U.S. patent application number 11/411992 was filed with the patent office on 2006-10-05 for methods and systems for reducing background signal in assays.
This patent application is currently assigned to Caliper Life Sciences, Inc.. Invention is credited to Theo T. Nikiforov, Aileen Zhou.
Application Number | 20060219557 11/411992 |
Document ID | / |
Family ID | 36576390 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060219557 |
Kind Code |
A1 |
Nikiforov; Theo T. ; et
al. |
October 5, 2006 |
Methods and systems for reducing background signal in assays
Abstract
Methods and systems of monitoring reactions, e.g., assays, that
filter out background signal from substrate, in detecting the
product. The methods and systems controllably move detectable
reaction product from a first location to a second location at
which the product is detected while controllably moving potentially
interfering substrate materials away from or not toward the second
location. Controllable movement of the different species is
accomplished through the controlled combination of bulk fluid flow
and differential electrophoresis of substrate and product to move
the product, but not the substrate past the detection region.
Inventors: |
Nikiforov; Theo T.; (San
Jose, CA) ; Zhou; Aileen; (San Leandro, CA) |
Correspondence
Address: |
CALIPER LIFE SCIENCES, INC.
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043-2234
US
|
Assignee: |
Caliper Life Sciences, Inc.
Mountain View
CA
|
Family ID: |
36576390 |
Appl. No.: |
11/411992 |
Filed: |
April 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10202487 |
Jul 24, 2002 |
7060171 |
|
|
11411992 |
Apr 25, 2006 |
|
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60309113 |
Jul 31, 2001 |
|
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Current U.S.
Class: |
204/451 |
Current CPC
Class: |
G01N 27/44726
20130101 |
Class at
Publication: |
204/451 |
International
Class: |
C07K 1/26 20060101
C07K001/26 |
Claims
1. A method of monitoring a reaction, comprising: providing a
quantity of a first reaction mixture at a first location in a
fluidic conduit, the first reaction mixture comprising: a first
reagent having a detectable label associated therewith; a second
reagent that reacts with the first reagent to produce a first
product having the detectable label associated therewith, the first
product having an electrophoretic mobility that is different from
the first reagent; providing a detection zone at a second location
in the fluidic conduit, the second location being different from
the first location; applying an electric field to the first
reaction mixture; controlling bulk fluid flow through the fluid
conduit, whereby under the electric field applied in the applying
step the first product moves from the first location to the second
location in a substantially greater ratio to the first reagent, as
compared to the ratio of product to first reagent at the first
location; and detecting the product at the second location.
2. The method of claim 1, wherein the ratio of product to first
reagent at the second location is greater than 2 times the ratio of
product to first reagent at the first location.
3. The method of claim 1, wherein the ratio of product to first
reagent at the second location is greater than 5 times the ratio of
product to first reagent at the first location.
4. The method of claim 1, wherein the ratio of product to first
reagent at the second location is greater than 10 times the ratio
of product to first reagent at the first location.
5. The method of claim 1, wherein the ratio of product to first
reagent at the second location is greater than 100 times the ratio
of product to first reagent at the first location.
6. The method of claim 1, wherein the ratio of product to first
reagent at the second location is greater than 500 times the ratio
of product to first reagent at the first location.
7. The method of claim 1, wherein during the controlling step, the
first reagent is directed from the first location substantially
away from the second location.
8. The method of claim 7, wherein at least 50% of the first reagent
at the first location is directed away from the second
location.
9. The method of claim 7, wherein at least 90% of the first reagent
at the first location is directed away from the second
location.
10. The method of claim 7, wherein at least 95% of the first
reagent at the first location is directed away from the second
location.
11. The method of claim 1, wherein the fluid conduit comprises a
microfluidic conduit.
12. The method of claim 11, wherein the fluid conduit comprises at
least first and second fluidly communicating fluid channels.
13. The method of claim 12, wherein the first location comprises a
location in the first fluid channel, and the second location
comprises a location in the second fluid channel.
14. The method of claim 12, wherein the first location comprises a
zone of intersection of the first fluid channel and the second
fluid channel and the second location comprises a zone in the
second fluid channel.
15. The method of claim 1, wherein the first reagent comprises a
phosphorylatable compound, and the second reagent comprises a
kinase enzyme.
16. The method of claim 1, wherein the first reagent comprises a
polypeptide and the second reagent comprises a protease enzyme
capable of cleaving the polypeptide.
17. The method of claim 1, wherein the first reagent comprises a
phosphorylated compound, and the second reagent comprises a
phosphatase enzyme.
18. A method of monitoring a reaction, comprising: providing a
fluid channel that comprises a first channel segment and a second
channel segment; introducing into the first channel segment a
substrate for the reaction having a detectable label associated
therewith and a first electrophoretic mobility, and a first reagent
that reacts with the substrate to produce a first product that
includes the detectable label and a second electrophoretic mobility
that is different from the first electrophoretic mobility; applying
an electric field between the first channel segment and the second
channel segment and controlling bulk fluid flow between the first
channel segment and the second channel segment, whereby the
substrate, but not the product is substantially directed away from
the second channel segment; and detecting the product in the second
channel segment.
19. A method of monitoring a reaction, comprising: providing a
fluid conduit having first and second zones that are at different
locations from each other; providing a quantity of a reaction
mixture in the first zone that comprises: a first reagent having a
detectable label and a first electrophoretic velocity in the
direction of the second zone of V.sub.S under an applied electric
field; a second reagent that reacts with the first reagent to
produce a product that includes the detectable label and has a
second electrophoretic velocity in the direction of the second zone
of V.sub.P under the applied electric field; applying the electric
field across a length of the fluid conduit between the first and
second zones; controlling bulk fluid movement in the direction from
the first zone to the second zone (F), whereby F+V.sub.P is greater
than 0 and F+V.sub.S is less than or equal to 0; and detecting the
product at the second zone.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/202,487, filed Jul. 24, 2002, which claims
the benefit of U.S. Provisional Patent Application No. 60/309,113,
filed Jul. 31, 2001, which is incorporated herein by reference in
its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] A number of advances have been made in the fields of
biotechnology and pharmaceutical research to increase the speed and
accuracy of analytical operations. In at least one major
advancement, technologies developed for the semiconductor industry
have been adapted to manufacture miniaturized integrated devices
that can be used to perform analytical operations much more
quickly, with much greater accuracy, and with less operator
involvement. These microfluidic devices have been commercially
adapted for genetic and protein analysis in the form of the Agilent
2100 Bioanalyzer and associated LabChip.RTM. microfluidic devices
and reagents developed by Caliper Life Sciences, Inc. Other
commercial applications for microfluidic devices and systems are in
the pharmaceutical industry where they are used in ultra
high-throughput screening analysis. These systems allow large
numbers of different pharmaceutical candidate compounds to be
screened against target assays in relatively short amounts of time,
to determine whether any of those compounds possess desirable
pharmacological activity. The resulting assays give improved data
quality and increased automation, while minimizing the amounts of
potentially very expensive reagents.
[0003] While microfluidic systems have been shown to improve the
speed and accuracy of screening assays, there are a number of areas
where the small size and enhanced speed of these systems can be a
handicap to a screening assay. This is the case, for example, where
a particular analyte is at a very low concentration in the fluid
that is being tested. In such instances, the small volumes of the
fluid that are present in a detection region of a microfluidic
device may contain only a few hundred molecules of interest. In
such cases, the amount of material present may fall below the
detection level of the particular system that is being employed.
Similarly, for assays that progress at relatively slow rates, the
speed of operation of microfluidic systems may cause some
difficulty in yielding enough product of the reaction so that it
can be readily and accurately detected. The present invention
provides some solutions for these problems, as well as others that
may be faced in microfluidic and other analytical systems.
BRIEF SUMMARY OF THE INVENTION
[0004] The present invention generally provides methods of
monitoring reactions, e.g., assays, that filter out background
signal from substrate, in detecting the product. In a first aspect,
the invention provides a method of monitoring a reaction that
comprises providing a quantity of a first reaction mixture a t a
first location in a fluidic conduit. The first reaction mixture
comprises a first reagent having a detectable label associated
therewith, and a second reagent that reacts with the first reagent
to produce a first product having the detectable label associated
therewith, and an electrophoretic mobility that is different from
the first reagent. A detection zone is provided at a second
location in the first fluidic conduit, wherein the second location
is different from the first location. An electric field is then
applied to the first reaction mixture and bulk fluid flow through
the fluid conduit is controlled, whereby under the applied electric
field, the first product moves from the first location to the
second location in a substantially greater ratio to the first
reagent, as compared to the ratio of product to first reagent at
the first location. The product is then detected at the second
location.
[0005] In a related aspect, a method is provided for monitoring a
reaction that comprises providing a fluid channel that comprises a
first channel segment and a second channel segment. Reagents are
introduced into the first channel segment that include a substrate
for the reaction having a detectable label associated therewith and
a first electrophoretic mobility, and a first reagent that reacts
with the substrate to produce a first product that includes the
detectable label and a second electrophoretic mobility that is
different from the first electrophoretic mobility. An electric
field is applied (initiated or maintained) between the first
channel segment and the second channel segment and the bulk fluid
flow is controlled between the first channel segment and the second
channel segment, whereby the substrate, but not the product is
substantially directed away from the second channel segment. The
product is then detected in the second channel segment.
[0006] In yet another related aspect, the invention provides a
method of monitoring a reaction that comprises providing a fluid
conduit having first and second zones that are at different
locations from each other. A quantity of a reaction mixture is
provided in the first zone that includes a first reagent having a
detectable label and a first electrophoretic velocity in the
direction of the second zone of V.sub.S under an applied electric
field, and a second reagent that reacts with the first reagent to
produce a product that includes the detectable label and as a
second electrophoretic velocity in the direction of the second zone
of V.sub.P under the applied electric field. The electric field is
applied across a length of the fluid conduit between the first and
second zones and bulk fluid movement in the direction from the
first zone to the second zone (F) is controlled, whereby F+V.sub.P
is greater than 0 and F+V.sub.S is less than or equal to 0. Again,
the product is detected at the second zone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a schematic illustration of a conventional
mobility shift assay method.
[0008] FIG. 2 is a schematic illustration of the mobility shift
assay methods of the present invention that filter out background
substrate signal from detected product.
[0009] FIG. 3 is a schematic illustration of an integrated channel
network approach to practicing the invention.
[0010] FIG. 4 is a schematic illustration of a microfluidic device
and integrated channel network for use in practicing the
invention.
[0011] FIG. 5 is a plot of fluorescence from an assay of injected
inhibitors using assay formats and methods of the invention.
DETAILED DESCRIPTION OF THE INVENTION
I. General
[0012] The present invention is generally directed to methods of
monitoring reactions, and particularly to assay methods that
exploit changes in electrophoretic mobility of the product relative
to the substrate in order to characterize the extent and/or speed
of the reaction in questions. While shifts in electrophoretic
mobility have been used to characterize reactions previously, the
present invention utilizes a controlled bulk fluid flow, in
conjunction with the shift in electrophoretic mobility to remove
any, or at least a substantial amount of background substrate
signal during the detection step in order to achieve higher
sensitivity and accuracy.
[0013] Typically, assay formats that utilize shifts in
electrophoretic mobility, also termed "mobility shift assays",
involve a simple electrophoretic separation of the substrate and
product after the reaction that alters the electrophoretic mobility
of the product relative to the substrate, where both substrate and
product are detected. As used herein, "electrophoretic mobility"
refers to the net mobility of a chemical species when subjected to
an electric field, which mobility is solely attributable to the
charge based movement of that species under the electric field.
Electrophoretic mobility is generally used as a relative term, in
that changes in electrophoretic mobility of, e.g., a product
relative to a substrate, generally denotes situations where
conditions that might effect the electrophoretic mobility of the
species that are external to the species, e.g., environmental
conditions such as pH, and salt concentration, remain substantially
unchanged.
[0014] FIG. 1 schematically illustrates a typical mobility shift
assay format as traditionally carried out in a capillary channel,
i.e., a capillary or channel of a microfluidic device. As shown, an
enzyme (E) and a labeled substrate (S) are introduced into a fluid
conduit, e.g., a capillary or a channel in a fluidic device. The
interaction of the substrate and the enzyme results in a product
(P) that has changed in its electrophoretic mobility relative to
the substrate, but still includes the detectable label from the
substrate. Under a constant applied electric field (E), this shift
in electrophoretic mobility (.mu.) results in a shift in the
electrophoretic velocity (V) of the product relative to the
substrate. Briefly, the electrophoretic mobility of a compound is
independent of the electric field applied, while the
electrophoretic velocity is dependent upon the applied electric
field, e.g., V=(.mu.)(E).
[0015] The shift in electrophoretic mobility, and consequently,
electrophoretic velocity is typically caused by a change in the
charge-to-: mass ratio of the product relative to the substrate. By
way of example, an uncharged substrate may have a highly charged
chemical group attached to it or removed from it by the action of
the enzyme that is being assayed, e.g., a phosphate group attached
by a kinase. In another example, a substrate may be acted upon by
the enzyme in question to cleave or otherwise separate the
substrate into two products. By providing a substrate with an
uneven charge distribution, it can result in at least one product
that has a substantially different charge-to-: mass ratio and thus,
a substantially different electrophoretic mobility and velocity
from the substrate. For example, a polypeptide can be provided with
a highly charged region that is away from the cleavage zone for a
protease of interest. Cleavage of the polypeptide then shifts the
electrophoretic mobility of each product (e.g., both fragments)
relative to the substrate polypeptide as a whole.
[0016] Referring again to FIG. 1, the reaction mixture is typically
flowed along the conduit while the reaction is occurring, or after
the reaction has occurred. This flow is typically as a result of
the bulk flow of the fluid through the conduit (as indicated by the
solid arrows), which results in equivalent flow of all of the
components of the reaction mixture (E, S and P). While moving, the
reaction mixture is subjected to an electric field, which then
results in a differential electrophoretic velocity of the substrate
and product (as indicated by the dashed arrow associated with the
product). The whole mixture is flowed past the detection point (as
indicated by the dashed ellipse). Because the product is moving at
a different rate (the sum of the bulk flow and the electrophoretic
velocity) from the substrate (moving with the bulk flow), it
results in detection of one labeled species as the product and
another that represents the substrate. Because separations are
never perfect, there can be some overlap between these peaks,
reducing the overall resolution of the assay format. In particular,
any substrate that is detected at the same time as the product is
detected results in a higher background level signal. Further, in
order to assure more sensitive detection, one may need to perform
the electrophoretic separation over a longer distance and for
longer periods of time, in order to ensure adequate separation
between the product and substrate.
[0017] The presence of substrate and product during the detection
step is particularly problematic in continuous flow assay methods
and systems, e.g., as described in U.S. Pat. Nos. 5,942,443 and
6,046,056, each of which is incorporated herein by reference in its
entirety for all purposes. In these continuous flow assays, in a
continuing stream of reaction components, e.g., enzyme and
substrate, is flowed along the analysis channel or conduit, which
react and produce a steady state amount of product by the time the
mixture reaches a detection point. When a test compound that has an
effect on the reaction, e.g., as an inhibitor, is introduced into
the flowing stream, it produces a perturbation in the steady state
amount of product. In typical fluorogenic assays, this perturbation
is readily measured as a shift in the amount of detectable signal.
In mobility shift assays, however, both the product and substrate
have the same signal. As such, an electric field is applied to the
mixture, resulting in localized concentration and depletion of
substrate and product to yield a representative signal profile. For
example, fluorescent product that moves faster under an electric
field will move ahead of the slower moving substrate. In the
absence of an inhibitor, this difference is undetectable, as the
overall flowing is constant. When an inhibitor is introduced in a
discrete plug into the flowing stream, it results in a reduction in
the amount of product, yielding a consequent reduction in the
amount of signal running ahead of the mixture. This also produces
an increase in the amount of slower, unreacted substrate running
with or behind the reaction mixture.
[0018] While these methods have proven effective, the existence of
a constant stream of both substrate and product through the conduit
results in a certain level of background fluorescence, from the
omnipresent substrate. In particular, while the overall reaction
produces an overall signal indicative of a perturbation in the
amount of product produced, that signal is overlaid on a constant
signal from a certain steady state level of substrate. This
constant signal reduces the overall sensitivity level of the assay,
e.g., the signal of interest, e.g., from the product, may be only a
small fraction of the overall fluorescence emanating from the
reaction mixture. This is complicated further in assay systems that
have relatively slow reaction kinetics, e.g., enzymes with slow
substrate turnover rates. In particular, the change in signal
resulting from a perturbation in the amount of product produced,
where very little product is produced, can be very small as
compared to the background signal from substrate, and can be lost
in the noise from the relatively high level of substrate signal. As
such, these slower reactions can be very difficult to analyze.
[0019] By filtering out background levels of substrate from the
detected product (and perturbations of the product) the methods of
the present invention increase the sensitivity of the overall
assay, and allow the assay method to be applied more readily to
assays that have substantially slower kinetics. The present
invention filters out the background signal emanating from
substrate by directing the substrate away from the detection zone,
while permitting the product to proceed through the detection zone.
In general, this is accomplished by electrophoresing one of the
substrate or the product in the desired direction. The differential
flow direction of the other species may be the result of opposite
electrophoretic velocity, or bulk fluid flow, or both.
[0020] An example of the methods of the present invention is
schematically illustrated in FIG. 2, in a format similar to that of
FIG. 1. In particular, as shown the enzyme E and substrate S are
introduced into the conduit. In this instance however, a different
bulk flow profile is used (as shown, bulk flow (F) is slightly away
from the detection zone), as shown by the solid arrows. When the
electric field is applied, the product still has a net overall flow
in the direction of the detector, as shown by the combination of
the solid and dashed arrows associated with the product (P), e.g.,
the sum of the electrophoretic velocity of the product (V.sub.P) (a
highly positive value) and the bulk flow (F) (slightly negative
value) is greater than zero. Again, the substrate has an overall
velocity that is the sum of the bulk flow and the electrophoretic
velocity of the substrate (V.sub.S) (which is shown as zero). In
this instance, however, the bulk flow is in a direction away from
the detector. Consequently, the substrate, which is moving with the
bulk flow is also directed away from the detector, e.g., the sum of
the electrophoretic velocity of the substrate toward the detector
(zero, as shown) and the bulk flow toward the detector (shown as a
negative value) is less than or equal to 0. As a result, only the
product moves past the detector. This eliminates any background
signal from the substrate that might reduce the sensitivity of the
overall assay. While the benefits of the methods of the invention
are clearest in small scale systems, e.g., microfluidics, where
assay sensitivity is at a premium, the methods are also useful in
enhancing the sensitivity of larger scale systems, e.g.,
non-microscale systems. Although described in terms of the bulk
flow being in a direction away from the detector, it will be
appreciated that bulk flow may be in the direction of the detector
where the product flows with the bulk flow, provided that the
substrate's overall mobility is away from the detection zone.
[0021] Although the above illustration is with respect to a
substrate that has no electrophoretic mobility under the applied
electric field, it will be appreciated that substrates that have
electrophoretic mobility can be used in these methods, whether that
mobility is in the same or in a different direction from the
product, provided that the electrophoretic velocity of the
substrate is sufficiently different from that of the product. For
example, where the substrate has some electrophoretic velocity,
e.g. in the direction of the detection zone, the bulk flow may be
adjusted to be of sufficient magnitude to overcome that
electrophoretic mobility, but not of sufficient magnitude to
overcome the electrophoretic mobility of the product. Where, on the
other hand, the substrate has an electrophoretic mobility in the
direction opposite to that of the product, then no bulk flow may be
required, and in fact, bulk flow toward the detection zone may be
used to increase the speed at which the product reaches the
detector, provided the bulk flow is not sufficient to overcome the
electrophoretic mobility of the substrate in the opposite
direction. Accordingly, the desired result is that the overall flow
of the substrate toward the detection zone, e.g., the sum of
electrophoretic velocity of the substrate (V.sub.S) and bulk flow
(F) is less than or equal to zero, while the overall flow of the
product toward the detection zone, e.g., the sum of the
electrophoretic velocity of the product (V.sub.P) and the bulk flow
(F) is greater than zero.
[0022] Although illustrated with respect to a single straight fluid
conduit, it will be appreciated that the methods of the present
invention are most useful in the context of integrated fluid
channel networks, e.g., lab-on-chip devices. The methods of the
invention are schematically illustrated in such an integrated
device in FIG. 3A. As shown, the device 300 includes a main
reaction channel 302 that is fluidly coupled to an external
sampling capillary (not shown) via port 310, for sampling test
compounds into the reaction channel 302. Reagent channels 304 and
306 are fluidly coupled to the main reaction channel 302, and bring
in reagents for the assay, e.g., enzyme and substrate,
respectively, from reservoirs 320 and 322. A detection channel 308
is provided fluidly connected to the main channel 302, and the
product of the assay reaction is directed preferentially down this
channel through detection zone 324 toward waste reservoir 326,
while the substrate continues to flow along the reaction channel
toward waste reservoir 330, or flows into optional auxiliary
channel 312 toward reservoir 328, which may be employed, e.g., to
allow application of an electric field that separates product and
substrate, and directs product into the detection channel 308.
Although illustrated with two reagent reservoirs 320 and 322, it
will be appreciated that in certain preferred aspects, additional
reservoirs and connecting channels may be provided for storing
additional reagents and/or buffers and diluent for the reagent
present in reservoirs 320 and 322.
[0023] Operation of the device illustrated in FIG. 3A, in the
methods of the invention, is schematically illustrated in FIG. 3B,
which illustrates a close-up of the intersection region of channels
302, 308 and 312 during operation. As can be seen, the reaction
mixture introduced into the main reaction channel includes an
enzyme (E) and a substrates (S), which react to produce the product
(P). The bulk flow of the reaction mixture is directed down the
entire length of the main reaction channel 302. An electric field
is applied through the intersection of the reaction channel and
detection channel 308 and auxiliary channel 312, e.g., via
electrodes 314 and 316, respectively. Because of its different
electrophoretic velocity under the applied electric field
(resulting from a shift in its electrophoretic mobility), the
product is driven from the reaction channel 302 into the detection
channel 308, by virtue of the applied electric field. The substrate
is either carried on through the reaction channel by the bulk flow
of the fluid, e.g., if the substrate is substantially uncharged, or
directed into auxiliary channel 312, by the electric field, as
indicated by the arrows, as a result of the combination of bulk
flow and the electrophoretic velocity of the substrate being net
away from the detection zone in the detection channel.
[0024] By controlling the bulk flow and the electric field, one can
optimally direct the product into the detection channel, while only
allowing only a small amount or no substrate into the detection
channel. In preferred aspects, this is accomplished by forcing the
substrate in a different direction from the product. However, it
will be appreciated that by adjusting the bulk flow of fluid
through the reaction channel, one can ensure that the substrate is
not exposed to the electric field for sufficient time or does not
have access to the entry to the detection channel for sufficient
time to permit substantial amounts of substrate to be directed into
the detection channel. This is slightly different from the net
movement of substrate away from the detection zone, as described
above, in that the substrate may have a net overall flow toward the
detection zone, but that this net overall flow toward the detection
zone is substantially reduced relative to the product. By thus
reducing the opportunity for the substrate to move in the direction
of the detection zone., e.g., by moving the substrate through an
intersection with the detection channel at sufficient rate to
minimize the entry of the substrate into the detection channel, one
can substantially reduce the interfering substrate being
detected.
[0025] As a result of the various methods of the invention, the
ratio of product to substrate in the detection channel is increased
substantially as compared to the ratio in the reaction channel.
Typically, the methods of the invention will result in at least a 2
fold, preferably, at least a 5 fold, and often a 10, 100 or 500
fold increase in the ratio of product to substrate in the detection
zone, as compared to that ratio within the reaction channel in the
absence of the applied electric field. In preferred aspects, the
labeled substrate is substantially directed away from the detection
zone, e.g., by continuing to be directed along the reaction channel
or by being directed into an auxiliary channel. By "substantially
directed away" from the detection zone is meant that at least 50%
of the labeled substrate is moved from the reaction channel or
location, e.g., the first location, in a direction in the conduit
or conduit network that is away from the detection zone, or second
location. In preferred aspects, at least 90%, more preferably, at
least 95% and in some cases, at least 99% of the labeled substrate
is moved in a direction other than through a channel toward the
detection zone.
[0026] As noted above, in the case where the product and substrate
are each charged, e.g., each having a different net positive charge
or net negative charge, then the bulk fluid flow within the
reaction channel may be adjusted to optimize for product direction
toward the detection zone, while minimizing the amount of substrate
that travels toward the detection zone. This optimization may be
carried out by performing control experiments, and/or by
calculating the mobilities of the substrate and product under the
applied electric field, and applying a bulk flow rate that achieves
the desired result.
II. Systems
[0027] A. System Elements
[0028] In accordance with preferred aspects of the present
invention, microfluidic channels or channel networks are operated
in conjunction with controller detector systems. In particular, the
channel or channel network is typically provided as an integral
device that includes one or more channel disposed in a solid body
structure. The device is placed into the controller/detector
instrument which typically includes a controller for controlling
the bulk fluid flow and electric field applied to the channel, as
well as a detector for detecting the product in the channel. The
overall system is also typically coupled to a computer for
recording data from the detector and for instructing the operation
of the controller. An example of an overall system for carrying out
the present invention is illustrated in FIG. 4. As shown, the
system includes a microfluidic device 400 that includes an
appropriate channel network disposed therein. The system includes a
flow controller 402 that comprises a pressure or vacuum source,
e.g., pump 414. The pressure source or pump 414 is shown coupled to
one reservoir of the microfluidic device via pressure or vacuum
line 410 and vacuum or pressure sealed coupling 412. The controller
also includes an electrical power source that is coupled to
reservoirs of the microfluidic device via electrodes 408, in order
to drive the electrophoretic movement of the assay components. The
system also includes a detector 404 as well as a computer or
processor 406 that is operably coupled to both the detector 404 and
the controller 402. The computer typically includes appropriate
programming to receive user input information and transfer that
information into instructions for the flow controller. The computer
also typically receives the data from the detector and manages that
data into a user understandable presentation.
[0029] B. Microfluidic Devices
[0030] The methods described herein are particularly useful, and
gain their greatest flexibility when used in conjunction with
integrated microfluidic devices. Microfluidic devices typically
comprise a unitary body structure in which is disposed a channel
network that includes one or more connected channels, as well as
fluid reservoirs for accessing and providing fluid for the various
channels of the device to carry out a desired operation. Typically,
such devices are fabricated as an aggregate of planar substrate
layers, where the channels are fabricated into the surface of one
or more of the facing substrates as grooves, wells, or
indentations. The mating of one substrate to the other covers the
grooves and seals them to form the channels of the device. One or
more of the substrates is also provided with apertures disposed
through it. These apertures are typically positioned so as to
provide access to the channels in the finished device. The
apertures also function as the reservoirs of the device for
introducing fluids into the channels, as well as providing access
for pressure sources and/or electrical connection ports for moving
materials through and among the various channels of the device. In
certain cases, a sampling element or pipettor is also provided
integrated into the body of the microfluidic device. The sampling
element typically comprises a capillary that includes a channel or
lumen disposed through it. The capillary is attached to the body of
the device so as to provide a fluid connection between the
capillary lumen and the channel network within the device.
[0031] The channels of the device typically include at least one
cross-sectional dimension, e.g., width, depth and/or diameter that
is within the microscale regime, which as used herein refers to a
dimension that is between about 0.1 and 500 .mu.m. In preferred
aspects, channels are generally rectangular or trapezoidal in shape
and have a depth of from about 2 to about 50 .mu.m, and a width of
between about 10 and about 100 .mu.m. Of course, channel dimensions
will often vary depending upon the desired application. For
example, channels that are intended to conduct particulate
containing fluids will typically have larger cross-sectional
dimensions to avoid clogging. Similarly, channel cross-sectional
dimensions and length are often varied to provide a desired bulk
flow characteristic. Specifically, fluidic resistance within
channels, and thus, the force required to achieve a particular flow
profile are dictated, in part, by the cross-sectional area, as well
as the length of the channels that make up the channel network. As
such, different flow profiles may include different cross-sectional
dimensions.
[0032] In addition, the channels of the device may be treated or
filled in order to adjust the electrophoretic properties of
materials moving through those channels. Treatments include surface
treatments, e.g., coatings (static or dynamic), surface
derivatization, e.g., silanization and derivatization, etc.
Fillings used in adjusting electrophoretic properties include
dynamic coating materials such as polydimethylacrylamide polymers
that modify the surface properties of the channels, as well as
present a sieving matrix for separations.
[0033] C. Controllers
[0034] The systems of the present invention typically include
electrical controllers for driving the electrophoretic movement of
materials, as well as controllers for driving bulk flow of fluid
through the channels of a microscale fluidic system. Electrical
controllers typically include an electrical power supply that is
capable of delivering set voltages or currents to or through the
channels of the device to drive electrophoresis within those
channels. Such power supplies are generally described in, e.g.,
U.S. Pat. No. 5,800,690, which is incorporated herein by reference
in its entirety for all purposes, and include those contained
within the Agilent 2100 Bioanalyzer system available commercially
from Agilent Technologies, or the Caliper AMS 90 analytical
instrument, available from Caliper Technologies Corp. (Mountain
View, Calif.).
[0035] As noted, the control systems also include an element for
controlling bulk flow of fluid within the microscale fluid
conduits. Such systems may employ electrokinetic forces, e.g.,
electroosmotic flow that may be dictated by the applied electric
field, as well as the surface properties of the conduit. Such
systems are advantageous in that a single control element, namely
the electrical controller, is all that is needed to control both
electrophoresis and bulk fluid flow.
[0036] However, in preferred aspects, a pressure based controller
is used to drive bulk fluid flow in the systems of the invention.
Such controllers typically include a pressure or vacuum source,
e.g., a pump that is coupled to one or more of the ports to the
fluid conduit. In the case of microfluidic systems this comprises a
pressure or vacuum line from the source or pump, that is coupled to
one or more of the reservoirs of the device, and thereby
communicates changes in pressure to the channels of the device. In
certain cases, multiple pressure sources and connections are used
to adjust the pressure at numerous different ports to the device,
simultaneously, in order to control precisely the fluid flow
through integrated channel networks. Briefly, pressure sources are
coupled to the reservoirs at the termini of the channels in an
integrated channel network. Examples of such multiple pressure
source controllers are described in U.S. application Ser. No.
09/792,435, filed Feb. 22, 2001, which is incorporated herein by
reference in its entirety for all purposes. By controlling relative
pressures at each of multiple reservoirs, one can precisely control
the rate and direction at which fluids move through such integrated
channel networks.
[0037] The controller systems of the invention typically include a
detection system integrated into a single base unit, e.g., such as
the Agilent 2100 Bioanalyzer. Such detection systems typically
comprise optical detectors, and preferably, fluorescence detectors,
again, like the Agilent 2100 Bioanalyzer.
III. Assays
[0038] As noted above, the methods and systems of the present
invention are particularly useful in carrying out assaying
reactions that are characterized by producing a reaction product
that has a different electrophoretic mobility from one or more of
the reactants or substrates that are combined or reacted to yield
that product. Of particular interest are reactions in which
differential detection of the product and reactants or substrates
is difficult due to the similarity in detectable properties of the
two, e.g., inherent optical properties of fluorescent labeling. In
one particularly preferred aspect, the present invention is applied
to assaying reactions that result in the addition or removal of
phosphate groups to or from a particular substrate. These
reactions, typically mediated by kinase or phosphatase enzymes,
change the nature of the electrical charge on the substrate
molecule by adding or removing a highly charged phosphate group to
or from the substrate molecule. Typically, assays for these
reactions typically include an optically detectable label affixed
to the substrate. Without more, both the product and substrate
would be indistinguishable, as they would include the same label.
However, such assays typically perform a separation function to
separate substrate from product, whereupon the product is detected.
Such separations include immunoaffinity separations, as well as
charge based separations, e.g., chromatographic or electrophoretic.
As can be readily appreciated from the instant disclosure, the
present invention relies upon the direction of the labeled, e.g.,
phosphorylated product in the case of a kinase assay, or the
unphosphorylated product in the case of a phosphatase assay, toward
the detection zone in a microfluidic channel, while the labeled
substrate, e.g., a phosphorylated or phosphorylatable substrate for
the given assay is directed or at least maintained in a position
away from the detection zone.
[0039] While phosphatase and kinase assays are particularly
benefited by the invention, a large number of other assays can also
be performed in the format that is compatible with the invention,
or configured to be compatible with that format. By way of example,
protease enzyme assays often rely upon the change in size of the
labeled substrate, e.g., a large polypeptide, as it is converted to
the product, e.g., fragment(s) of the large polypeptide. This
change in size is again typically determined by a size-based
separation, e.g., using gel exclusion or electrophoretic methods.
By configuring a labeled substrate to provide a labeled product
that has a different electrophoretic mobility, e.g., changing the
charge-to-mass ratio of the product relative to the substrate, one
can easily adapt that assay to the format of the invention. For
example, one can provide a substrate that has a substantially
neutral charge by allocating positively charged groups to one
region of the polypeptide, while oppositely charged groups are
allocated to another region, where the regions are separated by the
putative cleavage point of the enzyme of interest. Cleavage of the
large polypeptide (substantially neutral) than yields two fragments
that have high net charges, and thus substantially different
electrophoretic mobilities.
[0040] Similarly, nucleic acid assays may be configured whereupon
the reaction yields a product having a different charge: mass ratio
from the initial reagent(s). Typically, the charge: mass ratio of
naturally occurring nucleic acids is a constant, yielding a
constant electrophoretic mobility of all nucleic acids, regardless
of size. Electrophoretic separation is generally accomplished by
moving the nucleic acids through a sieving matrix which
accomplishes the size base separation. In the methods of the
present invention, nucleic acid analogs may be employed, e.g., in
hybridization probes, so as to significantly change the
electrophoretic mobility of, e.g., a hybridized double stranded
product relative to a single stranded substrate. In this example,
the hybrid would be differentially and preferentially directed
toward the detection zone over either a labeled target sequence or
labeled probe. For example, uncharged nucleic acid analogs, e.g.,
peptide nucleic acids, can be used as the probe. Upon
hybridization, the mass of the hybridized product is increased
relative to single stranded target sequence without any increase in
charge. In the case of a labeled target, this allows for
differential direction of the hybrid and the single stranded
target. Where the probe is labeled, the hybridization reaction
suddenly adds substantial charge to the labeled hybrid relative to
the labeled probe, again, giving a basis for differentially
directing the two in accordance with the invention. A variety of
other analogs are also useful in this aspect of the invention,
provided the analogs have the ability to yield a shift in the
charge to-: mass ratio of the product relative to the labeled
substrate. Such analogs include positively charged analogs such as
methyl phosphonate polymers and cationic nucleic acid analogs.
IV. Example
[0041] To illustrate the method, a protein kinase A (PKA)
inhibition assay was carried out on an 80A chip, whose schematic
layout is shown in FIG. 3A. The peptide substrate for this kinase,
Fl-LRRASLG-CONH.sub.2, was placed in well 320, and the enzyme in
well 322. A pressure of -0.5 psi was applied to well 330, while a
voltage gradient of 2450V was applied between wells 326 and 328
(2500V to the cathode in well 328, and 50V to the anode in well
326). The continuous pressure-driven flow of substrate and product
into the main reaction channel resulted in the conversion of a
fraction of the neutral substrate to a negatively charged,
phosphorylated product. Under the influence of the applied electric
field between wells 326 and 328 this negatively charged product was
electrophoretically separated from the neutral substrate, migrated
towards well 326 and was detected in the channel leading to this
well. The neutral substrate was unaffected by the applied
electrical field and migrated toward well 330. In the absence of
any PKA inhibitors, a constant fluorescent signal was observed at
the detection point. Upon injection of PKI, an inhibitor of protein
kinase A, through the capillary attached to the chip, a zone
containing a lower concentration of the reaction product was
obtained, and this was detected as a temporary decrease of the
constant fluorescent signal at the detection point. This decrease
of the fluorescent signal was proportional to the concentration of
the injected inhibitor, as seen in FIG. 5. Although not shown in
this example, the detection point could also be placed in the
channel leading to well 330, and in this case the injection of an
inhibitor would result in the appearance of peaks of fluorescence
intensity instead of dips shown in the example in FIG. 5. The use
of a neutral substrate and charged product demonstrates the ability
to differentially direct substrate and product in accordance with
the methods of the invention, as is indicated by the substantial
drop in fluorescent signal in the inhibited regions of the plot of
FIG. 5 (e.g., very little neutral substrate was present to produce
background fluorescence when the enzyme reaction that yielded
fluorescence was inhibited).
[0042] 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.
* * * * *