Methods and apparatus for in situ concentration and/or dilution of materials in microfluidic systems

The present invention is generally directed to methods, apparatus and systems for use in performing in situ dilution or concentration of a particular subject material in a microfluidic device or system. These methods and apparatus may generally be integrated with other microfluidic operations and/or systems, to perfom a number of different manipulations, wherein dilution or concentration, carried out within the context of the microfluidic device or system, is just one part.

BACKGROUND OF THE INVENTION 
Carrying out chemical or biochemical analyses, syntheses or preparations, 
even at the simplest levels, requires one to perform a large number of 
separate manipulations on the material components of that analysis, 
synthesis or preparation, including measuring, aliquoting, transferring, 
diluting, concentrating, separating, detecting etc. 
In developing microfluidic technologies, researchers have sought to 
miniaturize many of these manipulations and/or to integrate these 
manipulations within one or a few microscale devices. Many of the above 
described manipulations easily lend themselves to such miniaturization and 
integration. For example, the use of these microfluidic technologies has 
been described in a number of applications, including, e.g., amplification 
(U.S. Pat. Nos. 5,587,128 and 5,498,392) and separation of nucleic acids 
(Woolley et al., Proc. Nat'l. Acad. Sci. 91:11348-352 (1994) and 
hybridization analyses (WO 97/02357 to Anderson). 
Despite the application of microfluidic technologies to these 
manipulations, there are still a number of areas where that application is 
not so easily made. For example, the performance of large dilutions 
generally requires the combination of a small volume of the material that 
is desired to be diluted with a large volume of diluent. By definition, 
microfluidic systems have extremely small overall volumes, and are 
typically unable, or less able, to handle the larger volumes required for 
such dilutions. Further, such large dilutions also typically require the 
accurate, repeatable dispensing of extremely small volumes of the material 
to be diluted. However, most microfluidic technology is incapable of 
accurately dispensing fluid volumes substantially less than a microliter. 
Although the problems associated with the inability to aliquot extremely 
small volumes might generally be overcome by performing serial dilutions, 
such serial dilutions generally require devices with substantially larger 
volumes, e.g., tens or hundreds of microliters. Specifically, even if one 
assumes a lower limit of fluid handling of 100 nanoliters, a 1:10 dilution 
would require a device to handle at least a volume of 1 .mu.l. Further 
serial dilution steps only increase the required volume. 
It would therefor be desirable to provide microfluidic systems which are 
capable of performing each of the various manipulations required, and 
which are capable of doing so with a sufficiently small volume whereby, 
multiple operations can be integrated into a single low volume device or 
system and performed automatically and with a high degree of precision. Of 
particular interest would be a microfluidic device or system, as well as 
methods for using such devices and systems for performing in situ dilution 
or concentration of a particular material within a microfluidic format. 
The present invention meets these and many other needs. 
SUMMARY OF THE INVENTION 
The present invention is generally directed to methods, apparatus and 
systems for performing in situ concentration or dilution of a material in 
microfluidic devices or systems. In one aspect, the present invention 
provides microfluidic devices and systems for performing in situ dilution, 
and particularly in situ serial dilution, of a particular subject 
material. The devices and/or systems typically comprise a microfluidic 
device which has at least one main channel disposed therein, where the 
main channel has at least one microscale cross-sectional dimension. The 
devices and/or systems also typically comprise at least a first source of 
the subject material that is to be diluted, in fluid communication with 
the main channel at a first point along the length of the channel, at 
least a first source of diluent in fluid communication with the main 
channel at a second point along the length of the channel, and at least a 
first reservoir in fluid communication with the main channel at a third 
point along the length of the channel. The systems of the present 
invention further comprise a fluid direction for delivering diluent to the 
main channel to be combined with the subject material to form first 
diluted material, and for removing at least a portion of the first diluted 
material from the main channel to the reservoir. Additional diluent 
sources and reservoirs also may be supplied to further dilute the subject 
material. 
In a closely related aspect, the present invention provides a microfluidic 
system for continuously diluting a subject material within a microfluidic 
device. The devices and/or systems typically comprise a microfluidic 
device which has at least one main channel disposed therein, where the 
main channel has at least one microscale cross-sectional dimension. The 
device also typically comprises at least a first source of the subject 
material in fluid communication with the main channel at a first point 
along the length of the channel, at least a first source of diluent in 
fluid communication with the main channel at a second point along the 
length of the channel, and at least a first reservoir in fluid 
communication with the main channel at a third point along the length of 
the channel. The systems of the present invention further comprise a fluid 
direction system for continuously delivering diluent to the main channel 
to be combined with the subject material to form first diluted material, 
and continuously transporting a portion of the first diluted material from 
the main channel to the reservoir. 
The present invention also provides microfluidic systems for in situ 
concentration of a subject material within a microfluidic device. In this 
aspect, the system comprises a microfluidic device having a first channel 
disposed therein, which channel has first, second and third fluid regions 
disposed therein. The first fluid region typically comprises the subject 
material and has a first conductivity, whereas the second and third fluid 
regions are disposed within the first channel on both ends of the first 
fluid region. The second and third fluid regions have a second 
conductivity, where the second conductivity is greater than the first 
conductivity. The system also typically comprises an electroosmotic fluid 
direction system for transporting the first and second fluid regions along 
the first channel. 
In a further aspect, the present invention provides methods for in situ 
dilution of a subject material in a microfluidic device. The methods 
typically comprise combining a first volume of the subject material with a 
first volume of diluent in a first microscale channel to form a first 
diluted material. At least a first portion of the first diluted material 
is then transported out of the first channel. A second volume of diluent 
from a second diluent source is then delivered to the first microscale 
channel to combine the second volume of diluent with a second portion of 
the first diluted material to form a second diluted material. 
In a related aspect, the present invention provides a method for in situ 
dilution of a material in a microfluidic device, which method comprises 
combining a first volume of said material with a first volume of diluent 
in a first region of a microfluidic device to form a first diluted 
material. A portion of the first diluted material is then transported into 
a second region of the microfluidic device, i.e., a reservoir, where it is 
combined with a second volume of diluent to form a second diluted 
material. 
In a further aspect, the present invention provides a method for the in 
situ concentration of a material in a microscale channel. The method 
comprises introducing a first fluid containing the material into a 
microscale channel to provide a first fluid region within the channel. The 
first fluid has a first conductivity, and is bounded by second and third 
fluid regions, where the second and third fluid regions have a second 
conductivity which is greater than the first. A voltage gradient is then 
applied along the length of the microscale channel whereby the first, 
second and third fluid regions are transported along the length of the 
microscale channel with a first electroosmotic mobility, and whereby the 
material in the first fluid has an electrophoretic mobility different from 
the electroosmotic mobility, resulting in a concentration of the subject 
material at or near one end of the first fluid region.

DETAILED DESCRIPTION OF THE INVENTION 
I. Generally 
The present invention is generally directed to methods, apparatus and 
systems for use in performing in situ dilution or concentration of a 
particular subject material in a microfluidic device or system. These 
methods and apparatus may generally be integrated with other microfluidic 
operations and/or systems, to perfom a number of different manipulations, 
wherein dilution or concentration, carried out within the context of the 
microfluidic device or system, is just one part of the overall operation. 
The term "dilution," as used herein, generally encompasses the ordinary 
meaning of that term, namely, the reduction in the amount of a particular 
subject material per unit volume of a fluid containing that material, 
through the addition of a second fluid, or diluent, to a first fluid which 
contains the subject material, e.g., soluble chemical component, or a 
suspension or emulsion of a partially insoluble material, whereby the 
resulting concentration of the subject material is reduced over that of 
the first fluid. In terms of the present invention, the diluent may take 
on a variety of forms, including aqueous or nonaqueous fluids and/or it 
may include additional material components, e.g., soluble chemical 
components or suspensions or emulsions of at least partially insoluble 
components. As alluded to above, the subject material may comprise 
virtually any composition, including chemical compounds, either soluble or 
as suspensions or emulsions, biological material, either soluble or as 
suspensions (e.g., cells) or emulsions, and the like. By "serial dilution" 
is generally meant successive dilutions, as defined herein, wherein the 
subject material is diluted with diluent to form a first diluted material, 
which first diluted material is then diluted with a diluent again, to 
produce a second diluted material, etc. For example, one produces a first 
diluted material that is diluted 1:10 over the subject material. By then 
diluting at least a portion of this material 1:10, one produces a second 
diluted material that is a 1:100 dilution of the subject material. In 
general, the methods, devices and systems of the present invention are 
useful in diluting subject material greater than 10 fold (1:10), typically 
greater than 100 fold (1:100), preferably greater than 1000 fold (1:1000), 
and in many cases, greater than 10,000 fold (1:10,000), within a single 
integrated microfluidic device, which typically has an internal volume, 
e.g., channel volume, of less than 10 .mu.l and preferably less than 1 
.mu.l. 
The term "concentration" as used herein, generally refers to the ordinary 
meaning of that term, namely the increase in the amount of a particular 
subject material per unit volume of the fluid in which the material is 
disposed, e.g., wholly or partially dissolved, suspended, slurried, etc. 
As used herein, the term "microfluidic," or the term "microscale" when used 
to describe a fluidic element, such as a passage, chamber or conduit, 
generally refers to one or more fluid passages, chambers or conduits which 
have at least one internal cross-sectional dimension, e.g., depth or 
width, of between about 0.1 .mu.m and 500 .mu.m. In the devices of the 
present invenion, the microscale channels preferably have at least one 
cross-sectional dimension between about 0.1 .mu.m and 200 .mu.m, more 
preferably between about 0.1 .mu.m and 100 .mu.m, and often between about 
0.1 .mu.m and 20 .mu.m. Accordingly, the microfluidic devices or systems 
of the present invention typically include at least one microscale 
channel, and preferably at least two intersecting microscale channels 
disposed within a single body structure. 
The body structure may comprise a single component, or an aggregation of 
separate parts, e.g., capillaries, joints, chambers, layers, etc., which 
when appropriately mated or joined together, form the microfluidic device 
of the invention, e.g., containing the channels and/or chambers described 
herein. Typically, the microfluidic devices described herein will comprise 
a top portion, a bottom portion, and an interior portion, wherein the 
interior portion substantially defines the channels and chambers of the 
device. In preferred aspects, the bottom portion will comprise a solid 
substrate that is substantially planar in structure, and which has at 
least one substantially flat upper surface. A variety of substrate 
materials may be employed as the bottom portion. Typically, because the 
devices are microfabricated, substrate materials will generally be 
selected based upon their compatability with known microfabrication 
techniques, e.g., photolithography, wet chemical etching, laser ablation, 
air abrasion techniques, injection molding, embossing, and other 
techniques. The substrate materials are also generally selected for their 
compatability with the full range of conditions to which the microfluoidic 
devices may be exposed, including extremes of pH, temperature, salt 
concentration, and application of electric fields. Accordingly, in some 
preferred aspects, the substrate material may include materials normally 
associated with the semiconductor industry in which such microfabrication 
techniques are regularly employed, including, e.g., silica based 
substrates such as glass, quartz, silicon or polysilicon, as well as other 
substrate materials, such as gallium arsenide and the like. In the case of 
semiconductive materials, it will often be desirable to provide an 
insulating coating or layer, e.g., silicon oxide, over the substrate 
material, particularly where electric fields are to be applied. 
In additional preferred aspects, the substrate materials will comprise 
polymeric materials, e.g., plastics, such as polymethylmethacrylate 
(PMMA), polycarbonate, polytetrafluoroethylene (TEFLON.TM.), 
polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and the 
like. Such substrates are readily manufactured from microfabricated 
masters, using well known molding techniques, such as injection molding, 
embossing or stamping, or by polymerizing the polymeric precursor material 
within or against the mold or master. Such polymeric substrate materials 
are preferred for their ease of manufacture, low cost and disposability, 
as well as their general inertness to most extreme reaction conditions. 
Again, these polymeric materials may include treated surfaces, e.g., 
derivatized or coated surfaces, to enhance their utility in the 
microfluidic system, e.g., provide enhanced fluid direction, e.g., as 
described in U.S. patent application Ser. No. 081943,212 filed Apr. 14, 
1997 (Attorney Docket No. 17646-002610), and which is incorporated herein 
by reference in its entirety for all purposes. 
The channels and/or chambers of the microfluidic devices are typically 
fabricated into the upper surface of the substrate, or bottom portion, 
using the above described microfabrication techniques, as microscale 
grooves or indentations. The lower surface of the top portion of the 
microfluidic device, which top portion typically comprises a second planar 
substrate, is then overlaid upon and bonded to the surface of the bottom 
substrate, sealing the channels and/or chambers (the interior portion) of 
the device at the interface of these two components. Bonding of the top 
portion to the bottom portion may be carried out using a variety of known 
methods, depending upon the nature of the substrate material. For example, 
in the case of glass substrates, thermal bonding techniques may be used 
which employ elevated temperatures and pressure to bond the top portion of 
the device to the bottom portion. Polymeric substrates may be bonded using 
similar techniques, except that the temperatures used are generally lower 
to prevent excessive melting of the substrate material. Alternative 
methods may also be used to bond polymeric parts of the device together, 
including acoustic welding techniques, or the use of adhesives, e.g., UV 
curable adhesives, and the like. 
The microfluidic devices and systems of the present invention, e.g., which 
are capable of performing in situ dilution or concentration of a 
particular subject material, typically comprise a main microscale channel 
disposed within the body structure, which is used to transport, and in 
some embodments, mix the subject material and the diluent. Accordingly, 
the devices of the present invention comprise at least a first source of 
fluid containing a particular subject material in fluid communication with 
the main channel. For those aspects of the present invention directed to 
dilution of the subject material, a source of diluent is also generally 
provided in fluid communication with the main channel. Typically, the 
source of material and the source of diluent are provided as an integral 
part of the body structure, e.g., as wells or reservoirs within the body 
structure, e.g., the top portion of the device. Fluid communication 
between these sources and the main channel is generally via a connecting 
channel which is in fluid communication with the well or reservoir at one 
end, and in fluid communication with, e.g., intersecting, the main channel 
at the other end. 
In order to manipulate materials within the microfluidic devices described 
herein, the overall microfluidic systems of the present invention 
typically include a material direction system to manipulate selected 
materials within the various channels and/or chambers of the microfluidic 
device. Thus, by "material direction system" is meant a system which 
controls the movement and direction of fluids containing such materials 
within intersecting channel structures of a microfluidic device. 
Generally, such material direction systems employ pumps or pressure 
systems, and valves to affect fluid movement and direction in intersecting 
channels. A large number of microfabricated mechanical pumps and valves 
have been previously described in the art. Although such fluid direction 
elements may be useful in many aspects of the present invention, by and 
large, these elements are not preferred due to the complexity and cost of 
their manufacture. Further, the limits of microfabrication technology with 
respect to such pumps and valves, do not readily permit the manufacture of 
such elements that are capable of precisely handling sufficiently small 
volumes, e.g., volumes less than 1 .mu.l. Thus, in particularly preferred 
aspects, the microfluidic systems of the present invention employ 
electroosmotic material direction systems to affect direction and 
transport of fluid borne materials within the microfluidic devices and 
systems of the invention. "Electroosmotic material direction systems," as 
used herein, refer to material direction systems which employ controlled 
electroosmotic flow to affect fluid movement and direction in intersecting 
channel structures. In particular, such systems function by applying a 
voltage gradient across the length of 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 within that 
channel. Further, by concurrently regulating flow in two or more channels 
that meet at an intersection, one can direct fluid flow at that 
intersection. Such electroosmotic material direction systems and 
controllers are described in detail in, e.g., Published PCT Application 
No. 96/04547 to Ramsey et al., U.S. application Ser. No. 08/691,632, filed 
Aug. 2, 1996 and U.S. application Ser. No. 08/761,986, filed Dec. 6, 1996, 
each of which is incorporated herein by reference in its entirety for all 
purposes. 
II. Dilution 
As described above, in a first aspect, the present invention generally 
provides methods, devices and systems for performing the in situ dilution, 
and particularly serial dilution, of a subject material within a 
microfluidic device or system. 
Dilution of the subject material in microfluidic devices and systems 
according to the present invention, generally comprises transporting a 
first volume of fluid containing the subject material from the reservoir 
containing that subject material, into the main channel, and mixing that 
first volume with a second volume of diluent transported into the main 
channel from the reservoir of diluent, to produce a first diluted 
material. The first diluted material is then further diluted by combining 
a portion of the first diluted material with another volume of diluent to 
produce a second diluted material. These dilution steps may be repeated 
any number of times until the desired dilution is achieved. 
In preferred aspects, the process of mixing the fluid containing the 
subject material and the diluent preferably is carried out within the main 
channel. In particular, the first volume of the fluid containing the 
subject material is delivered from the source of the subject material into 
the main channel, while a second volume of diluent is transported from the 
diluent source into the main channel. The mixing of these two fluids 
within the main channel produces the first diluted material. This first 
diluted material is then combined with a further volume of diluent to 
produce a second diluted material, and so on. 
However, because volumes of microfluidic devices and/or systems are 
extremely small and therefore limited, in order to further dilute the 
first diluted material, e.g., in a serial dilution, one must transport at 
least a portion of the first diluted material out of the main channel, 
e.g., out of the device or into a waste reservoir, prior to delivering the 
further volume of diluent, in order to accommodate that additional volume 
of diluent. Once the first diluted material is transported out of the main 
channel, a further volume of diluent is delivered to the main channel and 
combined with the remaining portion of the first diluted material, which, 
upon mixing, produces a second diluted material which is the product of 
the first and second dilutions, e.g., if the first and second dilutions 
were each 10 fold, then the second diluted material would be a 100 fold 
dilution of the starting material. 
One example of a microfluidic device for carrying out in situ serial 
dilution as described herein, is schematically illustrated in FIG. 1. As 
shown, the device 100, includes a body structure 102 which has a main 
channel 104 disposed therein. As shown, a source of, or reservoir 
containing the subject material 106 is provided in fluid communication 
with the main channel. The illustrated device also includes multiple 
diluent sources 110, 114 and 118 in fluid communication with the main 
channel via diluent channels 122, 126 and 130, respectively. The device 
also includes reservoirs 112, 116 and 120 in fluid communication with the 
main channel via reservoir channels 124, 128 and 132, respectively. The 
main channel is also shown with a terminal waste reservoir 108 at its 
terminus. Although illustrated in terms of performing only dilutions of 
the subject material, it will be appreciated from the instant disclosure 
that this device or aspect of a device is readily integrated into a device 
or system which performs numerous other manipulations, including enzyme 
assays, immunoassays, screening assays, separations and the like. As a 
result, waste reservoir 108 may generally be subsituted with appropriate 
fluidic elements for performing further manipulations of the ultimately 
diluted material, e.g., analysis, reaction, detection, etc. 
In operation, a first volume of the subject material from reservoir 106 is 
flowed into the main channel 104. This volume of subject material is 
combined with a first volume of diluent which is flowed into main channel 
104 from diluent reservoir 110, via channel 122. While flowing down the 
main channel towards reservoir 108, the subject material and the diluent 
will mix to form a first diluted material. A portion of this first 
material is then directed from the first channel into reservoir channel 
124 and out to reservoir 112, while the remaining portion of first diluted 
material continues to flow down the length of the main channel towards 
reservoir 108. This remaining portion of first diluted material is then 
combined with a second volume of diluent delivered into main channel 104 
from diluent reservoir 114 via channel 126, whereupon, the second volume 
of diluent and first diluted material will mix to form a second diluted 
material flowing down the main channel towards reservoir 108. For ease of 
discussion, each step of directing a portion of the diluted material out 
of the main channel and delivering a volume of diluent into the channel to 
combine with the remaining portion of diluted material is referred to 
herein as a dilution stage. A portion of this second diluted material is 
then directed from the first channel into reservoir channel 128 and out to 
reservoir 116, while the remaining portion of second diluted material 
continues to flow down the main channel towards reservoir 108. The 
remaining portion of the second diluted material is then combined with a 
third volume of diluent delivered into main channel 104 from diluent 
reservoir 118 via channel 130, whereupon the third volume of diluent and 
the remaining portion of the second diluted material will mix to form a 
third diluted material. This third diluted material may then be subjected 
to a subsequent analysis, reaction, combination etc. However, in preferred 
aspects, and as shown in FIG. 1, a portion of the third diluted material 
in main channel 104 is transported out of the main channel and into 
reservoir 120, via channel 132. This allows the stepping down of the 
volume of third diluted material which must be subsequently handled. 
The steps of delivering diluent to the main channel and transporting 
portions of diluted material out of the main channel may be repeated as 
necessary in order to produce the dilution ratio desired. In addition, 
although described in terms of volumes of material and diluent, the 
devices of the present invention as they relate to dilution, preferably 
are run in a continuous flow arrangement. Specifically, subject material 
is flowed into the main channel in a stream at a first flow rate, while a 
stream of diluent is concurrently flowed into the main channel at a second 
flow rate, where the ratio of first flow rate to second flow rate are 
related to the desired dilution for that particular dilution step. These 
streams mix within the main channel to form a stream of first diluted 
material. Similarly, a portion of the stream of first diluted material is 
continuously flowed out of the main channel into the reservoir channel and 
out to the reservoir, while downstream, another stream of diluent is 
introduced into the main channel to mix with the remaining portion of the 
stream of first diluted material to produce second diluted material. These 
steps are repeated on down the main channel to form a continuous stream of 
material at the desired dilution as long as flow is maintained into and 
out of the main channel at the appropriate rates to affect the incremental 
dilutions at each dilution stage. 
The dilution methods and systems described herein are particularly useful 
in microfluidic systems which employ electroosmotic fluid direction 
systems. In these electrosmotic fluid direction systems, fluid flow is 
generally proportional to current flow. Accordingly, in performing 
dilutions using these systems, the dilution ratio is generally related to 
the ratio of current applied at the subject material reservoir to the 
current applied to the diluent reservoir. For relatively small dilutions, 
e.g., 1:10, these electroosmotic systems can carry out the desired 
dilution in one step, e.g., combining 1 part subject material to 9 parts 
diluent, or applying 1 .mu.A to the subject material reservoir and 9 .mu.A 
to the diluent reservoir. However, for dilutions somewhat greater than 
1:10, diffusional leakage of the subject material from its supply channel 
into the main channel limits further dilution, even where only extremely 
small currents are applied at the subject material reservoir. 
One potential solution to the problem of diffusional leakage is to perform 
multiple serial dilutions, e.g., perform three successive 10 fold 
dilutions. However, because fluid flow is related to current flow, the 
final dilution would require a current applied at the dilution well of 900 
.mu.A. Specifically, the first dilution would be carried out by applying 1 
.mu.A to the subect material reservoir while applying 9 .mu.A to the 
diluent reservoir, resulting in a 10 .mu.A current in the main channel. 
The second 1:10 dilution would then require a current of 90 .mu.A in the 
second diluent reservoir, and create a current of 100 .mu.A in the main 
channel. The final dilution would then require an applied current at the 
third diluent reservoir of 900 .mu.A, which would create a large number of 
adverse effects on the microfluidic system, electrolysis of the buffers, 
excessive heating of the fluid channels, etc. 
As described above, the methods and systems of the present invention 
combine the subject material with diluent to create a first diluted 
material. A portion of this diluted material is then removed from the main 
channel and the remaining portion is further diluted. This process allows 
one to perform serial dilutions as described above, the methods and 
systems of the present invention combine the subject material with diluent 
microfluidic systems, thereby obviating the problem of diffusional leakage 
at the intersections, as well as permitting much larger dilutions. 
Furthermore, this process allows for the use of electroosmotic fluid 
direction systems without requiring excessive current ratios, and thus, 
application of excessive currents. 
Referring to the system illustrated in FIG. 1, a particular subject 
material disposed in reservoir 106, is diluted up to 1,000 fold using 
these methods. In particular, a first 10 fold dilution of the subject 
material is achieved by applying 1 .mu.A to reservoir 106, while applying 
9 .mu.A to the first diluent reservoir 110, resulting in a 9 to 1 ratio of 
diluent flow to subject material flow. This also results in a 10 .mu.A 
current within the main channel 104, between its intersection with channel 
122 and its intersection with channel 124. A current of -9 .mu.A is 
applied at waste reservoir 112, which results in approximately 90% of the 
diluted material flowing to the waste reservoir from the main channel, and 
returns current in the main channel to 1 .mu.A between its intersection 
with channel 124 and its intersection with channel 126. Again, a 9 .mu.A 
current is applied in second diluent reservoir 114, resulting in a further 
1:10 dilution of the subject material within the main channel, or a total 
dilution of 1:100. Another -9 .mu.A current is then applied at waste 
reservoir 116, withdrawing approximately 90% of the second diluted 
material, and the final dilution step is supplied by applying 9 .mu.A at 
third diluent reservoir 118. A further waste reservoir 120 is also shown, 
for reducing the current at the final waste reservoir 108, back to 1 
.mu.A. Accordingly, a 1000 fold dilution only requires a 10 fold current 
range across the device. Further, because each dilution step is relatively 
modest, e.g., 1:10, these methods do not suffer from the problems of 
diffusional leakage at the combining intersection, because the smallest 
volume is easily controlled at these levels. 
An alternative structure that can be used in practicing the dilution 
methods described herein, is shown in FIG. 2. This alternate structure has 
the advantage of including a potentially unlimited number of dilution 
stages while only requiring six reservoirs at which electrical contact is 
made. As shown, the channel structure is shown fabricated in a substrate 
102 and again includes subject material reservoir 106, main channel 104 
and waste reservoir 108. In addition, the structure includes a main 
diluent channel 204 which has at its termini diluent reservoirs 202 and 
206, and main waste channel 210 which has at its termini waste reservoirs 
208 and 212. Main diluent channel 204 is in fluid communication with main 
channel 104 via a plurality of diluent connecting channels 214, 216 and 
218. Similarly, main waste channel 210 is in fluid communication with main 
channel 104 via waste connecting channels 220, 222 and 224, which are 
staggered from the diluent connecting channels, e.g., diluent connecting 
channel 214 intersects main channel 104 at a first point, and waste 
connecting channel 220 intersects the main channel 104 at a second point, 
that is further downstream from the first point. 
In operation, subject material from reservoir 106 is introduced into main 
channel 104, e.g., by applying a current along the length of channel 104. 
A first volume of diluent is introduced into the main channel from main 
diluent channel 204, via diluent connecting channel 214, to create a first 
diluted material, which continues to flow toward reservoir 108. After the 
subject material and diluent mix, a first portion of the first diluted 
material is transported out of main channel 104 into main waste channel 
210, via waste connecting channel 220. A second volume of diluent is then 
delivered to the main channel 104 from main diluent channel 204, via 
diluent connecting channel 216, whereupon it will mix with the remaining 
portion of first diluted material to form a second diluted material. A 
portion of this second diluted material is then transported out of channel 
104 into the main waste channel 210 via waste connecting channel 222. The 
process steps are then repeated, e.g., with diluent connecting channel 218 
and waste connecting channel 224, as necessary to achieve the desired 
dilution. As shown, the channel structure includes three dilution stages. 
However, as noted previously, the structure shown can include additional 
dilution stages without necessitating additional reservoirs. For example, 
in the figure shown, application of a first voltage to reservoirs 106, 202 
and 208, and a second voltage to reservoirs 108, 206 and 212 will cause 
material to move in parallel down each of main diluent channel 204, main 
channel 104, and main waste channel 210, with no dilution of the subject 
material in main channel 104. Specifically, no material will move among 
the various main channels. Increasing the potentials at reservoirs 202 and 
206, while symetrically reducing the potentials applied at reservoirs 208 
and 212, relative to the potentials applied at reservoirs 106 and 108, 
will cause dilution to occur in the main channel 104. Specifically, 
application of these voltage gradients will cause diluent to flow from 
main diluent channel 204 through diluent connecting channels 214, 216 and 
218, diluting the subject material present in the main channel at the 
intersection of the main channel and each of the diluent connecting 
channels. Similarly, material in the main channel, in addition to flowing 
toward reservoir 108, will also be flowing out of main channel 104 via 
waste connecting channels 220, 222 and 224 and thereby creating room for 
incoming diluent in a subsequent stage. 
In addition to the foregoing, it will be appreciated from the instant 
disclosure, that the length of the diluent and waste channels in FIGS. 1 
and 2 may be adjusted, in order to obviate the need for a wide range of 
applied voltages to achieve the desired currents. Specifically, higher 
voltages are required to achieve a certain desired current in a channel 
with higher resistance. By reducing the length of the channel, one also 
reduces the resistance along the length of that channel. 
As will be apparent from the disclosure of the invention herein, in many of 
the embodiments described, either the flow rates of the materials and 
diluents, or the length of the main channel between the diluent channels 
and the waste channels are selected whereby the subject material and the 
diluent are permitted to adequately mix before a portion of these 
materials is transported out of the main channel. These parameters will 
typically vary depending upon the diffusion rate for the material being 
transported, the length of the channel along which the material is being 
transported, and the flow velocity of the material being transported. 
Generally, however, these parameters are readily determinable with only 
routine experimentation, and may be readily optimized depending upon the 
overall system used. 
Both structures illustrated in FIGS. 1 and 2, provide for dilution of the 
subject material based upon the currents or voltages applied to the 
device, as well as the number of dilution modules or stages fabricated 
into the device. In a further modified aspect, the present invention also 
provides a dilution module that yields a given dilution of the subject 
material regardless of the current applied through the system, effectively 
`hard-wiring` a particular dilution factor into the device. 
A schematic illustration of one example of such a dilution module is shown 
in FIG. 3. As shown in FIG. 3, the device again includes a main channel 
104, which includes a narrowed region 302. Bypass channel 304 fluidly 
connects with main channel 104 above and below narrow region 302, i.e., 
upstream and downstream of the narrow region, and includes a diluent 
reservoir 306, disposed therein. In particular, diluent reservoir 306 is 
an integral part of the bypass channel 304. Reservoir 306 typically has a 
volume that is sufficiently larger than the volume of the channels, such 
that regardless of the flow of subject material into the reservoir via 
bypass channel 304, the effluent from the reservoir has an insignificant 
concentration of subject material. Because of the extremely small volumes 
of material required in these microfluidic systems, effective volumes of 
this diluent reservoir can be on the order of 1 to 10 .mu.l, and less, 
depending upon the flow rate used, the length of time the dilution is to 
run and the like. The increased volume of diluent reservoir 306 may be 
provided by increasing the length, width and/or depth of the reservoir 
relative to the channels of the device. 
The size of the dilution accomplished by this type of dilution module is 
dictated by the ratio of the resistance of channel 302 to the resistance 
of the loop defined by channels 304 and reservoir 306, regardless of the 
level of current flowing through the dilution module as a whole. In 
particular, as noted above, in electroosmotic flow systems, fluid flow is 
proportional to the level of current flow along a channel, which is 
inversely proportional to the resistance along a given channel. The 
resistance through a given fluid filled channel is inversely proportional 
to the cross sectional area, and directly proportional to the length of 
the channel through which current is passed. Accordingly, by adjusting the 
resistance along two potential current paths in a fluidic system, one can 
control the amount of electroosmotic fluid flow along each of the 
channels. For example, if fluid channels A and B begin and terminate in 
fluid communication, one can flow approximately 90% of fluid along path A 
by providing 90% of the current along this path. This is done by 
increasing the resistance along path B relative to path A. One simple 
method of increasing this resistance is to narrow the cross sectional 
dimensions of path B relative to path A. As can be appreciated, other 
methods may also be used to increase the resistance along path B, 
including increasing the length of path B relative to path A. 
Thus, as can be readily appreciated from the above, and with reference to 
FIG. 3, the ratio of subject material flowing out of main channel 104, and 
thus, the amount of diluent flowing into the main channel below the narrow 
region 302, to the amount of material flowing through the narrow region 
302, is proportional to the ratio of the resistance of the narrow region 
to that of the bypass region. This is the case regardless of the amount of 
current flowing through the system. Dilution occurs at the lower end of 
the narrow region, where subject material flowing into reservoir 306 
displaces an equal volume of diluent, which flows into the main channel at 
the lower end of the narrow region and mixes with the portion of subject 
material that traveled through the narrow region, to create a first 
diluted material. Because the diluent reservoir 306 has a sufficiently 
large volume, as compared with the amount of subject material flowing 
therein, the effluent is substantially entirely diluent. Rephrased, any 
amount of subject material coming through the reservoir is negligable when 
considered in light of the amount of subject material present in the first 
diluted material. Additional dilution modules can be included within a 
device, depending upon the level of dilution ultimately desired. Further, 
in those applications where one wishes to manipulate a number of different 
dilutions of a given subect material, e.g., in enzyme assays, binding 
assays, inhibitor screening assays, and the like, one can aliquot a sample 
of diluted material from the main channel after each dilution stage, for 
further manipulation, while the remainder of the material is subject to 
further dilution stages and manipulations. 
Another closely related example of a microfluidic system for performing in 
situ dilutions is schematically illustrated in FIG. 4. Again, the methods 
and devices described in this example perform serial dilutions of the 
subject material, and maintain the volume of material within the main 
channel by removing a portion of the diluted material from the channel 
following each step. This example differs from that described above, 
however, in that it includes a single diluent reservoir 402, but employs 
mixing chambers or regions, 406, 408 and 410, separate from the main 
channel 104, for mixing the diluent and subject material, in a number of 
steps. In particular, a first volume of the subject material is 
transported from reservoir 106 to reservoir 406, where it is mixed with a 
first volume of diluent transported to chamber 406 from reservoir 402, to 
form a first diluted material. Typically, the transport of the subject 
material and the first volume of diluent is carried out concurrently, 
although this is not necessary. Typically, the mixing chambers will have a 
substantially larger cross sectional dimension and/or depth than the 
channels of the system, to allow the introduction of larger volumes. A 
second volume of the first diluted material, e.g., typically some fraction 
of the total volume of the first diluted material, is then transported 
from mixing chamber 406 to mixing chamber 408, via main channel 104, 
whereupon it is mixed with a second volume of diluent to create a second 
diluted material. The second volume of first diluted material and second 
volume of diluent are selected depending upon the dilution that is desired 
for this step. Again, the transportation of the first diluted material and 
the second volume of diluent to mixing chamber 408 is typically carried 
out concurrently. This step is repeated in mixing chamber 410 and further 
as needed for the desired ultimate dilution. 
III. Concentration 
In another aspect, the present invention is directed to methods of 
performing in situ concentration of samples within microfluidic devices 
and systems, as well as being directed to the devices and systems 
themselves. In particular, the devices and systems concentrate a 
particular subject material electrophoretically, within the channels of 
the microfluidic device, and are therefore particularly suited for use 
with electroosmotic fluid direction systems, e.g., as described above. 
Specifically, in electroosmotic fluid direction, the presence of charged 
groups within a particular subject material or chemical specie can result 
in the electrophoretic mobility of that material within the system being 
different from the electroosmotic mobility of the fluid carrying that 
material. Depending upon the charge, this may result in the subject 
material moving through the channels of the system faster or slower than 
the fluid in which it is disposed. This shifting or electrophoretic 
biasing of the subject material within its fluid carrier can result in 
problems where one's goal is to transport the subject material from a 
first location in the microfluidic system to a second location in the 
system, substantially unchanged. In particular, these electroosmotic fluid 
direction systems function by applying a voltage gradient or electric 
field across the length of a channel along which fluid flow is desired. 
This same voltage gradient or electric field also causes the charged 
species within the fluid contained in the channel to electrophorese. Where 
the voltage gradient is greater, e.g., in lower conductivity fluids, the 
electrophoresis of the charged species is also greater, while lower 
voltage gradients, e.g., in high conductivity fluids, result in lower 
levels of electrophoresis. 
The present invention employs both low and high conductivity fluids in the 
same channel, to affect the concentration of the subject materal. 
Specifically, the subject material is generally provided in a fluid or 
buffer having a low relative conductivity, and dispensed as a discrete 
volume or fluid region, into a microscale channel, surrounded by fluid 
regions (also termed "spacer regions") of high relative conductivity. The 
fluid of high relative conductivity will typically have a conductivity 
that is at least two times the conductivity of the low relative 
conductivity fluid, and preferably, at least five times the conductivity 
of the low relative conductivity fluid, more preferably at least ten times 
the conductivity of the low relative conductivity fluid, and often at 
least twenty times the conductivity of the low relative conductivity 
fluid. Typically, the low conductivity fluid will have a conductivity in 
the range of from about 0.1 mS to about 10 mS. The high conductivity fluid 
typically has a conductivity in the range of from about 0.2 mS to about 
100 mS. 
When a voltage gradient is applied across the length of the channel, it 
will result in the movement of the subject material fluid along the 
channel by electroosmotic flow. The voltage gradient will also result in 
the electrophoresis of the charged subject material in the same or 
opposite direction of electroosmotic flow within the channel. The 
direction of electrophoretic mobility is determined by the charge on the 
subject material, e.g., either positive or negative. However, once the 
charged subject material reaches the interface of the low conductivity 
subject material region and the high conductivity spacer region, the 
electrophoresis of the charged material will be substantially reduced, 
because of the lack of a substantial voltage gradient across the high 
conductivity regions. This results in a "stacking" or concentration of the 
charged species at or near the interface of the two fluids. A portion of 
the fluid at or near this interface is then aliquoted from the remainder 
of the fluid in the channel at a substantially concentrated level, for 
further processing or analysis, e.g., separation. 
An example of a concentrator device, or concentrator elements of a device, 
for performing this type of concentration is schematically illustrated in 
FIG. 5. As shown in FIG. 5A, the concentrator includes a main channel 502, 
a sample introduction channel 504 in fluid communication with the main 
channel at a first intersection 506, a waste channel 508 in fluid 
communication with the main channel at a second intersection 510, and an 
aliquoting channel 512 in fluid communication with the main channel at a 
third intersection 514. The aliquoting channel may be used to shunt off 
the concentrated portion of the subject material. Alternatively, the 
stacked subject material may merely be subject to further manipulation as 
is. For example, in screening applications, e.g., employing inhibition 
assays, binding assays or the like, the concentration of the screened 
compound at the front of the fluid material that is loaded into the main 
channel will often be sufficient to ascertain screen results. Similarly, 
the concentration of species at the front edge of the material region may 
also act as a stacking process, e.g., like a stacking gel layer in 
SDS-PAGE or agarose sequencing gels, in order to provide greater 
resolution in separation applications, e.g., capillary electrophoresis. 
As shown the third intersection 514 is provided in the main channel 
somewhat dislocated from the first and second intersections 506 and 510, 
respectively, to provide adequate opportunity for the electrophoresis of 
the charged materials. Again, this distance will generally vary depending 
upon the nature of the material sought to be concentrated, e.g., its net 
charge, molecular weight, conformation, or other factors affecting the 
materials electrophoretic mobility within the channel. 
The concentrator illustrated in FIG. 5A also includes shallow regions 516 
and 518 within the sample loading and waste channels 504 and 508, 
respectively, where these channels reach their respective intersections 
with the main channel 502. These shallow regions generally prevent 
unwanted flow due to pressure effects associated with the electroosmotic 
pumping of different fluid regions within a microscale channel intersected 
by other channels, where those different fluid regions have different 
conductivities. This is described in substantial detail in U.S. patent 
application Ser. No. 08/760,446, filed Dec. 6, 1996, and incorporated 
herein by reference in its entirety for all purposes. 
Briefly, in the electroosmotic flow systems described herein, the presence 
of differentially mobile fluids (e.g., fluids having different 
conductivities and thus having different electroosmotic or electrokinetic 
mobilities in the particular system) in 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 conductivity fluids (e.g., high ionic 
concentration spacer regions and low ionic concentration material regions) 
in a given channel to produce electroosmotic flow. As the low ionic 
concentration regions within the channel tend to drop the most applied 
voltage across their length, they will tend to push the fluids through a 
channel. Conversely, high conductivity fluid regions within the channel 
provide 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 material regions), while the 
lowest pressure is typically found at the trailing or back edge of these 
low ionic concentration fluid regions. 
By reducing the depth of the channels intersecting the main channel, e.g., 
the sample introduction and waste channels 516 and 518, relative to the 
main channel 502, 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 with the depth of the channel for channels having an 
aspect ratio of &gt;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 &gt;5. Conversely, the pressure induced flow for 
the same channel will vary as the third power of the channel depth. Thus, 
the pressure build-up in a channel due to the simultaneous presence of 
fluid regions of differing ionic strength will vary as the square of the 
channel depth. 
Accordingly, by decreasing the depth of the intersecting second channel 
relative to the depth of the first or main channel by a factor of X, one 
can significantly reduce the pressure induced flow, e.g., by a factor of 
X.sup.3, while only slightly reducing the electroosmotically induced flow 
into that channel, 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 
magnitude, the pressure induced flow into that second channel 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 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 about two times as deep 
as the second channel, preferably greater than about 5 times as deep as 
the second channel, and often greater than about ten times as deep as the 
second channel. 
The operation of the concentrator element is schematically illustrated in 
FIG. 5B through 5E. As shown, the sample fluid containing the subject 
material and having a low conductivity (shown in grey) is loaded into the 
main channel by transporting the material from a sample channel 504 to 
waste channel 508 via the main channel 502, typically by applying a 
voltage gradient along this path. The direction of fluid flow is indicated 
by the arrows. As noted above, the sample fluid in the main channel 
(shaded region) has a low relative conductivity, and is typically bounded 
by fluid regions having relatively high conductivity (shown unshaded). 
Transport of the sample fluid into the main channel is typically carried 
out by application of a voltage gradient between reservoirs that are 
separately in fluid communication with the sample and waste channels. 
Switching the voltage gradient across the length of main channel 502, 
i.e., by applying different voltages at the termini of the main channel, 
then allows the transport of the subject material along that channel 
(Figure 5C). Concurrent with this transport is the electrophoretic 
stacking of the charged subject material within the subject material 
fluid, indicated by the darker shading at the front of the sample fluid 
plug being transported along the main channel (Figures 5C and 5D). 
Although illustrated in terms of the subject material being concentrated 
at the front of the subject material region, it will be readily 
appreciated that the charge on the subject material will affect the 
mobility of that material under an electic field. For example, if the 
subject material possesses a net charge opposite to that for which 
concentration is illustrated in FIG. 5B-5D, then the subject material will 
be concentrated at the back end of the subject material region, relative 
to the direction of electroosmotic flow. 
Finally, as shown in FIG. 5E, when the concentrated or stacked portion of 
the sample fluid reaches the third intersection, the concentrated portion 
can be shunted off from the remainder of the sample fluid plug by 
appropriate application of a voltage gradient along that path, effectively 
resulting in the concentration the subject material. In some cases, it is 
not necessary to shunt off the concentrated material in order to see the 
benefit of the concentration of the material. For example, where screening 
compounds of relatively low concentraton, the increased localized 
concentration of the compound at the front end of the low ionic 
concentration region resulting from the concentration methods described 
herein may be sufficient to produce an adequately detectable result. 
All publications and patent applications are herein incorporated by 
reference to the same extent as if each individual publication or patent 
application was specifically and individually indicated to be incorporated 
by reference. Although the present invention has been described in some 
detail by way of illustration and example for purposes of clarity and 
understanding, it will be apparent that certain changes and modifications 
may be practiced within the scope of the appended claims.