounds, i.e., having different pI, may be selected depending upon the pH sensitivity of the material being transported. Accordingly, as can be readily appreciated from the foregoing, the present invention is particularly useful
in E/O fluid direction systems where the materials to be transported include biological material, such as enzymes, substrates, ligands, receptors, or other elements of biological or biochemical systems, e.g., as those systems are defined in U.S. patent
application Ser. No. 08/761,575, previously incorporated herein by reference for all purposes.
Another method that can be used to affect the charge:size ratio of a charged molecule of interest in an E/O fluid direction system involves interacting that charged molecule of interest with another molecule or species such that the two molecules
form a complex having a different charge:size ratio. Merely by way of example, fluorescein is a molecule that carries two negative charges above neutral pH. The electrophoretic mobility of this molecule can be readily altered by adding an antibody,
such as anti-fluorescein to the solution. The resulting complex will have a substantially reduced electrophoretic mobility over that of fluorescein alone. Again, while this method is effective, it too carries a number of disadvantages. First, because
one must identify a compound that associates with the charged molecule of interest, a specifically associating compound must be identified for each charged molecular species in the fluid, and for each different fluid used in the system. Further, as is
the case with the fluorescein/anti-fluorescein complex described above, incorporation of an active molecule into a larger complex can have an adverse effect desired activity or function of that molecule, i.e., substantially reduced fluorescence.
The methods and systems of the present invention, on the other hand do not have these associated problems. For example, the function of zwitterionic compounds in reducing electrophoretic mobility of charged species is generally applicable, i.e.,
does not require a specific interaction between the charged species and the zwitterion. Further, the nature of this interaction results in little or no effect on the properties of the charged molecule of interest.
B. Increase In E/O Mobility
In addition to the advantages of reducing the electrophoretic mobility of charged species within fluids that are being transported using E/O fluid directions systems, incorporation of zwitterionic compounds in many systems can also have the
effect of increasing the E/O mobility in the fluid direction system, thereby further optimizing the apparent mobility of the material that is being transported.
In particular, incorporation of zwitterionic compounds within fluids being transported in E/O fluid direction systems has been shown to increase E/O mobility of those fluids. This effect is particularly apparent where those fluids include a
protein component or other larger charged molecular species.
II. Compounds Useful in Practicing the Invention
A wide variety of zwitterionic and related chemical compounds may be employed according to the present invention. For example, such compounds include, e.g., betaine, sulfobetaine, taurine, aminomethane sulfonic acid, zwitterionic amino acids,
such as glycine, alanine, .beta.-alanine, etc., and other zwitterionic compounds such as HEPES, MES, CAPS, tricine and the like. In particularly preferred aspects, non-detergent, low molecular weight su | | |
