U.S. patent application number 10/650174 was filed with the patent office on 2004-03-11 for microscale assays and microfluidic devices for transporter, gradient induced, and binding activities.
This patent application is currently assigned to Caliper Technologies Corp.. Invention is credited to Hodge, C. Nicholas, Parce, J. Wallace, Wada, H. Garrett.
Application Number | 20040048299 10/650174 |
Document ID | / |
Family ID | 27496186 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040048299 |
Kind Code |
A1 |
Parce, J. Wallace ; et
al. |
March 11, 2004 |
Microscale assays and microfluidic devices for transporter,
gradient induced, and binding activities
Abstract
Methods of monitoring transporter activity, gradient induced
activity, and binding activity in microscale systems, as well as
corresponding microscale devices, systems and kits are
provided.
Inventors: |
Parce, J. Wallace; (Palo
Alto, CA) ; Hodge, C. Nicholas; (Los Altos Hills,
CA) ; Wada, H. Garrett; (Atherton, CA) |
Correspondence
Address: |
CALIPER TECHNOLOGIES CORP
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043
US
|
Assignee: |
Caliper Technologies Corp.
Mountain View
CA
94043
|
Family ID: |
27496186 |
Appl. No.: |
10/650174 |
Filed: |
August 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10650174 |
Aug 28, 2003 |
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09579111 |
May 25, 2000 |
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6649358 |
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60155259 |
Jun 1, 1999 |
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60176001 |
Jan 12, 2000 |
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60176093 |
Jan 14, 2000 |
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60191784 |
Mar 24, 2000 |
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Current U.S.
Class: |
435/6.19 ;
435/287.2; 435/7.2 |
Current CPC
Class: |
G01N 21/65 20130101;
G01N 21/64 20130101; G01N 2333/70571 20130101; G01N 33/5302
20130101 |
Class at
Publication: |
435/006 ;
435/007.2; 435/287.2 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567; C12M 001/34 |
Claims
What is claimed is:
1. A method of detecting a binding activity, the method comprising:
flowing at least one first component or a set of first components
through a first channel concomitant with at least one second
component or a set of second components through the first channel,
wherein the at least one first component or the set of first
components diffuses more rapidly in solution than the at least one
second component or the set of second components, and wherein the
first channel comprises a mixing longitudinal segment, wherein the
at least one first component or the set of first components
diffuses substantially across the first channel in the mixing
longitudinal segment, and wherein the at least one second component
or the set of second components diffuses less than substantially
across the first channel in the mixing longitudinal segment,
wherein the at least one second component or the set of second
components binds to the at least one first component or the set of
first components; and, detecting a detectable signal that indicates
a final concentration of the at least one first component or the
set of first components that remains unbound after exiting from the
first channel, thereby detecting the binding activity.
2. The method of claim 1, further comprising the step of detecting
a detectable signal that indicates an initial concentration of the
at least one first component or the set of first components prior
to entry of the at least one first component or the set of first
components into the first channel.
3. The method of claim 1, wherein the at least one first component
or the set of first components is at least one ligand selected from
one or more of: an antigen, a set of antigens, a protein, a set of
proteins, a peptide, a set of peptides, a lipid, a set of lipids, a
carbohydrate, a set of carbohydrates, an inorganic molecule, a set
of inorganic molecules, an organic molecule, a set of organic
molecules, a drug, a set of drugs, a receptor ligand, a set of
receptor ligands, an antibody, a set of antibodies, a
neurotransmitter, a set of neurotransmitters, a cytokine, a set of
cytokines, a chemokine, a set of chemokines, a hormone and a set of
hormones.
4. The method of claim 1, wherein the binding activity is detected
at a temperature in the range of from about 10 to about 40.degree.
C.
5. The method of claim 1, wherein the binding activity is detected
at a temperature of about 25.degree. C.
6. The method of claim 1, wherein the binding activity is detected
at a temperature of about 37.degree. C.
7. The method of claim 1, wherein the at least one first component
or the set of first components diffuses in the range of from about
1.5 to about 100 times faster in solution than the at least one
second component or the set of second components.
8. The method of claim 1, wherein the at least one first component
or the set of first components diffuses about 50 times faster in
solution than the at least one second component or the set of
second components.
9. The method of claim 1, wherein an initial concentration of the
at least one first component or the set of first components prior
to entry into the first channel is in the range of from about 1 nM
to about 1 mM.
10. The method of claim 1, wherein an initial concentration of the
at least one first component or the set of first components prior
to entry into the first channel is about 10 .mu.M.
11. The method of claim 1, wherein the at least one first component
or the set of first components comprises a molecular weight in the
range of from about 200 to about 1000 daltons.
12. The method of claim 1, wherein the at least one first component
or the set of first components comprises a molecular weight of
about 400 daltons.
13. The method of claim 1, wherein the at least one first component
or the set of first components comprises a diffusional coefficient
in the range of from about 10.sup.-12 to about 10.sup.-4
cm.sup.2s.sup.-1.
14. The method of claim 1, wherein the at least one first component
or the set of first components comprises a diffusional coefficient
of about 10.sup.-6 cm.sup.2s.sup.-.
15. The method of claim 1, wherein the at least one second
component or the set of second components comprises one or more of:
an enzyme, a receptor, a cell, or a nucleic acid.
16. The method of claim 1, wherein the at least one second
component or the set of second components is an enzyme having a
concentration in the range of from about 1 nM to about 1 mM.
17. The method of claim 1, wherein the at least one second
component or the set of second components is an enzyme having a
concentration of about 10 .mu.M.
18. The method of claim 1, wherein the at least one second
component or the set of second components is an enzyme that
comprises a molecular weight in the range of from about 10 to about
200 kilodaltons.
19. The method of claim 1, wherein the at least one second
component or the set of second components is an enzyme that
comprises a molecular weight of about 30 kilodaltons.
20. The method of claim 1, wherein the at least one first component
or the set of first components and the at least one second
component or the set of second components are flowed using one or
more fluid direction components comprising one or more of: a fluid
pressure force modulator, an electrokinetic force modulator, a
capillary force modulator, and a fluid wicking element.
21. The method of claim 1, further comprising the step of flowing
the at least one first component or the set of first components
through the first channel, thereby providing a positive control for
detecting the detectable signal.
22. The method of claim 1, further comprising the step of flowing
the at least one second component or the set of second components
through the first channel, thereby providing a negative control for
detecting the detectable signal.
23. The method of claim 1, wherein the first channel is a
microchannel.
24. The method of claim 1, the method comprising concomitantly
flowing at least one modulator into contact with the at least one
second component or the set of second components in the first
channel, wherein the at least one modulator modulates the binding
of the at least one second component or the set of second
components to the at least one first component or the set of first
components.
25. The method of claim 24, wherein the at least one modulator
inhibits the binding of the at least one second component or the
set of second components to the at least one first component or the
set of first components.
26. The method of claim 24, wherein the detected binding activity
provides an indication of one or more of: the binding activity of
the at least one second component or the set of second components
and an ability of the at least one modulator to modulate the
binding activity of the at least one second component or the set of
second components.
27. The method of claim 1, wherein the detectable signals are
selected from: a refractive index, a cellular activity, a light
emission, a change in absorbance, a change in fluorescence, an
absorbance, a fluorescence, a color shift, a fluorescence resonance
energy transfer a radioactive emission, a change in pH, a change in
temperature, and a change in mass.
28. A kit comprising at least one first component or a set of first
components and at least one second component or a set of second
components, wherein the at least one first component or the set of
first components diffuses more rapidly in solution than the at
least one second component or the set of second components, wherein
when the at least one first component or the set of first
components and the at least one second component or the set of
second components are concomitantly flowed in a channel, the at
least one first component or the set of first components diffuses
substantially across the channel in a mixing longitudinal segment
of the channel, and wherein the at least one second component or
the set of second components diffuses less than substantially
across the channel in the mixing longitudinal segment of the
channel, wherein the at least one second component or the set of
second components binds to the at least one first component or the
set of first components; the kit further comprising one or more of:
a container for packaging the at least one first component or the
set of first components and the at least one second component or
the set of second components, instructions for practicing the
method of claim 1, one or more reagents for buffering or storing
the at least one first component or the set of first components and
the at least one second component or the set of second components,
and one or more test compounds.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. Nos. 09/579,111, filed May 25, 2000, which is related to
09/323,747, filed on Jun. 1, 1999 (converted to Provisional U.S.
Patent Application No. 60/155,259, to J. Wallace Parce, et al., and
entitled "Microscale Assay and Microfluidic Device for Transporter
Activity") and to Provisional U.S. Patent Application Nos.
60/176,001, filed Jan. 12, 2000; 60/176,093, filed Jan. 14, 2000;
and 60/191,784, filed Mar. 24, 2000, each to J. Wallace Parce, et
al. and each entitled "Microscale Assays and Microfluidic Devices
for Transporter, Gradient Induced, and Binding Activities." The
present application claims priority to and the benefit of each of
these earlier applications, pursuant to 35 U.S.C. .sctn. 19(e), as
well as any other applicable statute or rule. These prior
applications are incorporated herein in their entirety for all
purposes.
COPYRIGHT NOTIFICATION
[0002] Pursuant to 37 C.F.R. 1.71(e), Applicants note that a
portion of this disclosure contains material which is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or patent
disclosure, as it appears in the Patent and Trademark Office patent
file or records, but otherwise reserves all copyright rights
whatsoever.
BACKGROUND OF THE INVENTION
[0003] Model systems which mimic receptor binding and other
cell-based assays are of increasing importance in molecular
biology. Phenomena such as transporter activity, pre- and
post-synaptic cell interactions, chemotactic response,
enzyme-ligand binding and the like are of relevance to
pharmacology, clinical chemistry and basic research.
[0004] For example, one clinically relevant system involves the
interaction of cells at synapses. At least two general types of
synapses exist in nature. In electrical synapses, gap junctions
connect cells which are in communication. Gap junctions permit
direct transmission of electrical impulses from a presynaptic cell
to a postsynaptic cell.
[0005] In the more common chemical synapse, an axon terminal of a
presynaptic cell contains vesicles filled with a neurotransmitter,
such as epinephrine or acetylcholine, which is released by
exocytosis when a nerve impulse reaches the axon terminal. The
vesicles release their contents into the synaptic cleft and the
transmitter diffuses across the synaptic cleft. After a brief lag
time (e.g., about 0.5 ms) the transmitter binds to receptors on
postsynaptic cells. This typically causes a change in ion
permeability and electrical potential in the postsynaptic cell.
Excitatory signals induce an action potential in the postsynaptic
neuron. Inhibitory signals prevent production of an action
potential in the postsynaptic neuron. Both inhibitory and
excitatory signals can exist simultaneously in the same synaptic
cleft, depending on the cell types, neurotransmitters, etc.
Similarly, a postsynaptic cell can be in simultaneous contact with
multiple presynaptic cells, each of which can transmit both
excitatory and inhibitory signals to the postsynaptic cell.
[0006] The presence of transmitter in the synapse is regulated in a
variety of ways, thereby controlling the signal received by the
postsynaptic cell. For example, some cells and vesicles actively
transport transmitter out of the synapse, thereby reducing the
presence of the transmitter in the junction. Similarly, oxidases
and other enzymes degrade some neurotransmitters in the synapse.
Neurotransmitters can also diffuse away from the synapse. For a
review of neurotransmitter and transporter systems, see,
Neurotransmitter Transporters: Structure, Function and Regulation
(1997) M. E. A. Reith, ed. Human Press, Towata N.J., and the
references cited therein.
[0007] Transporters have a variety of important biological roles.
For example, the Na.sup.+/Cl.sup.- dependent transporters (e.g.,
the monoamine transporters, as well as betaine, creatine, GABA,
glycine, proline and taurine carriers) are the primary sites of
action for a variety of drugs of both therapeutic and abuse
potential. For example, among the monoamine transporters,
inhibition of the dopamine transporter (DAT) is linked to euphoric
and reinforcing properties of psychomotor stimulants such as
cocaine and amphetamines. The major classes of therapeutic
antidepressants act by inhibiting the norepinephrine and serotonin
transporters (NET and SERT) and many of these compounds have proved
clinically useful in the treatment of panic, stress, obsessive
compulsive disorders, and other conditions.
[0008] Chemotactic responses have also long been known to play
significant roles in various biological systems. Chemotaxis is the
capacity of a motile cell to respond to chemical changes in its
environment by directed movement. The migration of a motile cell
exhibiting a chemotactic response can be either up or down a
concentration gradient of a chemotactic factor. For example,
phagocytic cells like macrophages are attracted by and move toward
various substances generated in an immune response, whereas other
motile cells including certain bacteria can move either toward an
attractant (e.g., assorted sugars) or away from various repellents
(e.g., phenol). For further discussion of chemotaxis and related
components, including adhesion and chemotactic factors, see, Kuby,
Immunology, 3.sup.rd Ed. W. H. Freeman and Company, New York (1997)
and Stryer, Biochemistry, 4.sup.th Ed., W. H. Freeman and Company,
New York (1995).
[0009] Aside from methods and devices for modeling transporter
activity and chemotactic responses, general binding assays for
studying, e.g., enzyme-ligand binding interactions, receptor-ligand
binding interactions, and the like are also useful, e.g., in
modeling biological systems.
[0010] In general, existing in vitro systems for studying
transmitters, transporters, presynaptic and postsynaptic cells, and
other aspects of cell signaling do not provide ideal
high-throughput methods and devices for modeling and mimicking
transmitter diffusion, transporter activity, transmitter activity,
and the like. More generally, cell-cell signaling, which is central
to biological activity, is not ideally modeled using existing
technologies and additional high throughput methods of screening
for modulators of signaling activities are desirable. Furthermore,
progress in the study of chemotaxis and various binding activities
has also been impeded by in vitro assays that are tedious to
perform and whose results have been difficult to quantify. As such,
automated and quantitative assays for all of these important
biological processes are desirable.
[0011] The present invention provides these and other features by
providing high-throughput microscale systems for modeling
transporter activity, transmitter degradation activity, transmitter
activity, cell signaling, and detection of modulators (inhibitors
and enhancers) of transporter or transmitter degradation activity.
The present invention also relates to high-throughput systems for
modeling gradient induced activities, e.g., chemotactic responses,
and for assessing general binding activities. These and many other
features which will be apparent upon complete review of the
following disclosure.
SUMMARY OF THE INVENTION
[0012] The invention provides methods, devices, kits, reagents and
related materials for modeling various important biological
processes. For example, the present invention is optionally used to
determine the activity of transporter components such as
neurotransmitter transporters (for example, the neurotransmitter
acetylcholine is specifically internalized by cells via endocytosis
of acetylcholine from the synaptic cleft during the recovery period
following signal transmission). The invention is also optionally
used to assess gradient induced activities (e.g., study chemotactic
responses) and to evaluate the binding activity of, e.g., various
biological components. The methods are typically conducted in a
microscale format using a microfluidic system which includes or is
coupled to sources of the relevant assay components.
[0013] In the transporter-related methods and assays of the
invention, a first component which includes transporter activity is
flowed through a first channel. A second component which produces a
detectable signal upon exposure to a transportable molecule or set
of transportable molecules is flowed into the first channel. The
transportable molecule is flowed into the first channel and a
signal produced by contacting the second component with the
transportable molecule is then detected. Typically, the level of
signal product is inversely related to transporter activity.
[0014] A variety of formats for the methods are appropriate. The
first and second components are typically flowed sequentially (a
typical configuration) or simultaneously in the first channel. The
second component optionally is flowed into contact with the
transportable molecule in the presence or absence of the first
component (for example, if flowed in the absence of the first
component, the resulting signal serves as a positive control for
the signal produced by contacting the second component with the
transportable molecule).
[0015] Known activity modulators are optionally incorporated into
assay schemes as controls for modulation of a particular
transporter. For example, paraxetine, citalopram, fluxetine,
imipramine, amitriptyline, mazindol, cocaine, desipramine,
nomifensine, GBR12909, D-amphetamine, L-amphetamine, nortriptyline,
DA, MPP.sup.+, NE, and 5-HT are known inhibitors of the transport
of human monoamine clones. See, Neurotransmitter Transporters:
Structure Function and Regulation, e.g., at chapter 1 (1997) M. E.
A. Reith, ed. Human Press, Towata N.J., and the references cited
therein.
[0016] The first component is typically a cell or component with
similarity to a cell such as a cell membrane (or other lipid
membrane preparation) having transporter activity. Similarly, the
second component is typically, e.g., a cell, cell membrane (or
other lipid membrane preparation), or other biological or synthetic
moiety comprising a receptor for the transportable molecule which
is capable of producing a detectable signal upon exposure to a
transportable molecule (e.g., a transmitter). The first or second
components typically include a transporter or transmitter receptor
carrier moiety or set of carrier moieties which includes a receptor
or transporter. A "carrier" is a component comprising the specified
activity (e.g., transporter, transmitter, transmitter receptor,
etc.). Examples of carriers or carrier sets include cells,
liposomes, organelles, proteins, and protein-lipid complexes.
Examples of transportable molecules or set of transportable
molecules include proteins, sets of proteins, peptides, sets of
peptides, lipids, sets of lipids, carbohydrates, sets of
carbohydrates, organic molecules, sets of organic molecules, drugs,
sets of drugs, receptor ligands, sets of receptor ligands,
antibodies, sets of antibodies, neurotransmitters, sets of
neurotransmitters, cytokines, sets of cytokines, chemokines, sets
of chemokines, hormones, sets of hormones and a variety of other
biologically active and inactive molecules. In preferred aspects,
the first component is a carrier moiety or set of carrier moieties
comprising a transporter activity having neurotransporter
activity.
[0017] Preferred transporter components include cells which
specifically or non-specifically internalize transmitter molecules,
e.g., by specific or non-specific endocytosis, or pinocytosis.
These transporter molecules, such as the Na.sup.+/Cl.sup.-
dependent transporters (e.g., the monoamine transporters, as well
as betaine, creatine, GABA, glycine, proline and taurine carriers),
transport corresponding transportable molecules such as
acetylcholine, catecholamines (e.g., epinephrine, norepinephrine,
dopamine, serotonin, and other adrenergic neurotransmitters),
endorphins (e.g., a and .beta.-endorphin), enkephalins (e.g.,
Met-enkephalin or Leu-enkephalin), somatostatin, leutinizing
hormone-releasing hormone, thyrotropin-releasing hormone, substance
P, angiotensin I, angiotensin II, vasoactive intestinal peptide,
serotonin, and gamma-aminobutyric acid (GABA).
[0018] In one aspect of the invention, the transportable molecule
is flowed from a second channel into the first channel and the
second component is flowed from a third channel into the first
channel, where the first component, the second component and the
transportable molecule mix. For example, the second and third
channels optionally intersect the first channel in a mixing region,
where the first, second and third components diffuse into contact
in the mixing region. An advantage to this arrangement is that the
diffusion mimics diffusion of components in synapses in vivo,
providing a convenient way of modeling transport of biologically
active transportable molecules such as neurotransmitters. The first
and second components are optionally flowed sequentially, serially
or concomitantly. Potential transport modulatory compounds (e.g.,
inhibitors) are optionally flowed into contact with the first
component to test for an effect on transport of the transportable
molecule. For example, the modulatory compound is optionally flowed
into the first channel prior to introduction of the second
component and the transportable molecule, or concomitant with flow
of the transportable molecule, depending on the format of the
particular assay.
[0019] In one embodiment, concentration of the transportable
molecule is decreased in solution in the first channel as the first
component internalizes the transportable molecule. In other
aspects, the first component sequesters or otherwise inactivates
the transportable molecule.
[0020] A detectable signal produced by transport of the
transportable molecule, or inhibition of transport of the
transportable molecule provides an indication of, e.g., the
transporter activity present in the first component, or an ability
of the inhibitor to inhibit the transporter activity present in the
first component, or an ability of the second component to sequester
the transportable molecule. For example, the detectable signal is
optionally a cellular activity, a light emission, a radioactive
emission, a change in pH, a change in temperature, or the like. The
concentration of the first component, the transportable molecule,
or the second component (or any combination of these components),
as well as potential modulators is optionally varied in the first
channel and the resulting increase or decrease in signal strength
is typically measured.
[0021] The present invention also relates to methods of detecting a
gradient induced activity. The methods include providing a first
channel, e.g., a microchannel, with an internal surface including a
first and a second longitudinal segment. A first component (e.g.,
an adhesion factor) or a set of first components is typically
attached to a region of the first longitudinal segment and a second
component, such as a motile cell (e.g., a phagocytic, a protozoic,
a moneran cell, etc.) is generally attached to the first component
or to one or more members of the set of first components. The
second component is, e.g., optionally fluorescently labeled. A
gradient is typically formed from an edge of the second
longitudinal segment of the first channel, which induces the second
component to detach from the first component or from the one or
more members of the set of first components. Thereafter, a
detectable signal produced by the detached second component is
generally detected. The type of gradient utilized with these
methods optionally include a chemical composition gradient, a light
energy gradient, a magnetic gradient, a pH gradient, a dissolved
oxygen gradient, a temperature gradient, or the like.
[0022] The first component or set of first components are
optionally attached to the first channel by flowing the first
component or the set of first components over the first
longitudinal segment of the first channel concomitantly with
flowing a third component (e.g., a buffer) over the length of the
second longitudinal segment of the first channel. During this step,
at least some of the first component or set of first components
attaches to the first longitudinal segment. Thereafter, the third
component is flowed through the first channel to remove any
unattached first component. The second component is typically then
flowed through the first channel and in so doing, some of the
second component attaches to the attached first component. This
step is generally followed by flowing the third component through
the first channel to remove any unattached second component.
[0023] In one preferred embodiment, the chemical composition
gradient is established by concomitantly flowing a third component
(e.g., a buffer) and a fourth component (e.g., a chemotactic
factor) or a set of fourth components into the first channel in
which the fourth component or the set of fourth components forms
the gradient from an edge of the second longitudinal segment of the
first channel. For example, a buffer is typically flowed from a
second channel into the first channel and a chemotactic factor is
typically flowed from a third channel into the first channel in
which the buffer and the chemotactic factor mix in the first
channel to form the gradient of the chemotactic factor. In one
embodiment, the concentration of the fourth component or the set of
fourths components is highest along a length of the second
longitudinal segment that is farthest from the first longitudinal
segment and lowest along the length of the second longitudinal
segment that is nearest to the first longitudinal segment. In
another embodiment, the concentration of the fourth component or
the set of fourths components is lowest along a length of the
second longitudinal segment that is farthest from the first
longitudinal segment and highest along the length of the second
longitudinal segment that is nearest to the first longitudinal
segment.
[0024] As a negative control, e.g., a buffer is optionally flowed
into contact with the attached motile cell in the first channel to
assess the signal produced in the absence of the chemotactic
factor. Additionally, a positive control optionally includes
flowing, e.g., a chemotactic factor into contact with the attached
motile cell in the first channel to determine the signal produced
in the absence of a buffer.
[0025] The various components of these methods (e.g., the first,
second, third and the fourth or more components) are optionally
flowed, e.g., using a fluid direction component including, e.g., a
fluid pressure force modulator, an electrokinetic force modulator,
a capillary force modulator, a fluid wicking element, and/or the
like. The methods optionally further include flowing a modulator
into contact with the second component in the first channel prior
to introduction of the fourth component in which the modulator
modulates (e.g., activates or inhibits) detachment of the second
component from the first component or the set of first
components.
[0026] The detectable signal provides an indication of the gradient
induced activity present in the second component and/or an ability
of the modulator to modulate the gradient induced activity of the
second component. The detectable signal optionally includes a
refractive index, a cellular activity, a light emission, an
absorbance, a change in absorbance, a fluorescence, a change in
fluorescence, a color shift, a fluorescence resonance energy
transfer, a radioactive emission, a change in pH, a change in
temperature, a change in mass (e.g., by mass spectroscopy), or the
like. Additionally, the chemical composition gradient formed by the
fourth component or the set of fourth components in the first
channel is optionally varied and a resulting increase or decrease
in the detectable signal is optionally measured. Furthermore, the
concentration of the second component is optionally increased in
solution in the first channel as the gradient induces the second
component to detach from the first component or the set of first
components.
[0027] The present invention also relates to methods of detecting a
binding activity. For example, a first component is typically
flowed (e.g., in a first flow stream) through a first channel
(e.g., a microchannel) concomitantly with at least one second
component (e.g., in a second flow stream) or a set of second
components in which the second component (e.g., an enzyme or a
receptor) or the set thereof binds to the first component.
Thereafter, the methods include, e.g., detecting a detectable
signal that indicates a final concentration of the at least one
first component or the set of first components that remains unbound
after exiting from the first channel. Optionally, the methods
include detecting a detectable signal that indicates an initial
concentration of the at least one first component or the set of
first components prior to entry of the component or set thereof
into a first channel.
[0028] The first component or set of first components can diffuse
more rapidly in solution than the second component or set of second
components. Furthermore, the first channel generally includes a
mixing longitudinal segment in which, during the flowing step, the
first component or the set of first components diffuse
substantially across the first channel in the mixing longitudinal
segment, while the second component or the set of second components
typically diffuse less than substantially across the first channel
in the mixing longitudinal segment. The first and second components
are typically flowed through the first channel using fluid
direction components that optionally include, e.g., a fluid
pressure force modulator, an electrokinetic force modulator, a
capillary force modulator, a fluid wicking element, or the
like.
[0029] As a positive control for detecting a detectable signal, the
first component or the set of first components is optionally flowed
through the first channel, e.g., in the absence of the second
component or the set of second components. A negative control for
detecting a detectable signal optionally includes the step of
flowing the second component or the set of second components
through the first channel, e.g., in the absence of the first
component or the set of first components.
[0030] The methods also optionally include concomitantly flowing a
modulator into contact with the second component in the first
channel, in which the modulator modulates (e.g., activates or
inhibits) the binding of the second component to the first
component. The detected binding activity provides an indication of
the binding activity of the second component and/or an ability of
the modulator to modulate the binding activity of the second
component. Additionally, the first and second detectable signals
optionally include, e.g., a refractive index, a cellular activity,
a light emission, an absorbance, a change in absorbance, a
fluorescence, a change in fluorescence, a color shift, a
fluorescence resonance energy transfer, a radioactive emission, a
change in pH, a change in temperature, a change in mass (e.g., by
mass spectroscopy), or the like.
[0031] In one preferred embodiment, the invention provides methods
of detecting neurotransporter activity in a cell. In the methods, a
first cell or cell set which includes a transporter activity is
flowed in a first microscale channel. A selected neurotransmitter
and a second cell or second cell set comprising a receptor for the
neurotransmitter are flowed into the first channel. A signal
produced by contact of the second cell or cells of the second cell
set by the neurotransmitter is then detected, thereby determining
the rate of transport activity of the transporter in the first cell
or cells of the first cell set.
[0032] Optionally, the first cell or cell set is flowed into
contact with the neurotransmitter prior to contacting any remaining
neurotransmitter to the second cell or cell set. A transport
inhibitor is optionally added to the microchannel and the resulting
modulation in signal intensity produced by the second cell or cell
set is measured, thereby determining the activity of the inhibitor
on transport activity in the first cell set.
[0033] The invention provides devices and systems for practicing
the methods noted herein. For example, in one aspect, a device
which includes a body structure having at least a first, second and
third microscale channel fabricated therein is provided. The first
microscale channel typically includes a first component comprising
transporter activity which transports at least a first
transportable molecule. The second microscale channel intersects
the first microscale channel at a first channel intersection. The
second microscale channel typically includes a transportable
molecule. The third microscale channel intersects the first
microscale channel in a second channel intersection region. The
third microscale channel includes a second component which binds to
the first transportable molecule, causing emission of a detectable
signal.
[0034] The device optionally includes a source of a modulatory
agent such as an inhibitor which inhibits transport of the first
transportable molecule by the first component, or an activator
which enhances transport of the first transportable molecule.
Sources of the other assay components noted herein, such as carrier
moieties or sets of carrier moieties (including cells, liposomes,
organelles, proteins, protein-lipid complexes, etc.), transportable
molecules (proteins, sets of proteins, peptides, sets of peptides,
lipids, sets of lipids, carbohydrates, sets of carbohydrates,
organic molecules, sets of organic molecules, drugs, sets of drugs,
receptor ligands, sets of receptor ligands, antibodies, sets of
antibodies, neurotransmitters, sets of neurotransmitters,
cytokines, sets of cytokines, chemokines, sets of chemokines,
hormones, sets of hormones, etc.) are also optionally incorporated
into the devices herein.
[0035] Devices optionally incorporate additional elements such as
detectors for detecting signals produced in the first channel,
e.g., operably connected to a computer for data analysis, fluidic
controllers for directing fluid movement in the first channel, one
or more transparent detection window fluidly connected to the first
channel, robotic armatures for moving the body structure or sample
arrays. Systems and devices typically incorporate, or are used in
conjunction with, a computer having an instruction set for
controlling or processing a signal from the detector, the fluidic
controller, the robotic armature or other device or system
elements.
[0036] The first and second intersections are optionally opposed
across the first channel, or at least in a close proximal
relationship. This arrangement is advantageous for studying
biological diffusion properties of transporters, transportable
molecules and transport receptors across small distances, e.g., to
examine diffusion properties at, e.g., neural junctions between
nerve cells (e.g., axons and dendrites).
[0037] In one embodiment, during operation of the device, a mixture
of the first component, the second component and the transportable
molecule are flowed in the first channel and the device has a
detector for detecting a signal produced by the mixture. The
detector is typically positioned to detect a signal produced by the
mixture at multiple points in the first channel.
[0038] The present invention also includes a device or system
including a body structure that includes a first microscale channel
fabricated therein. The first microscale channel includes a first
component (e.g., an adhesion factor) or a set of first components
that includes a first attachment activity, in which the first
component or set thereof is attached to a region of a first
longitudinal segment of the first microscale channel. The first
microscale channel also includes a second component (e.g., a motile
cell) that includes a second attachment activity in which the
second component is attached to the first component or to one or
more members of the set of first components. The first microscale
channel also includes a third component (e.g., chemotactic factor)
or a set of third components that forms a gradient from an edge of
a second longitudinal segment of the channel, in which the gradient
induces the second component to detach from the first component or
the members of the set of first components to produce a detectable
signal.
[0039] In one embodiment, the depth of the first longitudinal
segment differs from the depth of the second longitudinal segment
in the first microscale channel of the device. Furthermore, the
second component (e.g., a phagocytic cell, a protozoic cell, a
moneran cell, etc.) are optionally fluorescently-labeled. The first
microscale channel also optionally includes a modulator (e.g., an
activator or an inhibitor) that modulates detachment of the second
component from the first component.
[0040] Additionally, the device or system typically includes a
detector in or proximal to the first microscale channel for
detecting a signal produced in the first microscale channel that is
operably connected to a computer. The device also generally include
a fluidic controller for directing fluid movement in the first
microscale channel, one or more transparent detection windows
fluidly connected to the first microscale channel, a robotic
armature for moving the body structure or sample plates relative to
the body structure, and/or a source of components (e.g., microwell
plates). When the device includes a computer, the computer
typically includes an instruction set for controlling or processing
a signal from the detector, the fluidic controller, and/or the
robotic armature.
[0041] During operation of the device, a mixture of the third
component (e.g., a chemotactic factor) or, e.g., the set of the
third components and a buffer are optionally flowed in the first
microscale channel and the device typically further includes a
detector for detecting a signal produced by detachment of the
second component induced by the third component in the mixture.
Alternatively, the device includes a detector for detecting a
signal produced by detachment of the second component induced by
the third component at multiple points in the first channel.
[0042] The present invention also relates to a device or system
that includes a body structure including at least a first, second,
and third microscale channel fabricated therein. The first
microscale channel includes a first and second component or sets
thereof in which the second component (e.g., an enzyme or a
receptor) binds to the first component. The second microscale
channel typically intersects the first microscale channel at a
first channel intersection and optionally the second microscale
channel includes a second detector in or proximal to the second
microscale channel for detecting an initial concentration of the
first component. The third microscale channel optionally intersects
the first microscale channel at a second channel intersection in
which the third microscale channel includes a first detector in or
proximal to the third microscale channel for detecting a final
concentration of the first component that remains unbound. The
first microscale channel of the device also optionally includes a
modulator (e.g., an inhibitor) that modulates the second component
from binding to the first component.
[0043] The first component or set of first components generally
diffuses more rapidly in solution than the second component or set
of second components. Furthermore, the first channel typically
includes a mixing longitudinal segment in which, during operation
of the device, the first component or the set of first components
diffuse substantially across the first channel in the mixing
longitudinal segment, while the second component or the set of
second components diffuse less than substantially across the first
channel in the mixing longitudinal segment.
[0044] The device also optionally includes a fluidic controller for
directing fluid movement in the first microscale channel, a
transparent detection window fluidly connected to the first
microscale channel, a robotic armature for moving the body
structure, and/or a source of components. When the device includes
a computer, the computer typically includes an instruction set for
controlling or processing a signal from the detector, the fluidic
controller, and/or the robotic armature.
[0045] The invention also provides kits for practicing the methods
and utilizing the devices noted herein. For example, the kits of
the invention optionally include a first component comprising a
transporter activity and a second component which is capable of
producing a signal upon exposure to a transportable molecule which
is transportable by the first component. The components generally
include a carrier moiety or set of carrier moieties, a container
for packaging the first or second component, instructions for
practicing the methods herein, e.g., using the devices noted
herein, one or more reagents for buffering or storing the first or
second component, one or more transportable molecule, one or more
test compound, a test compound library or the like.
[0046] Definitions
[0047] Unless otherwise indicated, the following definitions
supplement those in the art.
[0048] "Transporter activity" refers to movement of a transportable
molecule such as a transmitter across a biological barrier such as
a cell membrane. This is typically performed by specific
transporters or non-specific endocytosis of the transmitter or
other transportable molecule. See, e.g., Darnell et al. (1990)
Molecular Cell Biology, Second Edition Scientific American Books
New York, FIGS. 17-38 for an introduction to specific endocytosis
of a neurotransmitter. Additional details regarding
neurotransporters are found in Neurotransmitter Transporters:
Structure, Function and Regulation (1997) M. E. A Reith, ed. Human
Press, Towata N.J. An example class of transporters are the
biogenic amine transporters (e.g., those which transport
norepinephrine (NE), dopamine (DA), or serotonin (5-HT), referred
to as NET, DAT and SERT), which utilize ionic gradients of
Na.sup.+, K.sup.+ and Cl.sup.- ions to drive transport
reactions.
[0049] In addition to standard neurotransmitter transporters,
transporter activity as used herein optionally includes other
systems for translocating transmitters across a cell or other
membrane, such as translocation through ionophores or other
membrane translocation proteins such as permeases which facilitate
transport of materials by mechanisms other than endocytosis. See,
e.g., Darnell et al. (1990) Molecular Cell Biology Second Edition
Scientific American Books New York, Chapter 15 for an introduction
to permeases and other transport facilitating proteins.
[0050] A "transmitter" is a transportable molecule which can be
transported by the transporter, and/or which can trigger a
detectable change on a cell comprising a receptor for the
transmitter. Examples include neurotransmitters which transmit a
signal across a synaptic junction by binding to a receptor on a
post-synaptic cell, where the neurotransmitters are also
transported by neurotransmitter transporters.
[0051] A "longitudinal segment" includes a segment of a channel
(e.g., a microchannel) that extends over at least a substantial
portion of the length of the channel.
[0052] A "double Y" array is a configuration of channels (e.g.,
microchannels) in which at least four channels intersect with a
first channel, i.e., at least two channels intersect with one end
of the first channel and at least two channels intersect with the
other end of the first channel.
BRIEF DESCRIPTION OF THE DRAWING
[0053] FIG. 1 is a schematic of a microfluidic structure comprising
a flow of materials comprising transporter, transmitter, and
transmitter-receptor components.
[0054] FIG. 2, panels A, B and C are schematic drawings of an
integrated system of the invention, including a body structure,
microfabricated elements, and a pipettor channel.
[0055] FIG. 3 is a schematic drawing of the integrated system of
FIG. 2, further depicting incorporation of a microwell plate, a
computer, a detector and a voltage/pressure controller.
[0056] FIG. 4 depicts one embodiment of the device for detecting a
gradient induced activity.
[0057] FIG. 5 shows a cross-sectional view of a microchannel from
an additional embodiment of the device for detecting a gradient
induced activity.
[0058] FIG. 6 depicts a microfluidic device for detecting binding
activity.
[0059] FIG. 7 is a schematic showing an interface between a
microfluidic device and electrospray ionization mass
spectrometer.
[0060] FIG. 8 is a schematic showing an interface between a
microfluidic device used in binding affinity assays and
electrospray ionization mass spectrometer.
[0061] FIG. 9 illustrates a microchannel configuration for
detecting binding activity.
[0062] FIG. 10 illustrates a microchannel configuration for
detecting competitive binding activity.
DETAILED DISCUSSION OF THE INVENTION
[0063] The present invention provides, inter alia, methods and
microfluidic systems for modeling various biological processes. For
example, the invention is optionally used to model nerve impulse
transmissions across synaptic clefts to assess the activity of
assorted transporter components, e.g., neurotransmitter
transporters. Other processes that are generally modeled using the
present invention, include gradient induced activities, e.g.,
chemotactic responses of motile cells. The invention additionally
includes methods and devices for analyzing the binding activities
of many different biological constituents, such as enzyme-substrate
interactions, receptor-ligand interactions, or other binding
interactions. Furthermore, the effect of modulators of these
activities also optionally evaluated.
[0064] In general, existing techniques for studying transmitters,
transporters, and other aspects of cell signaling lack sufficient
throughput for modeling transmitter diffusion, transporter
activity, transmitter activity, and the like. Additionally,
progress in the study of gradient induced responses and binding
activities has also been hindered by in vitro systems that also
lack high-throughput and whose results have been difficult to
quantify. As such, the automated and quantitative assays of the
present invention for modeling all of these important biological
processes are desirable.
[0065] The following provides details regarding the high-throughput
microscale systems of the present invention for use in modeling
transporter activity, transmitter degradation activity, transmitter
activity, cell signaling, and detection of modulators (inhibitors
and enhancers) of transporter or transmitter degradation activity.
It also provides details relating to high-throughput systems for
modeling gradient induced activities and general binding
activities. These and many other features which will be apparent
upon complete review of the following disclosure.
[0066] Transporter Activity
[0067] Specific endocytosis of neurotransmitters permits recycling
of transmitters released by, e.g., presynaptic cells into a
synaptic cleft. In addition to conserving transmitter, this
recycling of transmitters regulates the level of a transmitter in
the synapse. Thus, transporter activity is central to many
cell-signaling biological activities. See, Neurotransmitter
Transporters: Structure Function and Regulation (1997) M. E. A.
Reith, ed. Human Press, Towata N.J.; and Neurotransmitter Methods:
Methods in Molecular Biology Volume 72 (1997) R. Rayne, ed. Human
Press, Towata, N.J., for a review of transporter and transmitter
biology.
[0068] The transport of transportable moieties such as certain
neurotransmitters serves as an activity regulation point for a
variety of cell-signaling events. For example, by sequestering
transmitters using ion-dependent transporter activities to drive
transport of transmitters, cells comprising transporter activity
decrease the concentration of local available transmitter
molecules, thereby reducing the amount of transmitter available to
bind to cells which have transmitter receptors. Similarly, the
local degradation of transmitters, e.g., through activity of an
enzyme such as an oxydase or esterase typically have a related
biological effect.
[0069] In addition to using endocytosis mechanisms such as those
driven by transporters which utilize ionic gradients of Na.sup.+,
K.sup.+ and Cl.sup.- ions to drive transport reactions, cells can
transport transmitters through the activity of transport
facilitating proteins. Transport facilitating proteins can also
transport materials into cells. Transport facilitating proteins in
biological systems facilitate transport of transportable moieties
in at least two different ways. First, many transport facilitating
proteins facilitate endocytosis of specific transportable
components. Second, some permeases (optionally considered to be
transporters herein) assist in passive diffusion of ions or
molecules across a membrane by providing a path for the ion or
molecule in the membrane. In diffusion, the rate of transport is
directly proportional to the concentration gradient across the
membrane. Some transporters engage in active transport in which
metabolic energy is used to move transportable ions or molecules
across a membrane or other biological barrier, including against
the concentration gradient of the transportable component. Cellular
energy sources and/or ion gradients across cell membranes are used
to drive active transport strategies.
[0070] The present invention relates to a new assay system for
monitoring transporter/ transmitter activities in vitro. In one
format, cells or other components which comprise transporter
activity are flowed through a first microfluidic channel as shown
in FIG. 1. Transmitter 100 is flowed (flow direction is indicated
by arrows 102) into the first channel from a second channel, while
cells or other components comprising receptors to the transmitter
are flowed from a third channel into the first channel. If
cells/components with transporter activity 104 actively take up
transmitter 100, the concentration of transmitter 100 is decreased
in the channel as transmitter 100 diffuses across the channel and
into contact with cells/components with transmitter receptor 106,
thereby reducing transmitter 100 available to bind cells/components
with transmitter receptor 106. Typically, activity of transmitter
100 on cells/components with transmitter receptor 106 is assessed
by monitoring a transmitter-dependent activity in cells/components
with transmitter receptor 106. A variety of receptor-transmitter
activities are known. See, Neurotransmitter Transporters: Structure
Function and Regulation (1997) M. E. A. Reith, ed. Human Press,
Towata N.J., and the references cited therein.
[0071] If a transporter modulator is added upstream or concurrent
with the transmitter, the modulator can increase (i.e., when the
modulator is an activator of transport function) or decrease (i.e.,
when the modulator is an inhibitor of transport function) the
activity of the transporter. Thus, one preferred assay monitors the
activity of the transporter in the presence of the modulator by
monitoring a transmitter-dependent activity in the cells/components
comprising the transmitter receptor. Because of the high-throughput
nature of this microscale assay, a large library of compounds can
be screened for transporter modulatory activity.
[0072] Specific Assay Formats for Transporter Activity Assays
[0073] FIG. 1 shows one basic assay format for the present
invention. As depicted, cells or other components comprising
transporter activity are flowed in a first channel. Transmitter is
flowed into the first channel from a second channel, where it
contacts the cells or other components comprising transporter
activity. The cells comprising transporter activity transport the
transmitter, thereby localizing the transmitter within the cells or
other components comprising transporter activity. Transmitter which
is not localized by transport diffuses into contact with cells or
other components which include receptors for the transmitter.
Following binding of the transmitter to the cells or other
components which include receptors for the transmitter, a
detectable signal is produced.
[0074] An advantage of this assay format is that it mimics the
biological activity of the relevant components (e.g., as noted
above) in the assay. For example, the cells comprising transporter
activity are analogous to a pre-synaptic cell comprising
transporter activity, while the cells comprising a transmitter
receptor are analogous to a post-synaptic cell which is activated
by the transmitter. The diffusion of the transmitter across the
first channel and into contact with the cells comprising a
transmitter receptor mimics diffusion of the transmitter across a
synapse.
[0075] Variations on the above assay are optionally made, depending
on the components to be assayed, microfluidic system available,
preferences of the investigator and the like. For example, although
depicted with cells or components comprising transporter activity,
other activities which eliminate the ability of the transmitter to
bind to the cells comprising a transmitter receptor are optionally
substituted and the effects of modulators can be tested on these
activities. The activity of an enzyme which inactivates the
transmitter is optionally monitored by substitution of the enzyme
for the cells comprising transporter activity, as noted above.
[0076] In a configuration desirable for screening potential
transport modulator compounds, a modulator is flowed into contact
with the cells comprising transporter activity, before, during or
after flowing the transmitter into proximity with the cells in the
first channel. The transport inhibitory or activating effect of the
modulator is typically monitored by assaying for any change in the
signal produced by the cell, as compared to signal produced in the
absence of the modulator.
[0077] The modulator is optionally flowed with the transmitter, the
cells comprising the transporter activity, the cells with the
transmitter receptor, or from a separate source of modulator.
Commonly, potential modulators will be arranged in libraries which
are accessed by the microfluidic system, such as in microwell
plates or dried on substrates. Knapp et al. "Closed Loop
Biochemical Analyzers" (WO 98/45481; PCT/US98/06723) provide a
variety of such library accessing strategies for microfluidic
systems.
[0078] When the modulator is flowed from a source distinct from the
other assay components, the microfluidic system will, commonly,
include an additional modulator flow channel which intersects the
flow path of the cells which comprise transporter activity. The
modulator compounds are typically incubated with the cells while
under flow in a channel, or separately in a chamber or well. The
modulator compounds are also, commonly, incubated with the cells
prior to introduction into the first channel, e.g., in a microtiter
dish, or a well integrated into the body structure comprising the
microscale channel.
[0079] Commonly, reagents facilitating transport are flowed
concomitantly or separately with the components comprising
transport activity, depending on the intended assay. For example,
buffer comprising ions such as sodium, potassium or calcium are
flowed into contact with components comprising transport activities
which depend on the presence of these ions for transporter
function. For a description of common ion-dependent transporters,
and a description of other reagent and buffer conditions for
transporter activity, see, Neurotransmitter Transporters:
Structure, Function and Regulation (1997) M. E. A. Reith, ed. Human
Press, Towata N.J., and the references cited therein.
[0080] Transmitters, Transporters and Transmitter Receptors
[0081] A wide variety of signal transduction systems have been
extensively characterized and are optionally applied to the assays
of the invention. The basic transmitter, transporter and
transporter receptor elements (or cells or other components
comprising these activities) are commercially available or can be
obtained using known techniques. For example, the 1998 CALBIOCHEM
Signal Transduction Catalog and Technical resource lists over 2100
commercially available products related to signal transduction,
including G-protein related products, calcium metabolism related
products, cytokines, growth factors and hormones, cell adhesion/
extracellular matrix tools, protein kinases, protein phosphatases,
enzymes, substrates and probes, enzyme activators, enzyme
inhibitors, reagents for nitric oxide research, oxidative stress
and free radicals, ionophores, neurochemicals, neurotoxins, and
neurotrophins, immunophillins, bioactive lipids, cell cycle and
apopotosis components, and the like. Similarly, a number of texts
describe transmitter and transporter systems, as well as providing
information relevant to assays, cell growth and supplies of cells,
and other features relevant to the present invention, including:
Neurotransmitter Transporters: Structure, Function and Regulation
(1997) M. E. A. Reith, ed. Human Press, Towata N.J.;
Neurotransmitter Methods: Methods in Molecular Biology Volume 72
(1997) R. Rayne, ed. Human Press, Towata, N.J.; Neuropeptide
Protocols: Methods in Molecular Biology Volume 73: Irvine and
Williams, eds., Human Press, Towata N.J.; Neurochemistry: A
Practical Approach, 2.sup.nd edition (1997) Turner and Bachelard,
eds., Oxford Press, Oxford England; and, Neural Cell Culture: A
Practical Approach (1996) Cohen and Wilkins, eds. Oxford Press,
Oxford England.
[0082] One of skill can adapt available cells and reagents to the
present invention by providing the cells and reagents to the
microfluidic systems herein as noted. Assay conditions and buffer
and reagent parameters utilizing transport modulators,
transmitters, transporters and the like are typically selected
based upon established activity levels, transmitter-transporter
pairings, concentrations and kinetic information for known
transporters and transmitters, modified by the addition of a
selected modulator to the system. Initial screens of a particular
putative modulator are optionally conducted at a single
concentration of modulator in the system, or at multiple modulator
concentrations. Typically, compounds which have modulatory activity
based upon an initial screen are titrated into contact with the
transporter (or other assay component, as appropriate), in
increasing or decreasing amounts, to establish a dose-response
curve for the modulator.
[0083] Rather than simply using cells which naturally comprise
transporters and transmitter receptors in the assays noted herein,
recombinant cells are also optionally constructed which include
desired transporters or transmitter receptors. This is advantageous
because certain cells can be easily maintained in culture using
established methods.
[0084] Methods of making recombinant cells and expressing cellular
proteins such as transport facilitating proteins and transmitter
receptors are well known in the art. For an introduction to
recombinant methods, see, Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology volume 152 Academic
Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular
Cloning--A Laboratory Manual (2nd ed.), Vol. 1-3, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook"); and
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 1999) ("Ausubel"). Culture of mammalian cell lines and
cultured cells from tissue or blood samples is well known in the
art. Freshney (Culture of Animal Cells, a Manual of Basic
Technique, Third Edition, Wiley-Liss, New York (1994)) and the
references cited therein provides a general guide to the culture of
animal cells. Culture of plant cells is described in Payne et al.
(1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley
& Sons, Inc., New York, N.Y. Additional information on cell
culture, including prokaryotic cell culture, is found in Ausubel,
Sambrook and Berger, supra. Cell culture media are described in
Atlas and Parks (eds) The Handbook of Microbiological Media (1993)
CRC Press, Boca Raton, Fla. Additional information is found in
commercial literature such as the Life Science Research Cell
Culture catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.)
and, e.g., the Plant Culture Catalogue and supplement (1997) also
from Sigma-Aldrich, Inc (St Louis, Mo.). Additional details on the
cloning and expression of transporters is found in Neurotransmitter
Transporters: Structure, Function and Regulation (1997) M. E. A.
Reith, ed. Human Press, Towata N.J.; Neurotransmitter Methods:
Methods in Molecular Biology Volume 72 (1997) R. Rayne, ed. Human
Press, Towata, N.J.; Neuropeptide Protocols: Methods in Molecular
Biology Volume 73: Irvine and Williams, eds., Human Press, Towata
N.J.; Neurochemistry: A Practical Approach, 2.sup.nd edition (1997)
Turner and Bachelard, eds., Oxford Press, Oxford England; and,
Neural Cell Culture: A Practical Approach (1996) Cohen and Wilkins,
eds. Oxford Press, Oxford England.
[0085] Modulators
[0086] Essentially any molecule can be tested for transporter
modulatory activity, gradient induced modulatory activity, or
binding modulatory activity. Further, essentially any chemical
compound can be used as a potential modulator in the assays of the
invention, although most often compounds which can be dissolved in
aqueous or organic (e.g., DMSO-based) solutions are used to
facilitate flow in microscale systems. The assays herein are
designed to screen large chemical libraries by automating the assay
steps and providing compounds from any convenient source to the
assays. It will be appreciated that there are many suppliers of
chemical and biological compounds, including Sigma (St. Louis,
Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.),
Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the
like.
[0087] In one preferred embodiment, high throughput screening
methods involve providing a combinatorial library containing a
large number of potential transport activity modulator compounds
("potential modulator compounds"). Such "combinatorial chemical
libraries" are screened in one or more assays, as described herein,
to identify those library members (particular chemical species or
subclasses) that display a desired characteristic activity. The
compounds thus identified can serve as conventional "lead
compounds" or can themselves be used as potential or actual
therapeutics for treating conditions amenable to treatment by
modulating transporter activities. As noted, a variety of diseases
are treated by administering transport modulators, including:
panic, stress, obsessive compulsive disorders, depression, chronic
pain and many other physical and psychological conditions. See,
Neurotransmitter Transporters: Structure, Function and Regulation
(1997) M. E. A. Reith, ed. Human Press, Towata N.J., and the
references cited therein.
[0088] A typical combinatorial chemical library is a collection of
diverse chemical compounds generated by either chemical synthesis
or biological synthesis, by combining a number of chemical
"building blocks" such as reagents. For example, a linear
combinatorial chemical library such as a polypeptide library is
formed by combining a set of chemical building blocks (amino acids)
in every possible way, or a selected way for a given compound
length (i.e., the number of amino acids in a polypeptide compound).
Millions of chemical compounds can be synthesized through such
combinatorial mixing of chemical building blocks.
[0089] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries are also optionally used. Such chemistries
include, but are not limited to: peptoids (PCT Publication No. WO
91/19735), encoded peptides (PCT Publication WO 93/20242), random
bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines
(U.S. Pat. No. 5,288,514), diversomers such as hydantoins,
benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci.
USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al.,
J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics
with .alpha.-D-glucose scaffolding (Hirschmann et al., J. Amer.
Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of
small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661
(1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)),
and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658
(1994)), nucleic acid libraries (see, Berger and Kimmel, Guide to
Molecular Cloning Techniques Methods in Enzymology, volume 152,
Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.
(1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3,
Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.,
(Sambrook); and Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(e.g., current through 1999, e.g., at least through supplement 37)
(Ausubel)), peptide nucleic acid libraries (see, e.g., U.S. Pat.
No. 5,539,083), antibody libraries (see, e.g., Vaughn et al.,
Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287),
carbohydrate libraries (see, e.g., Liang et al., Science,
274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic
molecule libraries (see, e.g., benzodiazepines, Baum C&EN,
January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. Nos. 5,506,337; benzodiazepines, 5,288,514,
and the like).
[0090] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pass., Martek
Biosciences, Columbia, Md., etc.).
[0091] Control reactions which measure the activity of the selected
transporter which does not include a modulator are optional, as the
assays are highly uniform. Such optional control reactions are
generally appropriate, however, and increase the reliability of the
assay(s). Accordingly, in a preferred embodiment, the methods of
the invention include a control reaction (or reactions). For each
of the assay formats described, "no modulator" control reactions
which do not include a modulator provide a background level of
transporter activity.
[0092] In some assays, it is desirable to have positive controls to
ensure that the components of the assays are working properly. At
least two types of positive controls are appropriate. First, a
known transport modulator is optionally flowed into contact with
components comprising transport activities, and the resulting
affects on transporter activity monitored. Examples of known
modulators include paraxetine, citalopram, fluxetine, imipramine,
amitriptyline, mazindol, cocaine, desipramine, nomifensine,
GBR12909, D-amphetamine, L-amphetamine, nortriptyline, DA,
MPP.sup.+, NE, 5-HT, .beta.-PMA, TIA, 4.beta.-PDBu, ConA, Mezerein,
Histamine, Impromidine, Dimaprit, PEA, SNP, EGTA, Thapsigargin,
Genistein, MHC, NECA, N6-CPA, 8-Br-cGMP, SNAP, Hydroxylamine,
Calmidazolium, LY-83583, Methylene blue, CGS9343B, DOI,
Interferon-alpha, and combinations thereof. See, Reith, supra.
[0093] Second, a known general inhibitor of cellular activity is
optionally added, and the resulting decrease in transporter
activity similarly detected. It will be appreciated that modulators
are also optionally combined in assays with known transport
activators or inhibitors to find modulators which inhibit
activation or repression of transporter activity by the known
activator or inhibitor.
[0094] Gradient Induced Activity
[0095] Model systems that mimic biological processes such as
gradient induced activities are of increasing importance, e.g., in
pharmacology, immunology, and the like. However, as mentioned
above, progress in the study of gradient induced activities (e.g.,
chemotaxis) has been impeded by in vitro assays that are, inter
alia, tedious to perform. Furthermore, the results produced by
these assays have been difficult to quantify. The present invention
addresses these shortcomings in the prior art by providing
automated and quantitative assays that are optionally used to model
essentially any gradient induced activity.
[0096] In particular, embodiments of the present invention that are
used to assay gradient induced activities (e.g., chemotactic
responses) generally include simultaneously flowing an adhesion
factor and a buffer into a microchannel such that the adhesion
factor binds to at least a substantial portion of a longitudinal
segment of the microchannel. Thereafter, a suspension of motile
cells (e.g., fluorescently-labeled cells) is typically flowed into
the channel in which some of the cells generally adhere to the
adhesion factor. Unbound cells are optionally washed away.
Following these preparatory steps, a gradient of, e.g., a selected
test compound is typically established in the channel by flowing
the compound into the channel simultaneously with a buffer. If the
test compound induces a response, cells will migrate off the
adhesion factor and be swept down the channel in a fluid stream
that pass through a detector where a detectable signal produced by
the cells (e.g., fluorescence) is detected.
[0097] As discussed, the present invention relates in part to
methods of detecting a gradient-induced activity. The methods
include providing a first channel (e.g., a microchannel) with an
internal surface that typically includes a first and a second
longitudinal segment. Longitudinal segments extend lengthwise
within the channel. Optionally, the internal surface includes three
or more longitudinal segments. A longitudinal segment optionally
extends over at least a substantial portion of one, two, three or
all four walls of a channel that includes a square, rectangular, or
other polygonal cross-sectional area. Alternatively, a longitudinal
segment optionally includes, e.g., about {fraction (1/10000)},
{fraction (1/1000)}, {fraction (1/100)}, {fraction (1/10)}, 1/5,
1/4, 1/3, 1/2, or more or less of the axial cross-sectional area of
the channel and extends over at least a substantial portion of the
length of the channel. Other variations are conceivable so long as
the longitudinal segment extends over a substantial portion of the
length of the channel and includes a portion of the internal
cross-sectional perimeter and/or internal cross-sectional area of
the channel.
[0098] For example, an adhesion factor is generally attached to the
first longitudinal segment and a motile cell (e.g., a phagocytic, a
protozoic, a moneran, or other cell) is typically attached to the
adhesion factor. Thereafter, a gradient is typically established in
the second longitudinal segment of the first channel. The gradient
can induce the motile cell to detach from the adhesion factor to
produce a detectable signal that is detected. Various types of
gradients are suitable for use with these methods including a
chemical gradient, a light energy gradient, a magnetic gradient, a
pH gradient, a dissolved oxygen gradient, a temperature gradient,
or the like.
[0099] Many adhesion factors are known and are optionally used in
detecting gradient induced activities, including those from the
cadherin family (e.g., B-cadherin, E-cadherin, N-cadherin,
P-cadherin, etc.), the selectin family (e.g., L-selectin,
P-selectin, E-selectin, etc.), the mucin-like family (e.g.,
GlyCAM-1, CD34, PSGL-1, MAdCAM-1, etc.), the integrin family (e.g.,
.alpha.4.beta.1,VLA-4, LPAM-1, .alpha.4.beta.7, LPAM-1,
.alpha.6.beta.1, VLA-6, .alpha.L.beta.2, LFA-1, .alpha.M.beta.2,
Mac-1, .alpha.X.beta.2, CR4, p150/95, etc.), the immunoglobulin
superfamily (ICAM-1, ICAM-2, ICAM-3, VCAM-1, LFA-2, CD-2, LFA-3,
CD58, MAdCAM-1, etc.), or the like. There are also many known
chemotactic factors that are suitable for use in these methods
including an antigen, a set of antigens, a protein, a set of
proteins, a peptide (e.g., an acetyl-proline peptide, a fibrino
peptide, an N-formyl peptide, etc.), a set of peptides, a lipid
(e.g., a prostaglandin, a prostacyclin, a thromboxane, a
leukotriene, PAF etc.), a set of lipids, a carbohydrate (e.g., a
maltose, a galactose, a glucose, a ribose, etc.), a set of
carbohydrates, an inorganic molecule (e.g., a chlorinated benzene,
a phosphate, etc.), a set of inorganic molecules, an organic
molecule (e.g., citrate, ATP, etc.), a set of organic molecules, a
drug, a set of drugs, a receptor ligand, a set of receptor ligands,
an antibody, a set of antibodies, a neurotransmitter, a set of
neurotransmitters, a cytokine, a set of cytokines, a chemokine
(e.g., monocyte chemoattractant protein-1, monocyte chemoattractant
protein-2, monocyte chemoattractant protein-3, monocyte
inflammatory protein-1 alpha, monocyte inflammatory protein-1 beta,
RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262, MIP-1b,
T39765, NAP-2, ENA-78, IL-1, IL-6, IL-8, Gro-a, Gro-b, Gro-c,
IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, etc.), a set of chemokines, a
hormone, a set of hormones, or the like.
[0100] Chemotaxis Detection
[0101] In one embodiment of the methods of detecting a gradient
induced activity, the applicable gradient is a chemical gradient.
Chemotaxis is the migration of motile cells up a concentration
gradient of a chemotactic factor. Chemotaxis plays a major role in
the recruitment of the appropriate immune cells to the site of an
infection. Although it has long been known to be an important
biological function, in vitro assays for chemotaxis are tedious and
difficult to quantify. As such, an automated and quantitative assay
for chemotaxis would be very useful.
[0102] There are many references that can be consulted regarding
chemotaxis-related subject matter appropriate to the methods
disclosed herein, including chemotactic agents and target cells.
These references include Agree, A. (1994) "Lymphocyte Recirculation
and Homing: Roles of Adhesion Molecules and Chemoattractants"
Trends Cell Biol. 4:326, Bargatze, R. F. et al. (1995) "Distinct
Roles of L-Selectin and Integrins .alpha.4.beta.7 and LFA-1in
Lymphocyte Homing to Peyer's Patch-HEV In Situ: The Multistep Model
Confirmed and Refined" Immunity 3:99, Grey, H. et al. (1989) "How T
Cells See Antigen" Sci. Am. 261(5):56, Bradley, L. M. and Watson,
S. R. (1996) "Lymphocyte Migration Into Tissue: The Paradigm
Derived From CD4 Subsets" Curr. Opin. Immunol. 8:312; Dianzani, U.
and Malavasi, F. (1995) "Lymphocyte Adhesion to Endothelium" Crit.
Rev. Immunol. 15:167, Hogg, N. and Berlin, C. (1995) "Structure and
Function of Adhesion Receptors in Leukocyte Trafficking" Immunol.
Today 16, Hynes, R. O. (1994) "The Impact of Molecular Biology on
Models for Cell Adhesion" Bioessays 16:663; and Hynes, R. O. (1992)
"Contact and Adhesive Specificities in the Associations,
Migrations, and Targeting of Cells and Axons" Cell 16:303. See
also, Kuby, Immunology, 3.sup.rd Ed. W. H. Freeman and Company, New
York (1997), Pigott and Power, The Adhesion Molecule: Facts Book,
Academic Press Inc., San Diego (1993), and Rot, The Molecular
Biology of Leukocyte Chemotaxis, Chapman and Hall, London
(1998).
[0103] The present invention includes the material handling
advantages of performing analysis and quantification of chemotaxis
in a microfluidic processor or chip. Any arrangement of one or more
microchannels (i.e., microscale channels) in such a processor are
optionally used so long as a response to a gradient can be
detected. The microfluidic devices or systems prepared in
accordance with the invention typically include at least one
microchannel, usually at least two intersecting microchannels, and
often, three or more intersecting microchannels disposed within a
single body structure. Channel intersections can exist in a number
of formats, including cross intersections, "T" intersections, "Y"
intersections, or any number of other structures in which at least
two channels can be in fluid communication.
[0104] In one embodiment shown in FIG. 4, simple double Y
microfluidic processor 400 is optionally used to quantify
chemotaxis. Motile cells 406 require specific adhesion factors or
molecules 404 on a surface in order to move along that surface. The
appropriate adhesion factor is generally flowed down the left side
of the double Y structure with concomitant flow of buffer down the
right side of the structure. For many adhesion factors, nonspecific
binding to glass is sufficient to get cells to adhere to the coated
surface. The adhesion factor is then typically washed out,
resulting in a channel in the middle of the chip where there is
adhesion factor bound to the left side, but none on the right side
(of course, this arrangement is optionally reversed, or a
top/bottom arrangement of components is optionally used). Cells are
then typically flowed in and followed by a wash with buffer. The
cells will attach to the left side of the channel where there is
adhesion factor, but the right side of the channel will not contain
cells.
[0105] By balancing the flows from the two top reservoirs with
those to the two bottom reservoirs, two parallel streams are formed
which contact each other in the central channel and diverge into
the two wastes. Introduction of chemotactic factor 408 into the
upper right channel and buffer into the upper left channel will
cause a gradient of chemotactic factor to form across the channel,
with a high concentration on the right and a low concentration on
the left. If the cells respond to the chemotactic factor they
migrate to the right side on the channel. When they migrate off of
the adhesion factor, they are swept away in the flowing stream, and
travel down the bottom right channel past a cell detection region
to a waste well. The rate at which cells pass detector 402 as a
function of the established concentration gradient of chemotactic
factor allows for quantification of chemotaxis, and the study of
modulators, e.g., inhibitors, of chemotaxis by adding those
modulators to the flowing stream.
[0106] FIG. 5 depicts an alternative mode of detecting a
chemotactic response in which motile cells 504 are fluorescently
labeled with label 508 (e.g., fluorescein, rhodamine, and the
like). Additionally, the bottom of channel 500 is optionally
sloped, i.e., deeper on one side than on the other. In turn, as
cells migrate off of adhesion factor 506 towards chemotactic factor
502, they go into or out of the detector's plane of focus, and thus
increase or decrease their detected fluorescence.
[0107] Binding Affinity Detection
[0108] Binding interactions are replete in nature. For example,
immune responses can involve membrane-bound (e.g., on B cells) or
non-membrane-bound antibodies binding to specific antigens. As
mentioned above, synaptic junctions typically involve transporters
or receptors on post-synaptic neurons, e.g., binding to various
neurotransmitters. Additionally, myriad enzyme-substrate binding
interactions are known. The study of these interactions has been
hampered by the lack of simple and reliable assays. However, the
present invention provides methods and devices that are optionally
used to easily assess the binding affinity or association constant
(K.sub.a) of essentially any small or rapidly diffusing molecule
(i.e., a ligand or a target) with essentially any large or slowly
diffusing molecule or complex of molecules (i.e., a binding
agent).
[0109] The binding affinity assays of the present invention provide
various advantages. For example, binding affinity is optionally
analyzed without labeling binding agents or ligands, e.g., with
colorimetric, fluorescent, radioactive, or other tags.
Additionally, the assays include very low consumption of ligand and
binding agents. Typically, the maximum consumption of ligand is
about one picomole, e.g., with a ligand flow rate of 1 nl/sec, a
run time of 30 seconds, and a ligand concentration of 40 .mu.M
(i.e., 1 nl/sec * 30 sec * 40 .mu.M), while the maximum binding
agent consumption is also about one picomole, e.g., with a binding
agent flow rate of 1 nl/sec, a run time of 10 seconds, and a
binding agent concentration of 100 .mu.M (i.e., 1 nl/sec * 30 sec *
100 .mu.M). Other advantages include the ability to assess the
K.sub.as of a series of ligands for a given binding agent or
target, or the K.sub.as of a series of binding agents with a given
ligand in rapid succession under the control of an automated or
integrated system. Integrated systems are discussed in greater
detail below. Furthermore, resulting K.sub.a values are
thermodynamically meaningful, accurate, and reproducible.
[0110] In particular, the binding assays of the present invention
optionally include detecting an initial concentration of a selected
labeled or unlabeled test ligand prior to flowing the ligand into a
microchannel with a binding agent (e.g., a biological receptor, an
enzyme, a receptor, a protein, a cell, a membrane, a lipid, a
nucleic acid, etc.) which is typically much larger in size than the
ligand. In the laminar flow streams of the microfluidic devices of
the invention, diffusion between streams is generally only
significant for the smaller ligand molecules. As such, when the
ligand and binding agent are flowed from separate channels into a
single microchannel, only the ligand will substantially diffuse
into the stream flowing the binding agent. This "one-way" diffusion
is optionally further ensured by adjusting the length of the
channel in which simultaneous flow occurs. Thereafter, the ligand
flow stream is typically flowed into another channel and through a
detector to determine a final ligand concentration. The difference
between the initial and final ligand concentrations are typically
used to derive an indication of binding activity.
[0111] As discussed, the present invention also includes methods
relating to the detection of various binding activities, including
unlabeled protein-ligands. Although these methods are applicable to
any detection mode, absorption or refractive index, and mass
spectroscopic detection are preferred modes, because no labeling is
necessary. As shown in FIG. 6, ligand 604 is generally flowed into
first channel 600 from second channel 614. Binding agent 602 (e.g.,
an enzyme, a receptor, a cell, a nucleic acid, etc.) is typically
also be flowed into first channel 600 from third channel 612
concomitant with ligand 604. Although not shown, a buffer is
optionally flowed into first channel 600 from third channel 612,
while binding agent 602 is alternatively flowed into first channel
600 from one or more other channels which intersect with first
channel 600 downstream from third channel 612 and second channel
614. See also, FIG. 8. As depicted, ligand 604 is typically smaller
(i.e., lower molecular weight, smaller surface area, etc.) than
binding agent 602 such that ligand 604 generally diffuses more
rapidly in solution than binding agent 602. In turn, as ligand 604
and binding agent 602 flow in first channel 600, only ligand 604
diffuses substantially across first channel 600, while binding
agent 602 remains substantially undiffused as it flows through
first channel 600.
[0112] When ligand 604 diffuses across first channel 600, binding
agent 602 can bind to ligand 604 to form binding agent-ligand
complex 616. (FIG. 6). Once ligand 604, binding agent 602, and
binding agent-ligand complex 616 flow to the end of first channel
600, they exit first channel 600 via fourth channel 610. Only
ligand 604 also exits first channel 600 via fifth channel 608 and
therein detector 606 typically detects a detectable signal that
indicates a final concentration of ligand 604 that remains unbound.
Although not shown, detectors are also optionally located in or
proximal to channels 610, 612, and/or 614, or are fluidly coupled
to the channels (e.g., in the case of mass spectroscopy). An
advantage of these methods is that they enable one to directly
extract the percent ligand 604 bound to binding agent 602 without
knowing anything about the spectral properties of ligand 604 and
without labeling binding agent 602.
[0113] As shown in FIG. 6, the binding assay provided by the
present invention optionally utilizes a double Y array on a chip.
This figure illustrates a simple example, other embodiments
include, e.g., multiple interconnected double Y microchannel
configurations, e.g., for use in extracting selected molecules or
other compounds. In any case, as shown in FIG. 6, a first step
typically involves calibrating ligand 604 concentration to 100
percent. In the embodiments where ligand concentrations are
detected both prior to entry into (i.e., D.sub.1) and after exit
from (i.e., D.sub.2) first channel 600, this is optionally
accomplished by flowing the initial concentration of ligand 604,
[L].sub.I, to be used in the particular experiment, through first
channel 600 in the absence of binding agent 602 to be used (i.e.,
[E]=0). The ratio of signals (D.sub.2/D.sub.1) which is the
calibrated signal, D.sub.cal, is then typically set to 100%. To
measure binding, one generally first flows the concentration of
binding agent 602 to be used in the experiment through the first
channel (i.e., [E].sub.I) until it equilibrates (i.e., for
t.sub.eqb). Thereafter, one typically flows [L].sub.I through the
first channel and measures the concentration of L at each detector
and calculates D.sub.exp=D.sub.2/D.sub.1. The mole fraction of L
bound to E is generally estimated as follows:
[1-(D.sub.exp/D.sub.cal]* 100% .varies.(% bound). Furthermore, one
typically assumes fast k.sub.on and k.sub.off for the initial
evaluation.
[0114] There are various parameters that are generally applicable
to the methods of detecting binding activity. For example, the
binding activity is optionally detected at a temperature in the
range of from about 0 to about 100.degree. C. (e.g., for certain
thermophilic cell based applications), or e.g., commonly in the
range of from about 10 to about 40.degree. C. for typical
physiological assays (e.g., those at about 37.degree. C.), in the
range of from about 20 to about 30.degree. C. (e.g., for room
temperature assays), or e.g., at about 20-25.degree. C. The first
component or the set of first components typically diffuses in the
range of from about 1.5 to about 100 or more times (e.g., 3, 5, 10,
15, 25, 40, 60, 75, or more times) faster in solution than the
second component or the set of second components. For example, the
first component or the set of first components typically diffuses
about 50 times faster in solution than the second component or the
set of second components. In addition, the initial concentration of
the ligand prior to entry into the first channel is optionally,
e.g., in the range of from about 1 nM to about 1 mM, in the range
of from about 10.sup.-2 .mu.M to about 100 .mu.M, or at about 10
.mu.M depending on the assay at issue. Also, the ligand optionally
has a molecular weight in the range of from about 200 to about 2000
daltons, in the range of from about 300 to about 1500 daltons,
e.g., from about 500 to about 1200 daltons, or is about 1000
daltons. The ligand also typically has a diffusional coefficient in
the range of from about 10.sup.-12 to about 10.sup.-4
cm.sup.2s.sup.-1, in the range of from about 10.sup.-7 to about
10.sup.-5 cm.sup.2s.sup.-1, or has one at about 10.sup.-6
cm.sup.2s.sup.-1. Additionally, when the binding agent to be used
is an enzyme, it typically has a concentration in the range of from
about 1 nM to about 1 mM, in the range of from about 10.sup.-2
.mu.M to about 100 .mu.M, or is at about 10 .mu.M depending, e.g.,
on the activity of the enzyme relative to the substrate of
interest. Furthermore, an enzymatic binding agent also typically
includes a molecular weight in the range of from about 10 to about
200 kilodaltons, in the range of from about 20 to about 40
kilodaltons, or is about 30 kilodaltons.
[0115] FIG. 8 is a schematic of an interface between a microfluidic
device that is optionally used to perform binding affinity assays
and an electrospray ionization (ESI) mass spectrometer. As shown,
binding assay system 800 includes a microfluidic device that
includes a channel network and capillary channel 810 extending from
the body of the device. Capillary channel 810 is typically used to
draw samples or reagents (e.g., binding agents, ligands, buffers,
or the like) from, e.g., the wells of a microwell plate into the
device. Although not shown, the body structure optionally includes
more than one capillary channel. During operation, buffer is
optionally flowed, e.g., from buffer well 802, while binding agent
(e.g., proteins, receptors, etc.) is optionally flowed from binding
agent well 804. The flow of binding agent from binding agent well
804 is optionally switchable on and off. As also shown, the
microfluidic device also interfaces with waste line 806 through
which fluids are optionally directed to waste. Examples of device
operation are discussed below. The system also includes, e.g., time
of flight (TOF) mass analyzer 812, electrospray ionization ion
source 814, system inlet 816, and detector 818 which are under
vacuum 808. Vacuum 808 is typically set to allow flow rates in the
range of from about 0.5 to about 5 nL per second. A preferred flow
rate is about 1 nL/s. As mentioned, operational flow rates and
channel dimensions are optionally designed such that the binding
agent diffuses less than half the width of the main assay channel
by the time it reaches the end of the channel and is directed to
waste. Binding assay system 800 also includes a switch for rapidly
switching between flow from buffer well 802 and flow from binding
agent well 804.
[0116] As mentioned, essentially any molecule is optionally used as
a ligand for these methods. For example, a ligand is optionally
selected from amongst the chemotactic factors specified supra.
EXAMPLE 1
[0117] Operational Procedure
[0118] An example of an operational procedure is as follows:
[0119] 1. Prepare a microtiter plate (any density) with positive
control, negative control, unknown samples and wash solution.
[0120] 2. Prepare binding agent (e.g., a protein) in stabilizing
buffer at, e.g., 10 .mu.M and add to binding agent reservoir on the
microfluidic device.
[0121] 3. With no flow from binding agent reservoir, sip from wash
solution well until total ion current at positive control mass is
stable (baseline calibration) (Refer to this as T.sub.1).
[0122] 4. Move capillary channel to positive control and sip until
ion current at positive control mass is stable. (100% calibration,
approximately 5 .mu.M concentration assuming 50% flow into
detection channel).
[0123] 5. Pulse 10% binding agent into main channel for time equal
to, e.g., 2.times. total travel time from binding agent entry point
until detection point and repeat in 10% increments until 100%.
[0124] 6. Observe ion current for positive control mass over this
time period. Should drop to 1% of value observed in step (4). Refer
to time for drop to less than 10% as T.sub.2.
[0125] 7. Turn off binding agent flow and move capillary channel to
wash well. Sip until ion current for positive control mass returns
baseline value. Refer to this as time T.sub.3.
[0126] 8. Move capillary channel to negative control well and sip
for time T.sub.1. Set mass spectrometer to monitor ion current for
most intense observed mass. This mass should correspond to negative
control mass.
[0127] 9. Pulse protein for time T.sub.2. Ion current for negative
control mass should not decrease by more than 5%.
[0128] 10. Turn off binding agent flow.
[0129] 11. Move capillary channel to wash well and sip for time
T.sub.3. Ion current should return to baseline value.
EXAMPLE 2
[0130] Operational Procedure for Detecting Binding Affinities of
Unknown Samples to Binding Agent
[0131] An example of an operational procedure for detecting binding
affinities of unknown samples (e.g., ligands) to binding agents
(e.g., protein) is as follows:
[0132] A. Perform the operational procedure described above, (i.e.,
steps (1)-(11)) using positive control, negative control and wash
solution to determine T.sub.1, T.sub.2, and T.sub.3. Record the
baseline value determined in step (3) and refer to this value as
V.sub.1.
[0133] B. Move capillary channel to well containing an unknown
sample and sip for time T.sub.1. Set MS to monitor ion current for
most intense observed mass. Record ion current at end of T.sub.1
and refer to it as V.sub.2.
[0134] C. Pulse protein for time T.sub.2 and record change in ion
current at end of T.sub.2. Refer to this value as V.sub.3.
[0135] D. Turn off binding agent flow.
[0136] E. Move capillary channel to wash well and sip for time
T.sub.3. Record baseline ion current and refer to this value as
V.sub.4.
[0137] F. Repeat steps (B)-(E) for each well containing an unknown
sample. At end of every row on the microtiter plate, sip from
positive control and repeat steps (B)-(E).
[0138] G. If A.sub.init=B.sub.init, then calculate the approximate
binding affinity of unknown samples by the equation
K.sub.a=[A.sub.init-(V.sub.3/-
V.sub.2)A.sub.init]/[(V.sub.3/V.sub.2)A.sub.init].sup.2. If
A.sub.init and B.sub.init are different values, the method shown in
Example 3, below, is used to calculate the K.sub.a.
[0139] H. Calculate the signal to noise ratio from V.sub.1 and
V.sub.2. Calculate the confidence interval from the values V.sub.1,
V.sub.2, and V.sub.3 using the positive control at the end of each
well.
EXAMPLE 3
[0140] Calculations for Bimolecular Binding Reactions
[0141] The following describes the calculation of various values,
e.g., binding agent and ligand concentrations and association
constants associated with the bimolecular binding interactions
detected in the binding affinity assays of the present invention.
For example, the concentration of a species A (e.g., a ligand) in
the presence of binding agent B at equilibrium is:
[A]=[AB]/K.sub.a[B]
[0142] Where [A], [B], and [AB] are the equilibrium concentrations
of A, B and the complex AB, respectively, and K.sub.a is the
association constant of A with B. If A and B are present in the
same initial concentrations, A.sub.init=B.sub.init, then
[A]=([A.sub.init]-[A])/K.sub.a[A], and
K.sub.a(A.sup.2)-A+A.sub.init=0
[0143] Solving for the positive root gives
[-1+sqrt(1+4*A.sub.init*K.sub.a)]/2*K.sub.a
[0144] When, e.g., K.sub.a=10 .mu.M and A.sub.init=B.sub.init=10
.mu.M, the concentration of A at equilibrium is 6.18 .mu.M. When
A.sub.init=1 .mu.M, A=0.618 .mu.M. For additional calculated values
see Table 1, below. These data provide an estimate of the detection
sensitivity that may be necessary for accurate approximations of
binding affinities by the methods disclosed herein.
1TABLE 1 Values of K.sub.d, K.sub.a, and A (final concentration of
detected ligand) when A.sub.init = B.sub.init = 1 .mu.M. K.sub.d
K.sub.a A 1ee-9 1ee9 0.031127 1ee-8 1ee8 0.095125 1ee-7 1ee7
0.270156 1ee-6 1ee6 0.618034 1ee-5 1ee5 0.91608 1ee-4 1ee4 0.990195
1ee-3 1ee3 0.999002
EXAMPLE 4
[0145] Experimental Procedures for Detecting Unknown Sample Binding
Affinity for a Target
[0146] The following includes examples of experimental procedures
for detecting the binding affinity of an unknown sample (U) (e.g.,
a ligand) for a target (T) (e.g., a binding agent).
[0147] Device Preparation
[0148] The initial step involves preparing a device such as the one
depicted in FIG. 9, so that upon introduction of T 900 from target
channel 902, T 900 does not substantially diffuse across main
channel 908. The direction of fluid flow is indicated by arrow 914.
For example, if the diffusion constant of T 900 is
5.times.10.sup.-7, the diffusion constant of U 906 is
5.times.10.sup.-5, and the flow rate is 1 mm/sec, then suitable
dimensions of main channel 908 on the device are, e.g., 20 mm in
length, 100 .mu.m in width, and 100 .mu.m in depth. Additionally,
detection channel 904 (which includes detector 916 disposed
proximate thereto) and binding channel 910 intersect main channel
908, as shown. In main channel 908, so configured, T 900 and any
bound U 906, will diffuse on average approximately 45 .mu.m across
main channel 908 by the time they reach binding channel 910 and, in
turn, will exit via binding channel 910. By comparison, unbound U
906 will diffuse across main channel 908 on average approximately
4.5 times more than T 900, and will exit via both detection channel
904 and binding channel 910. Unknown sample channel 912 optionally
intersects the center of main channel 908 (as shown), as a sipper
capillary which extends from the device, as a channel entering from
a side of main channel 908 (as shown in FIG. 10), or any other
configuration, e.g., in which T 900 exits main channel 908
substantially only from binding channel 910.
[0149] Experimental Protocol
[0150] An example experimental protocol is as follows:
[0151] (a) Prepare a multi-well microtiter plate with unknown
samples in a compatible solvent such that a concentration
[U.sub.init] (e.g., 10 .mu.M) is introduced into the main
channel.
[0152] (b) Prepare a wash well containing stabilizing buffer either
on or off the microtiter plate.
[0153] (c) Place a target in stabilizing buffer in a reservoir on
the chip such that a concentration [T.sub.init] (e.g., 10 .mu.M) is
introduced into the main channel. This concentration calculation
takes into account the fact that the target does not diffuse all
the way across the channel.
[0154] (d) In the absence of flow from the target reservoir, sip
using, e.g., a sipper capillary from wash well until detector
signal is stable (baseline calibration). A signal corresponding to
approximately 0% unknown sample is observed; record this signal
intensity as V.sub.1. For example, a mass selective detector or an
UV absorbance detector is optionally used.
[0155] (e) Move sipper capillary to an unknown sample well and sip
until signal at detector is stable (e.g., at the maximum observed
ion current in a mass detector, or the maximum intensity peak in a
UV detector). This is the 100% calibration step. Record this signal
as V.sub.2.
[0156] (f) Pulse the target into the main channel for a time long
enough for the target to bind to the unknown sample and to
stabilize the unknown sample signal (optimally 1 to 60 seconds).
Record the value of the signal at the detector as V.sub.3.
Optionally for higher throughput screening, the target is pulsed
for just long enough to detect a detectable change in the unknown
sample signal.
[0157] (g) Calculate the concentration of free (i.e., unbound)
unknown [U] at the end of the main or reaction channel, as follows:
[U]=(V.sub.3-V.sub.1/V.sub.2-V.sub.1)*[U.sub.init].
[0158] (h) Calculate the approximate concentration of free target
at the end of the reaction channel, as follows:
[T]=(V.sub.3-V.sub.1/V.sub.2-V.s- ub.1)*[T.sub.init].
[0159] (i) Calculate the approximate affinity constant K.sub.a of
the unknown for the target using the following equation:
K=[UT]/([U][T]), where [UT] is the approximate concentration of the
unknown-target complex at the end of the reaction channel.
Substituting the equations from steps (h) and (i) gives
K.sub.a=([U.sub.init]-[U])/[U.sub.init]*([T.sub.init]-[-
U.sub.init]+[U]).
[0160] Thus, for example if [U.sub.init] is 10 .mu.M and
[T.sub.init] is 10 .mu.M, and the observed signals are
V.sub.1=2000, V.sub.2=10,000, and V.sub.3=6,000 units, then the
calculated approximate affinity constant of U for T is
2.times.10.sup.-5. A more precise K.sub.a is optionally calculated
by taking several measurements using this experimental protocol at
several concentrations of U and T. As a further option, many
parallel channels are used with short target-unknown contact times
to perform high-throughput screening of many samples simultaneously
(e.g., 1 to >1,000,000 compounds). For additional discussion of
parallel screening techniques, see, e.g., U.S. Pat. No. 6,046,056
to Parce, et al., entitled "High Throughput Screening Assay Systems
in Microscale Fluidic Devices," which issued Apr. 4, 2000.
[0161] Competitive Binding
[0162] A related mode of detecting competitive binding activity of
a ligand with a target provides another example of the uses to
which the methods disclosed herein are optionally put. In cases in
which a ligand that binds to the target or binding agent of
interest is already known or identifiable, the diffusion-based
methods described herein are optionally used to determine the
affinity of an unknown compound for the target of interest by
displacement of the ligand. For example, in one embodiment, the
binding assay described above is initially used to identify a known
ligand, and this ligand is used in the following method to quickly
determine the affinity of an unknown sample for the target.
[0163] In the example below and with reference to FIG. 10, the
following steps are taken to determine the ability of unknown
sample 1000 to compete with known sample 1002 for binding to target
1004 (and thus the affinity of unknown sample 1000 for target 1004
at the site of binding of known sample 1002):
[0164] (1) The flow rate and the chip are of the dimensions
described above (see, Device Preparation), such that:
[0165] (i) The substantially larger molecular weight target (T)
1004 enters from target sample channel 1006 and diffuses only far
enough across main channel 1008 so that the majority of T 1004
exits main channel 1008 via binding channel 1010. The direction of
fluid movement is indicated by arrow 1018;
[0166] (ii) The substantially smaller molecular weight unknown (U)
1000 enters main channel 1008 from unknown sample channel 1014 and
diffuses rapidly across the entire main channel 1008, exiting via
both detection channel 1012 (which includes detector 1020 disposed
proximate thereto) and binding channel 1010. Unknown sample channel
1014 is optionally a side arm (as shown), a sipper capillary, a
channel entering from the center of main channel 1008 (as shown in
FIG. 9), or any other configuration that provides, e.g., adequate
separation of T 1004 from U 1000; and,
[0167] (iii) Known sample (K) 1002, which includes a known positive
binding affinity for T 1004, enters from known sample channel 1016
and diffuses across main channel 1008 according to its diffusion
constant D. The placement of known sample channel 1016 and the flow
rate are chosen such that K 1002 exits via both binding channel
1010 and detection channel 1012 and with sufficient time to bind to
T 1004 (optimally 1 to 60 seconds). Any diffusion constant, and
therefore any molecular weight, for K 1002 is compatible with this
embodiment as long as these conditions are met.
[0168] (2) A detector placed at any point along detection channel
1012 is tuned to detect the concentration of K 1002 in detection
channel 1012. For example, a mass selective detector set at the
molecular weight of K 1002 is optionally used.
[0169] (3) T 1004 and K 1002 are continuously flowed for a long
enough period to obtain a stable signal for K 1002 at detector 1020
(optimally 1 to 60 seconds). The concentration of K 1002 observed
at detector 1020 will depend on the initial concentration of K
1002, the initial concentration of T 1004, and the binding constant
between the two, K.sub.K-T.
[0170] (4) U 1000 is pulsed for a sufficient period to allow
binding of U 1000 (if the unknown has a measurable binding
affinity) to T 1004, and to allow the new signal for K 1002 to
stabilize at detector 1020 (optimally 1 to 60 seconds).
[0171] (5) The flow of U 1000 is discontinued, and buffer is flowed
along with the continuing flow of K 1002 and T 1004 until the
signal for K 1002 stabilizes again.
[0172] (6) Optionally, the cycle is repeated for other unknown
samples.
[0173] (7) To determine the binding constant of U 1000 for T 1004,
the magnitude of the signal from Part (4) is subtracted from the
magnitude of the signal from Part (3) to obtain the difference in
signal (DS) in the presence and absence of target 1004. This
difference in signal is proportional to the binding constant of U
1000 for T 1004 (K.sub.U-T) provided that U 1000 competes for the
same binding site on T 1004 that is occupied by K 1002.
[0174] Example of experimental conditions are as follows:
[0175] Initial main channel concentration of T=10 .mu.M
[0176] Initial main channel concentration of K=10 .mu.M
[0177] Initial main channel concentration of U=10 .mu.M
[0178] Diffusion constant of target: 5.times.10.sup.-7
[0179] Diffusion constant of unknown sample: 5.times.10.sup.-5
[0180] Diffusion constant of known ligand 1.times.10.sup.-6
[0181] Dimensions of main channel as described above (see, Device
Preparation), with known sample entering 5 mm downstream of the
beginning of the main channel.
[0182] A more precise K.sub.a is optionally calculated by taking
several measurements, as indicated above, at several concentrations
of U, K and T, and numerically solving the equation for two ligands
competing for one target. As an additional option, many parallel
channels are used with short target-unknown contact times to
perform high-throughput screening of many samples (e.g. 1 to
>1,000,000 compounds). For further discussion of parallel
screening techniques, see, e.g., U.S. Pat. No. 6,046,056 to Parce,
et al., entitled "High Throughput Screening Assay Systems in
Microscale Fluidic Devices," which issued Apr. 4, 2000.
[0183] If there is concern that unknown U may have a substantial
affinity for known K, or that the presence of U may disrupt the
signal at the detector due to K, one optionally adds a step in
which the signal from K is detected at the detector in the absence
of T, but in the presence and absence of U. In this way the effect
of U on the detection of K is optionally subtracted and thereby
disregarded in calculating the affinity of U for T.
[0184] Flow of Transporters, Transmitters, Chemotactic Agents,
Cells, Modulators and other Components in Microscale Systems
[0185] A variety of microscale systems which can be adapted to the
present invention by incorporating transporter components,
transmitter components, chemotactic agents, cells, modulators and
the like are available. Microfluidic devices which can be adapted
to the present invention by the addition of transporter assay
components are described in various PCT applications and issued
U.S. Patents by the inventors and their coworkers, including U.S.
Pat. Nos. 5,699,157 (J. Wallace Parce) issued Dec. 16, 1997,
5,779,868 (J. Wallace Parce et al.) issued Jul. 14, 1998, 5,800,690
(Calvin Y. H. Chow et al.) issued Sep. 01, 1998, 5,842,787 (Anne R.
Kopf-Sill et al.) issued Dec. 01, 1998, 5,852,495 (J. Wallace
Parce) issued Dec. 22, 1998, 5,869,004 (J. Wallace Parce et al.)
issued Feb. 09, 1999, 5,876,675 (Colin B. Kennedy) issued Mar. 02,
1999, 5,880,071 (J. Wallace Parce et al.) issued Mar. 09, 1999,
5,882,465 (Richard J. McReynolds) issued Mar. 16, 1999, 5,885,470
(J. Wallace Parce et al.) issued Mar. 23, 1999, 5,942,443 (J.
Wallace Parce et al.) issued Aug. 24, 1999, 5,948,227 (Robert S.
Dubrow) issued Sep. 07, 1999, 5,955,028 (Calvin Y. H. Chow) issued
Sep. 21, 1999, 5,957,579 (Anne R. Kopf-Sill et al.) issued Sep. 28,
1999, 5,958,203 (J. Wallace Parce et al.) issued Sep. 28, 1999,
5,958,694 (Theo T. Nikiforov) issued Sep. 28, 1999, and 5,959,291
(Morten J. Jensen) issued Sep. 28, 1999; and published PCT
applications, such as, WO 98/00231, WO 98/00705, WO 98/00707, WO
98/02728, WO 98/05424, WO 98/22811, WO 98/45481, WO 98/45929, WO
98/46438, and WO 98/49548, WO 98/55852, WO 98/56505, WO 98/56956,
WO 99/00649, WO 99/10735, WO 99/12016, WO 99/16162, WO 99/19056, WO
99/19516, WO 99/29497, WO 99/31495, WO 99/34205, WO 99/43432, and
WO 99/44217.
[0186] For example, pioneering technology providing cell based
microscale assays are set forth in Parce et al. "High Throughput
Screening Assay Systems in Microscale Fluidic Devices" WO 98/00231
and, e.g., in Ser. No. 60/128,643 filed Apr. 4, 1999, entitled
"Manipulation of Microparticles In Microfluidic Systems," by Mehta
et al. Complete integrated systems with fluid handling, signal
detection, sample storage and sample accessing are available. For
example, Parce et al. "High Throughput Screening Assay Systems in
Microscale Fluidic Devices" WO 98/00231 provide pioneering
technology for the integration of microfluidics and sample
selection and manipulation.
[0187] In general, cells, modulators and other components can be
flowed in a microscale system by electrokinetic (including either
electroosmotic or electrophoretic) techniques, or using
pressure-based flow mechanisms, or combinations thereof.
[0188] Cells in particular are desirably flowed using
pressure-based flow mechanisms. Pressure forces can be applied to
microscale elements to achieve fluid movement using any of a
variety of techniques. Fluid flow (and flow of materials suspended
or solubilized within the fluid, including cells or other
particles) is optionally regulated by pressure based mechanisms
such as those based upon fluid displacement, e.g., using a piston,
pressure diaphragm, vacuum pump, probe or the like to displace
liquid and raise or lower the pressure at a site in the
microfluidic system. The pressure is optionally pneumatic, e.g., a
pressurized gas, or uses hydraulic forces, e.g., pressurized
liquid, or alternatively, uses a positive displacement mechanism,
i.e., a plunger fitted into a material reservoir, for forcing
material through a channel or other conduit, or is a combination of
such forces.
[0189] In other embodiments, a vacuum source is applied to a
reservoir or well at one end of a channel to draw the suspension
through the channel. Pressure or vacuum sources are optionally
supplied external to the device or system, e.g., external vacuum or
pressure pumps sealably fitted to the inlet or outlet of the
channel, or they are internal to the device, e.g., microfabricated
pumps integrated into the device and operably linked to the
channel. Examples of microfabricated pumps have been widely
described in the art. See, e.g., published International
Application No. WO 97/02357.
[0190] Hydrostatic, wicking and capillary forces can also be used
to provide pressure for fluid flow of materials such as cells. See,
e.g., U.S. Pat. No. 6,416,642. In these methods, an adsorbent
material or branched capillary structure is placed in fluidic
contact with a region where pressure is applied, thereby causing
fluid to move towards the adsorbent material or branched capillary
structure.
[0191] Mechanisms for reducing adsorption of materials during
fluid-based flow are described in U.S. Pat. No. 6,458,259. In
brief, adsorption of cells, transmitters, chemotactic factors,
potential modulators and other materials to channel walls or other
microscale components during pressure-based flow can be reduced by
applying an electric field such as an alternating current to the
material during flow.
[0192] Mechanisms for focusing cells and other components into the
center of microscale flow paths, which is useful in increasing
assay throughput by regularizing flow velocity is described in U.S.
Pat. No. 6,506,609. In brief, cells are focused into the center of
a channel by forcing fluid flow from opposing side channels into
the main channel comprising the cells, or by other fluid
manipulations. Diffusible materials such as the transmitters of the
present invention are also optionally washed from cells as
described by Wada et al. during flow of the cells, i.e., by
sequentially flowing buffer into a channel in which cells are
flowed and flowing the buffer back out of the channel.
[0193] In an alternate embodiment, microfluidic systems can be
incorporated into centrifuge rotor devices, which are spun in a
centrifuge. Fluids and particles travel through the device due to
gravitational and centripetal/centrifugal pressure forces.
[0194] One method of achieving transport or movement of
transmitters, chemotactic factors, enzymes, receptors, ligands,
modulators, and even cells (particularly transmitters and
modulators) through microfluidic channels is by electrokinetic
material transport. "Electrokinetic material transport systems," as
used herein, include systems that transport and direct materials
within a microchannel and/or chamber containing structure, through
the application of electrical fields to the materials, thereby
causing material movement through and among the channel and/or
chambers, i.e., cations will move toward a negative electrode,
while anions will move toward a positive electrode. For example,
movement of fluids toward or away from a cathode or anode can cause
movement of transmitters, cells, chemotactic factors, enzymes,
receptors, ligands, modulators, etc. suspended within the fluid.
Similarly, the transmitters, cells, chemotactic factors, enzymes,
receptors, ligands, modulators, etc. can be charged, in which case
they will move toward an oppositely charged electrode (indeed, in
this case, it is possible to achieve fluid flow in one direction
while achieving particle flow in the opposite direction). In this
embodiment, the fluid can be immobile or flowing and can comprise a
matrix as in electrophoresis.
[0195] In general, electrokinetic material transport and direction
systems also include those systems that rely upon the
electrophoretic mobility of charged species within the electric
field applied to the structure. Such systems are more particularly
referred to as electrophoretic material transport systems. For
electrophoretic applications, the walls of interior channels of the
electrokinetic transport system are optionally charged or
uncharged. Typical electrokinetic transport systems are made of
glass, charged polymers, and uncharged polymers. The interior
channels are optionally coated with a material which alters the
surface charge of the channel.
[0196] A variety of electrokinetic controllers and systems are
described, e.g., in Ramsey WO 96/04547, Parce et al. WO 98/46438
and Dubrow et al., WO 98/49548, as well as a variety of other
references noted herein.
[0197] Use of electrokinetic transport to control material movement
in interconnected channel structures was described, e.g., in WO
96/04547 and U.S. Pat. No. 5,858,195 to Ramsey. An exemplary
controller is described in U.S. Pat. No. 5,800,690. Modulating
voltages are concomitantly applied to the various reservoirs to
affect a desired fluid flow characteristic, e.g., continuous or
discontinuous (e.g., a regularly pulsed field causing the sample to
oscillate direction of travel) flow of labeled components toward a
waste reservoir. Particularly, modulation of the voltages applied
at the various reservoirs can move and direct fluid flow through
the interconnected channel structure of the device.
[0198] Sources of Assay Components and Integration with
Microfluidic Formats
[0199] Sources of transporter containing components such as cells
or cell fractions, sources of transmitters, adhesion factors,
chemotactic factors, enzymes, receptors, ligands, and sources of
components such as cells or cell fractions comprising transmitter
receptors can be fluidly coupled to the microchannels noted herein
in any of a variety of ways. In particular, those systems
comprising sources of materials set forth in Knapp et al. "Closed
Loop Biochemical Analyzers" (WO 98/45481; PCT/US98/06723) and Parce
et al. "High Throughput Screening Assay Systems in Microscale
Fluidic Devices" WO 98/00231 and, e.g., in Ser. No. 60/128,643
filed Apr. 4, 1999, entitled "Manipulation of Microparticles In
Microfluidic Systems," by Mehta et al. are applicable.
[0200] In these systems, a "pipettor channel" (a channel in which
components can be moved from a source to a microscale element such
as a second channel or reservoir) is temporarily or permanently
coupled to a source of material. The source can be internal or
external to a microfluidic device comprising the pipettor channel.
Example sources include microwell plates, membranes or other solid
substrates comprising lyophilized components, wells or reservoirs
in the body of the microscale device itself and others.
[0201] For example, the source of a cell type, component, or
modulator reagent can be a microwell plate external to the body
structure, having, e.g., at least one well with the selected cell
type or reagent. Alternatively, a well disposed on the surface of
the body structure comprising the selected cell type, component, or
reagent, a reservoir disposed within the body structure comprising
the selected cell type, component or reagent; a container external
to the body structure comprising at least one compartment
comprising the selected particle type or reagent, or a solid phase
structure comprising the selected cell type or reagent in
lyophilized or otherwise dried form.
[0202] A loading channel region is optionally fluidly coupled to a
pipettor channel with a port external to the body structure, e.g.,
as depicted in FIGS. 2 and 3. The loading channel can be coupled to
an electropipettor channel with a port external to the body
structure, a pressure-based pipettor channel with a port external
to the body structure, a pipettor channel with a port internal to
the body structure, an internal channel within the body structure
fluidly coupled to a well on the surface of the body structure, an
internal channel within the body structure fluidly coupled to a
well within the body structure, or the like. Example configurations
are depicted in the figures herein.
[0203] As described more fully herein, the integrated microfluidic
system of the invention can include a very wide variety of storage
elements for storing reagents to be assessed. These include well
plates, matrices, membranes and the like. The reagents are stored
in liquids (e.g., in a well on a microtiter plate), or in
lyophilized form (e.g., dried on a membrane or in a porous matrix),
and can be transported to an array component of the microfluidic
device using conventional robotics, or using an electropipettor or
pressure pipettor channel fluidly coupled to a reaction or reagent
channel of the microfluidic system.
[0204] In general, the test modulator compounds are separately
introduced into the assay systems described herein, or at least
introduced in relatively manageable pools of modulator materials.
The relative level of a particular cellular transport function is
then assessed in the presence of the test compound, and this
relative level of function is then compared to a control system,
which lacks an introduced test modulator compound. Increases or
decreases in relative cellular function are indicative that the
test compound is an enhancer or an inhibitor of the particular
cellular function, respectively.
[0205] Detectors and Integrated Systems
[0206] Although the devices and systems specifically illustrated
herein are generally described in terms of the performance of a few
or one particular operation, it will be readily appreciated from
this disclosure that the flexibility of these systems permits easy
integration of additional operations into these devices. For
example, the devices and systems described will optionally include
structures, reagents and systems for performing virtually any
number of operations both upstream and downstream from the
operations specifically described herein. Such upstream operations
include sample handling and preparation operations, e.g., cell
separation, extraction, purification, amplification, cellular
activation, labeling reactions, dilution, aliquotting, and the
like. Similarly, downstream operations may include similar
operations, including, e.g., separation of sample components,
labeling of components, assays and detection operations,
electrokinetic or pressure-based injection of components into
contact with particle sets, or materials released from particle
sets, or the like.
[0207] Assay and detection operations include, without limitation,
cell fluorescence assays, cell activity assays, probe interrogation
assays, e.g., nucleic acid hybridization assays utilizing
individual probes, free or tethered within the channels or chambers
of the device and/or probe arrays having large numbers of
different, discretely positioned probes, receptor/ligand assays,
immunoassays, and the like. Any of these elements can be fixed to
array members, or fixed, e.g., to channel walls, or the like.
[0208] Instrumentation
[0209] In the present invention, the materials such as cells are
optionally monitored and/or detected so that an activity can be
determined. Depending on the label signal measurements, decisions
are can be made regarding subsequent fluidic operations, e.g.,
whether to assay a particular modulator in detail to determine
kinetic information.
[0210] The systems described herein generally include microfluidic
devices, as described above, in conjunction with additional
instrumentation for controlling fluid transport, flow rate and
direction within the devices, detection instrumentation for
detecting or sensing results of the operations performed by the
system, processors, e.g., computers, for instructing the
controlling instrumentation in accordance with preprogrammed
instructions, receiving data from the detection instrumentation,
and for analyzing, storing and interpreting the data, and providing
the data and interpretations in a readily accessible reporting
format.
[0211] Controllers
[0212] A variety of controlling instrumentation is optionally
utilized in conjunction with the microfluidic devices described
above, for controlling the transport and direction of fluids and/or
materials within the devices of the present invention, e.g., by
pressure-based or electrokinetic control.
[0213] For example, in many cases, fluid transport and direction
are controlled in whole or in part, using pressure based flow
systems that incorporate external or internal pressure sources to
drive fluid flow. Internal sources include microfabricated pumps,
e.g., diaphragm pumps, thermal pumps, lamb wave pumps and the like
that have been described in the art. See, e.g., U.S. Pat. Nos.
5,271,724, 5,277,556, and 5,375,979 and Published PCT Application
Nos. WO 94/05414 and WO 97/02357. As noted above, the systems
described herein can also utilize electrokinetic material direction
and transport systems. Preferably, external pressure sources are
used, and applied to ports at channel termini. These applied
pressures, or vacuums, generate pressure differentials across the
lengths of channels to drive fluid flow through them. In the
interconnected channel networks described herein, differential flow
rates on volumes are optionally accomplished by applying different
pressures or vacuums at multiple ports, or preferably, by applying
a single vacuum at a common waste port and configuring the various
channels with appropriate resistance to yield desired flow rates.
Example systems are described in U.S. Ser. No. 09/238,467 filed
Jan. 28, 1999.
[0214] Typically, the controller systems are appropriately
configured to receive or interface with a microfluidic device or
system element as described herein. For example, the controller
and/or detector, optionally includes a stage upon which the device
of the invention is mounted to facilitate appropriate interfacing
between the controller and/or detector and the device. Typically,
the stage includes an appropriate mounting/alignment structural
element, such as a nesting well, alignment pins and/or holes,
asymmetric edge structures (to facilitate proper device alignment),
and the like. Many such configurations are described in the
references cited herein.
[0215] The controlling instrumentation discussed above is also used
to provide for electrokinetic injection or withdrawal of material
downstream of the region of interest to control an upstream flow
rate. The same instrumentation and techniques described above are
also utilized to inject a fluid into a downstream port to function
as a flow control element.
[0216] Detector
[0217] The devices herein optionally include signal detectors,
e.g., which detect fluorescence, phosphorescence, radioactivity,
pH, charge, absorbance, refractive index, luminescence,
temperature, magnetism or the like. Fluorescent detection is
preferred.
[0218] More specifically, the detectors used in the devices and
systems of the present invention can include, e.g. an optical
detector, a microscope, a CCD array, a photomultiplier tube, a
photodiode, an emission spectroscope, a fluorescence spectroscope,
a phosphorescence spectroscope, a luminescence spectroscope, a
spectrophotometer, a photometer, a nuclear magnetic resonance
spectrometer, an electron paramagnetic resonance spectrometer, an
electron spin resonance spectroscope, a turbidimeter, a
nephelometer, a Raman spectroscope, a refractometer, an
interferometer, an x-ray diffraction analyzer, an electron
diffraction analyzer, a polarimeter, an optical rotary dispersion
analyzer, a circular dichroism spectrometer, a potentiometer, a
chronopotentiometer, a coulometer, an amperometer, a conductometer,
a gravimeter, a mass spectrometer, a thermal gravimeter, a
titrimeter, a differential scanning calorimeter, a radioactive
activation analyzer, a radioactive isotopic dilution analyzer, and
the like. Especially preferred detectors for use in the methods and
devices of the invention include optical detectors and, e.g.,
electrospray ionization mass spectrometers, which can, e.g., be
proximal to or coupled to one or more microscale channels of a
device. Detector embodiments including mass spectrometer elements
are discussed further below.
[0219] The detector(s) optionally monitors one or a plurality of
signals from upstream and/or downstream of an assay mixing point in
which, e.g., a ligand and an enzyme or a receptor, or a transmitter
and a cell or other component with a transmitter receptor and the
cell or other component with transporter activity are mixed. For
example, the detector can monitor a plurality of optical signals
which correspond in position to "real time" assay results.
[0220] Example detectors include photomultiplier tubes, a CCD
array, a scanning detector, a galvo-scann or the like. Cells or
other components which emit a detectable signal can be flowed past
the detector, or, alternatively, the detector can move relative to
the array to determine cell position (or, the detector can
simultaneously monitor a number of spatial positions corresponding
to channel regions, e.g., as in a CCD array).
[0221] The detector can include or be operably linked to a
computer, e.g., which has software for converting detector signal
information into assay result information (e.g., kinetic data of
modulator activity), or the like.
[0222] Signals from arrays are optionally calibrated, e.g., by
calibrating the microfluidic system by monitoring a signal from a
known source.
[0223] A microfluidic system can also employ multiple different
detection systems for monitoring the output of the system.
Detection systems of the present invention are used to detect and
monitor the materials in a particular channel region (or other
reaction detection region). Once detected, the flow rate and
velocity of cells in the channels is also optionally measured and
controlled as described above. As described in PCT Publication WO
98/56956, correction of kinetic information based upon flow
velocity can be used to provide accurate kinetic information.
[0224] Examples of detection systems include optical sensors,
temperature sensors, pressure sensors, pH sensors, conductivity
sensors, mass sensors, and the like. Each of these types of sensors
is readily incorporated into the microfluidic systems described
herein. In these systems, such detectors are placed either within
or adjacent to the microfluidic device or one or more channels,
chambers or conduits of the device, such that the detector is
within sensory communication with the device, channel, or chamber.
The phrase "within sensory communication" of a particular region or
element, as used herein, generally refers to the placement of the
detector in a position such that the detector is capable of
detecting the property of the microfluidic device, a portion of the
microfluidic device, or the contents of a portion of the
microfluidic device, for which that detector was intended. For
example, a pH sensor placed in sensory communication with a
microscale channel is capable of determining the pH of a fluid
disposed in that channel. Similarly, a temperature sensor placed in
sensory communication with the body of a microfluidic device is
capable of determining the temperature of the device itself.
[0225] Particularly preferred detection systems include optical
detection systems for detecting an optical property of a material
within the channels and/or chambers of the microfluidic devices
that are incorporated into the microfluidic systems described
herein. Such optical detection systems are typically placed
adjacent to a microscale channel of a microfluidic device, and are
in sensory communication with the channel via an optical detection
window that is disposed across the channel or chamber of the
device. Optical detection systems include systems that are capable
of measuring the light emitted from material within the channel,
the transmissivity or absorbance of the material, as well as the
materials spectral characteristics. In preferred aspects, the
detector measures an amount of light emitted from the material,
such as a fluorescent or chemiluminescent material. As such, the
detection system will typically include collection optics for
gathering a light based signal transmitted through the detection
window, and transmitting that signal to an appropriate light
detector. Microscope objectives of varying power, field diameter,
and focal length are readily utilized as at least a portion of this
optical train. The light detectors are optionally photodiodes,
avalanche photodiodes, photomultiplier tubes, diode arrays, or in
some cases, imaging systems, such as charged coupled devices (CCDs)
and the like. In preferred aspects, photodiodes are utilized, at
least in part, as the light detectors. The detection system is
typically coupled to a computer (described in greater detail
below), via an analog to digital or digital to analog converter,
for transmitting detected light data to the computer for analysis,
storage and data manipulation.
[0226] In the case of fluorescent materials such as labeled cells,
the detector typically includes a light source which produces light
at an appropriate wavelength for activating the fluorescent
material, as well as optics for directing the light source through
the detection window to the material contained in the channel or
chamber. The light source can be any number of light sources that
provides an appropriate wavelength, including lasers, laser diodes
and LEDs. Other light sources required for other detection systems.
For example, broad band light sources are typically used in light
scattering/transmissivity detection schemes, and the like.
Typically, light selection parameters are well known to those of
skill in the art.
[0227] The detector can exist as a separate unit, but is preferably
integrated with the controller system, into a single instrument.
Integration of these functions into a single unit facilitates
connection of these instruments with the computer (described
below), by permitting the use of few or a single communication
port(s) for transmitting information between the controller, the
detector and the computer.
[0228] Mass Spectrometry
[0229] Mass spectrometry is a widely used analytical technique that
can be used to provide information about, e.g., the isotopic ratios
of atoms in samples, the structures of various molecules, including
biologically important molecules (e.g., transporter molecules,
transmitters, enzymes, receptors, chemotactic factors, and the
like), and the qualitative and quantitative composition of complex
mixtures. Common mass spectrometer systems include a system inlet,
an ion source, a mass analyzer, and a detector which are under
vacuum. The detector is typically operably connected to a signal
processor and a computer. Desorption ion sources for use in the
present invention, include field desorption (FD), electrospray
ionization (ESI), chemical ionization, matrix-assisted
desorption/ionization (MALDI), plasma desorption (PD), fast atom
bombardment (FAB), secondary ion mass spectrometry (SIMS), and
thermospray ionization (TS). As mentioned, ESI sources are
especially preferred.
[0230] Mass spectrometry is well-known in the art. References
specifically addressing the interfacing of mass spectrometers with
microfluidic devices include, e.g., Karger, et al., U.S. Pat. No.
5,571,398, "PRECISE CAPILLARY ELECTROPHORETIC INTERFACE FOR SAMPLE
COLLECTION OR ANALYSIS" and Karger, et al. U.S. Pat. No. 5,872,010
"MICROSCALE FLUID HANDLING SYSTEM." General sources of information
about mass spectrometry include, e.g., Skoog, et al. Principles of
Instrumental Analysis (5.sup.th Ed.) Hardcourt Brace & Company,
Orlando (1998). In general, mass spectrometers are well suited to
interface with microfluidic devices, because the usual input into a
microfluidic system is a capillary channel. In the present
invention, this mass spectrometry capillary is simply fluidly
coupled to channel in the microscale system. Methods of affixing
external capillaries to microscale systems include various bonding
and/or drilling operations, as described, e.g., in Parce, et al.,
U.S. Pat. No. 5,972,187 "ELECTROPIPETTOR AND COMPENSATION MEANS FOR
ELECTROPHORETIC BIAS." One particular advantage of coupling mass
spectrometers to microfluidic systems is that the ionization
chamber of a mass spectrometer is usually under vacuum. This vacuum
can be used as a negative pressure source for the microfluidic
system, providing a driving mechanism for the system.
[0231] FIG. 7 is a schematic of an interface between a microfluidic
device and an ESI mass spectrometer. The interfaced system 700 is
optionally used, e.g., to identify and/or determine the
concentration of various molecules (e.g., drug-like organic
molecules) in the effluent in one or more microchannels. Such
effluent is typically drawn from device 702 into ion source 704
using the negative pressure of ion source 704. A shown, time of
flight (TOF) mass analyzer 706 with detector 708 is included in
this system. In the systems of the present invention, the molecular
weights of samples to be analyzed can be in the range of from about
150 Kd to about 800 Kd, e.g., about 500 Kd. Effluent concentrations
can be, e.g., in the range of about 1 .mu.M to about 10 .mu.M,
e.g., about 5 .mu.M.
[0232] Computer
[0233] As noted above, either or both of the controller system
and/or the detection system are coupled to an appropriately
programmed processor or computer which functions to instruct the
operation of these instruments in accordance with preprogrammed or
user input instructions, receive data and information from these
instruments, and interpret, manipulate and report this information
to the user. As such, the computer is typically appropriately
coupled to one or both of these instruments (e.g., including an
analog to digital or digital to analog converter as needed).
[0234] The computer typically includes appropriate software for
receiving user instructions, either in the form of user input into
a set parameter fields, e.g., in a GUI, or in the form of
preprogrammed instructions, e.g., preprogrammed for a variety of
different specific operations. The software then converts these
instructions to appropriate language for instructing the operation
of the fluid direction and transport controller to carry out the
desired operation. The computer then receives the data from the one
or more sensors/detectors included within the system, and
interprets the data, either provides it in a user understood
format, or uses that data to initiate further controller
instructions, in accordance with the programming, e.g., such as in
monitoring and control of flow rates, temperatures, applied
voltages, and the like.
[0235] In the present invention, the computer typically includes
software for the monitoring of materials in the channels.
Additionally the software is optionally used to control
electrokinetic or pressure modulated injection or withdrawal of
material. The injection or withdrawal is used to modulate the flow
rate as described above.
EXAMPLE SYSTEM
[0236] FIG. 2, panels A, B and C and FIG. 3 provide additional
details regarding example integrated systems of the invention. As
shown, body structure 202 has main channel 204 fabricated therein.
Cells with transporter activity are flowed from reservoir 214,
e.g., by applying a vacuum at vacuum source 216 (and/or at any of
the reservoirs or wells noted below) through main channel 204.
Cells with transmitter receptor, or transmitter, or a potential
modulator or a different material such as a buffer or label can be
flowed from wells 210 or 212 and into main channel 204. Cells with
transmitter receptor, or transmitter, or a potential modulator or
any additional material can be flowed from wells 206 or 208, or
materials can be flowed into these wells, e.g., when they are used
as waste wells, or when they are coupled to a vacuum source. Flow
from wells 214, 212, 210, 206, or 208 can be performed by
modulating fluid pressure, or by electrokinetic approaches as
described. Instead of the arrangement of channels depicted in FIG.
2, an arrangement with opposing channels, as depicted in FIG. 1 can
be substituted.
[0237] Transmitter, transporters, or material with transmitter
receptors can be flowed from the enumerated wells, or can be flowed
from a source external to body 202. As depicted, the integrated
system can include pipettor channel 220, e.g., protruding from body
202, for accessing an outside source of reagents. For example, as
further depicted in system 300 as shown in FIG. 3, pipettor channel
220 can access microwell plate 308 which includes cells,
transmitters, transporters, activity modulators, controls, or the
like, in the wells of the plate. For example, a library of
potential inhibitor compounds can be stored in the wells of plate
308 for easy access by the system. Inhibitors or other reagents
relevant to the assays can be flowed into channel 204 through
pipettor channel 220. Detector 306 is in sensory communication with
channel 204, detecting signals resulting, e.g., from the
interaction of a transmitter with a transmitter receptor, as
described above. Detector 306 is operably linked to Computer 304,
which digitizes, stores and manipulates signal information detected
by detector 306. Voltage/pressure controller 302 controls voltage,
pressure, or both, e.g., at the wells of the system, or at vacuum
couplings fluidly coupled to channel 204 (or the other channels
noted above). Optionally, as depicted, computer 304 controls
voltage/pressure controller 302. In one set of embodiments,
computer 304 uses signal information to select further reaction
parameters. For example, upon detecting transporter inhibition by a
potential modulator in a well from plate 308, the computer
optionally directs withdrawal of additional aliquots of the
potential modulator through pipettor channel 220, e.g., to deliver
different concentrations of the potential modulator to the assay,
e.g., to determine kinetic data (such as a dose-response curve) for
the potential modulator.
[0238] Kits
[0239] Generally, the microfluidic devices described herein are
optionally packaged to include reagents for performing the device's
preferred function. For example, the kits can include any of
microfluidic devices described along with assay components,
reagents, sample materials, control materials, or the like. Such
kits also typically include appropriate instructions for using the
devices and reagents, and in cases where reagents are not
predisposed in the devices themselves, with appropriate
instructions for introducing the reagents into the channels and/or
chambers of the device. In this latter case, these kits optionally
include special ancillary devices for introducing materials into
the microfluidic systems, e.g., appropriately configured
syringes/pumps, or the like (in one preferred embodiment, the
device itself comprises a pipettor element, such as an
electropipettor for introducing material into channels and chambers
within the device). In the former case, such kits typically include
a microfluidic device with necessary reagents predisposed in the
channels/chambers of the device. Generally, such reagents are
provided in a stabilized form, so as to prevent degradation or
other loss during prolonged storage, e.g., from leakage. A number
of stabilizing processes are widely used for reagents that are to
be stored, such as the inclusion of chemical stabilizers (i.e.,
enzymatic inhibitors, microcides/bacteriostats, anticoagulants),
the physical stabilization of the material, e.g., through
immobilization on a solid support, entrapment in a matrix (i.e., a
gel), lyophilization, or the like.
[0240] The kits of the present invention can optionally include a
first, second, and third component. The first component, e.g., an
adhesion factor, can include a first attachment activity. The
second component, e.g., a motile cell, can include a second
attachment activity and be capable of detaching from the first
component in response to the third component, e.g., a chemotactic
factor, that is capable of forming a gradient. These kits can also
include a container for packaging the at least one first, second,
and third components, instructions for practicing the methods
herein, reagents for buffering or storing the at least one first,
second, and third components, and/or one or more test
compounds.
[0241] A kit can optionally include a first component (e.g., an
enzyme or receptor) or a set of first components and a second
component (e.g., a ligand) or a set of second components, in which
the first component or the set of first components can diffuse more
rapidly in solution than the second component or the set of second
components. The first component or the set thereof can
substantially diffuse across a channel in a mixing longitudinal
segment when the first and second components or the sets thereof
are concomitantly flowed in the channel. However, the second
component or the set thereof typically diffuse less than
substantially across the first channel in the mixing longitudinal
segment. Furthermore, the second component or the set thereof can
bind to the first component or the set of first components. The kit
can also include a container for packaging the first and second
components or the sets thereof, instructions for practicing the
method herein, reagents for buffering or storing the at least one
first and second components or the sets thereof, and one or more
test compounds.
[0242] Kits also optionally include packaging materials or
containers for holding microfluidic device, system or reagent
elements.
[0243] The discussion above is generally applicable to the aspects
and embodiments of the invention described in the claims.
[0244] Moreover, modifications can be made to the method and
apparatus described herein without departing from the spirit and
scope of the invention as claimed, and the invention can be put to
a number of different uses including the following:
[0245] The use of a microfluidic system for performing the
transporter, gradient, and binding assays set forth herein.
[0246] The use of a microfluidic system as described herein,
wherein a biochemical system flows through one of said channels
substantially continuously, providing for, e.g., sequential testing
of a plurality of transporter or ligand compounds.
[0247] The use of electrokinetic injection in a microfluidic device
as described herein to modulate or achieve flow of transporters,
transmitters or other assay components in channels of a microscale
device, optionally in conjunction with pressure-based flow
mechanisms.
[0248] The optional use of a combination of adsorbent materials,
electrokinetic injection and pressure based flow elements in a
microfluidic device as described herein to modulate or achieve flow
of materials e.g., in the channels of the device.
[0249] An assay utilizing a use of any one of the microfluidic
systems or substrates described herein.
[0250] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above may be used in various
combinations. All publications, patent documents, and other
references cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication or patent document were individually so
denoted.
* * * * *