Note: Descriptions are shown in the official language in which they were submitted.
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Reconfigurable microfluidic systems: Scalable, multiplexed immunoassays
[01]
[02]
[03)
Technical Field
,[04] The disclosure is generally related to rnicrofluidic systems.
Background
[05] Microfluidic systems manipulate microliter and smaller scale volumes
of fluids. Ink-
jet printing and biochemical assays are two prominent applications of
microfluidics among
many others. The ability to move, control and mix tiny quantities of liquids
is valuable in
biochemistry since it permits more experiments to be done with a given amount
of starting
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material. The increased surface¨to¨volume ratio associated with microfluidic
channels as
compared to traditional microwell plates also speeds up surface reactions upon
which some
kinds of assays are based.
[06] Despite the profound advances in microfluidics achieved over the last
30 years, there
is room for improvement. It is still a challenge, for example to make
microfluidic valves that
open and shut as reliably as conventional size valves. New approaches to
interfaces
between microfluidic devices and microwell plates are needed. Finally,
microfluidic assays
need to be made scalable so that hundreds or thousands of assays can be
performed in
parallel on one chip.
Brief Description of the Drawings
[07] Fig. 1 is diagram of a reconfigurable microfluidic device, seen in
cross section.
[08] Fig. 2 illustrates loading the device of Fig. 1 from an external fluid
source.
[09] Fig. 3 illustrates unloading the device of Fig. 1 to an external fluid
store.
[10] Figs. 4A, 4B and 4C are diagrams illustrating operation of the device
of Fig. 1, seen in
plan view.
[11] Fig. 5 is a graph of fluid volume transferred between a reservoir and
a node of a
device similar that of Fig. 1.
[12] Fig. 6 is a diagram of steps 0 through 6 illustrating operation of a
reconfigurable
microfluidic device, seen in plan view.
[13] Fig. 7 is a diagram of a reconfigurable microfluidic device, seen in
cross section,
including ports for clearing microfluidic channels.
[14] Fig. 8 is a graph of absorbance representing results of an automated
dilution
experiment.
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[15] Fig. 9 is a diagram of a reconfigurable microfluidic system, including
a pressure
sequencer.
[16] Figs. 10A (cross sectional view) and 108 (plan view) are diagrams
illustrating a gas
flow manifold in a reconfigurable microfluidic device.
[17] Fig. 11 is a diagram of a reconfigurable microfluidic device for
single channel
immunoassays, seen in plan view.
[18] Fig. 12 is a diagram of a reconfigurable microfluidic device for
single channel
immunoassays, seen in plan view.
[19] Fig. 13 is a diagram of a reconfigurable microfluidic device for
single channel
immunoassays, seen in plan view.
[20] Fig. 14 is a diagram of a reconfigurable microfluidic device for
single channel
immunoassays, seen in plan view.
[21] Fig. 15 is a diagram of a reconfigurable microfluidic device for
single channel
immunoassays, seen in plan view.
[22] Fig, 16 is a diagram of a reconfigurable microfluidic device for
single channel
immunoassays, seen in plan view.
[23] Fig. 17 is a graph of competitive ELISA absorbance data.
[24] Fig. 18 is a graph of competitive ELISA normalized absorbance data.
[25] Fig. 19 is a diagram of a reconfigurable microfluidic device for
multichannel
immunoassays, seen in plan view.
[26] Fig. 20 is a diagram of a reconfigurable microfluidic device for
multichannel
immunoassays, seen in plan view.
[27] Fig. 21 is a diagram of a reconfigurable microfluidic device for
multichannel
immunoassays, seen in plan view.
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[28] Fig. 22 is a diagram of a reconfigurable microfluidic device for
multichannel
immunoassays, seen in plan view.
[29] Fig. 23 is a diagram of a reconfigurable microfluidic device for
multichannel
immunoassays, seen in plan view.
[30] Fig. 24 is a diagram of a reconfigurable microfluidic device for
multichannel
immunoassays, seen in plan view.
[31] Fig. 25 is a conceptual diagram of a reconfigurable microfluidic
device for 95-channel
immunoassays.
[32] Fig. 26 in parts A and B illustrates a microfluidic switched
interaction region.
[33] Fig. 27 illustrates multiple microfluidic switched interaction regions
in series.
[34] Fig. 28 is a diagram of a reconfigurable microfluidic device for
multiplexed
immunoassays.
[35] Fig. 29 is a diagram of a reconfigurable microfluidic device for
multiplexed
immunoassays.
[36] Fig. 30 is a diagram of a reconfigurable microfluidic device for
multiplexed
immunoassays.
[37] Fig. 31 is a diagram of a reconfigurable microfluidic device for
multiplexed
immunoassays.
[38] Fig. 32 is a diagram of a reconfigurable microfluidic device for
multiplexed
immunoassays.
[39] Fig. 33 is a diagram of a reconfigurable microfluidic device for
multiplexed
immunoassays.
[40] Fig. 34 shows the reconfigurable microfluidic device of Figs. 28¨ 33
with the addition
of optional nodes.
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[41] Fig. 35 is a diagram of a microfluidic device for multiplexed
immunoassays based on
microvalves.
Detailed Description
[42] Reconfigurable microfluidic systems are based on networks of
microfluidic cavities
connected by hydrophobic microfluidic channels. Each cavity is classified as
either a
reservoir or a node, and includes a pressure port via which gas pressure may
be applied.
Sequences of gas pressures, applied to reservoirs and nodes according to a
fluid transfer
rule, enable fluid to be moved from any reservoir to any other reservoir in a
system.
Systems may be configured with multiple switched interaction regions connected
in series
for scalable, multiplexed immunoassays. Multiple, switched interaction regions
may also be
implemented with microvalves.
[43] Reconfigurable microfluidic systems may be designed from these basic
components
¨ reservoirs, nodes and channels ¨ to perform many different microfluidic
tasks including
homogenous arid inhomogeneous assays and microwell plate interfacing. The
systems are
scalable to any number of fluid inputs and outputs, and they can manipulate
very small fluid
volumes necessary for multiplexing samples with analytes to perform multiple
simultaneous
assays.
[44] A microfluidic cavity is an internal volume for accumulating fluid in
a microfluidic
device. A reservoir is a microfluidic cavity that is connected to only one
microfluidic
channel. A node is a microfluidic cavity that is connected to more than one
microfluidic
channel. Finally, a channel is a microfluidic passageway between nodes or
reservoirs. Each
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channel in a reconfigurable microfluidic system connects at most two cavities.
Said another
way, there are no channel intersections.
[45] Nodes are designed to present lower resistance to fluid flow than are
channels. The
fluid flow resistance of a cavity or channel is inversely proportional to the
square of its cross
sectional area. Therefore the difference in flow resistance between a channel
and a
reservoir, or between a channel and a node, may be engineered via different
cross sectional
areas.
[46] Reservoirs store fluids; e.g. samples or reagents. Nodes, on the other
hand, do not
store fluid, except temporarily during a sequence of fluid transfer steps.
Provisions for
automated loading fluid into, or unloading fluid from, a reservoir may be
provided, with a
small plastic tube extending from a reservoir to a glass bottle being a simple
example.
[47] Reconfigurable microfluidic systems may be implemented in a variety of
ways as
long as: reservoirs, nodes, channels and pressure ports are provided;
resistance to fluid flow
is greater in the channels than in the nodes; and the channels are hydrophobic
to prevent
fluid flow when pressures at the two ends of a channel are equal or nearly so.
A typical
implementation includes a substrate layer, a hydrophobic fluid layer, and a
pneumatic layer.
[48] Fig. 1 is diagram of a reconfigurable microfluidic device, seen in
cross section. In Fig.
1, microfluidic device 105 includes a substrate layer 110, a hydrophobic
fluidic layer 115,
and a pneumatic layer 120. Cavities in the hydrophobic fluidic layer are
labeled 'A', 'B' and
'C'. Cavities A and B are connected by channel 125 while cavities B and C are
connected by
channel 130. Cavities A and C are classified as reservoirs because they are
connected to
only one channel each. Cavity B is classified as a node because it is
connected to more than
one channel: B is connected to both channel 125 and channel 130.
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[49] Pressure sources 135, 140 and 145 are connected to reservoir A, node B
and
reservoir C, respectively, via gas tubes 150, 155 and 160 respectively. Each
of the three
pressure sources is capable of providing at least two different pressures: a
high pressure and
a low pressure. Labels 'H' and 'L' in the figure refer to the capability of a
pressure source to
provide a high or low pressure. Pressure source 135 is also capable of
providing a pressure
that is less than atmospheric pressure; i.e. a partial vacuum. Label 'V in the
figure refers to
this capability. As an example, high pressure may be about 2 kPa, low pressure
may be
about 0 kPa, and partial vacuum pressure may be about ¨6 kPa, where all
pressures are
gauge pressures.
[50] Several different ways of making a structure like microfluidic device
105 are possible.
As a first example, substrate 110 may be made of glass, polydimethylsiloxane
(PDMS),
polyethylene terephtha late (PET), or plastic. Hydrophobic fluidic layer 115
may be made
from PDMS. A mold for casting PDMS to define hydrophobic microfluidic channels
may be
produced with a programmable cutter for vinyl decals or defined
photolithographically in an
epoxy-based negative photoresist such as SU-8. After patterned PDMS is cured
and
removed from a mold, it may be bonded to a flat substrate. Pneumatic layer 120
may also
be made from PDMS. Gas tubes may be made from polyetheretherketone (PEEK)
tubing
which forms convenient seals when inserted in appropriately sized holes in
PDMS.
Hydrophobic materials that are suitable alternatives to PDMS include
fluorinated ethylene
, propylene (FEP) and polytetrafluoroethylene (PTFE).
[51] In example devices, the cross-sectional dimensions of channels 125 and
130 were
about 100 ii.rn by about 300 itm. The sizes of reservoirs A and C, and of node
B were
between about 2 mm and about 4 mm in diameter. The distance between reservoir
A and
node B was between about 5 mm and about 10 mm; the distance between node B and
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reservoir C was about the same. The cross-sectional areas of the cavities in
typical devices
are approximately 100 to 400 times greater than the cross-sectional areas of
the channels.
Therefore the flow resistance of the channels is about 10,000 to 160,000 times
greater than
the flow resistance of the cavities. Alternative designs for channels and
cavities lead to the
flow resistance of channels being about 100 times greater or about 1,000 times
greater than
the flow resistance of cavities.
[52] A second way to make a structure like microfluidic device 105 is hot
embossing a
hydrophobic thermoplastic polymer such as cyclic olefin copolymer (COC)
followed by
solvent¨assisted lamination to form enclosed, hydrophobic channels. A third
way to make a
structure like microfluidic device 105 is injection molding a hydrophobic
polymer such as
COC. Finally, hydrophilic rnicrofluidic channels, formed in polycarbonate for
example, may
be made hydrophobic via chemical surface treatment. There are, no doubt, other
ways to
make a structure containing cavities connected by hydrophobic microfluidic
channels.
[53] Fig. 2 illustrates loading the device of Fig. 1 from an external fluid
source. In Fig. 2,
reference numbers 105 ¨ 160 refer to the same items as in Fig. 1. In Fig. 2,
however,
pressure sources 135, 140 and 145 supply partial vacuum, low pressure and low
pressure,
respectively. Supply tube 165 connects reservoir A to an external fluid source
170 that is at
atmospheric pressure. When a partial vacuum is applied to reservoir A by
pressure source
135 via gas tube 150, fluid is withdrawn from fluid source 170 and accumulated
in reservoir
A. Fluid does not flow from reservoir A to node B in this situation because
the gas pressure
applied to node B is higher than the gas pressure applied to reservoir A.
[54] Fig. 3 illustrates unloading the device of Fig. 1 to an external fluid
store. In Fig. 3,
reference numbers 105 ¨ 160 refer to the same items as in Fig. 1. In Fig. 3,
however,
pressure sources 135, 140 and 145 supply low pressure, high pressure and high
pressure,
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respectively. Drain tube 175 connects reservoir C to an external fluid store
180. The fluid
store is at atmospheric pressure. When high pressure is applied to reservoir C
by pressure
source 145 via gas tube 160, fluid is expelled from reservoir C and
accumulated in fluid store
180. Fluid does not flow from reservoir C to node B in this situation because
the gas
pressure applied to node B is the same as the gas pressure applied to
reservoir C.
[55] In reconfigurable microfluidic systems, fluid flow through
microfluidic channels is
controlled by gas pressure differences applied to reservoirs and nodes. Fluid
flow through a
hydrophobic channel exhibits a pronounced threshold effect. At first, no fluid
flows as the
pressure difference from one end of the channel to the other is increased.
However, once a
threshold pressure difference is reached, fluid flow rate through the channel
increases in
proportion to applied pressure difference. The hydrophobicity of channels sets
the
threshold pressure difference, and the difference between "high" and "low"
pressures used
in a system is designed to be greater than the hydrophobic threshold pressure.
Thus, when
the pressure is "high" at one end of a channel and "low" at the other end,
fluid flows rapidly
in the channel.
[56] The hydrophobic threshold pressure of hydrophobic channels keeps fluid
in nodes
and reservoirs from leaking into the channels when no pressure differences are
applied.
The threshold pressure is designed to be great enough to prevent fluid flow
that might be
driven by the hydrodynamic pressure caused by the weight of fluid in a
reservoir or node, or
by residual pressure differences that might exist when applied pressures are
switched
between "high" and "low". Thus a "hydrophobic channel" is defined as one that
exhibits a
pressure threshold that prevents fluid from leaking into the channel when the
pressure
difference between the two ends of the channel is less than a design
pressure.. In an
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example reconfigurable microfluidic system, channels were designed to have
about 1 kPa
hydrophobic threshold pressure.
[57] Fluid transfer between reservoirs and nodes is accomplished by
switching pressures
applied to each reservoir and node in a system according to a specific
pattern. The
following terminology aids discussion of a fluid transfer rule for
reconfigurable microfluidic
systems. The origin is a reservoir or node from which fluid is to be
transferred. The
destination is the reservoir or node to which fluid is to be transferred. Two
gas pressures
are needed: high pressure and low pressure.
[58] A fluid transfer rule for reconfigurable microfluidic systems may be
summarized in
the following steps:
[59] Step 0: Apply low pressure to all cavities.
[60] Step 1: Apply high pressure to the origin and any cavity connected to
the origin by a
channel, other than the destination. Apply low pressure to the destination and
any cavity
connected to the destination, other than the origin.
[61] Step 2 (optional): Switch origin back to low pressure. The purpose of
this optional
step is to ensure an air gap (i.e. section without fluid) exists in all
channels after Step 1. This
optional step is useful when transferring less than all of the fluid that is
in the origin cavity at
Step 0.
[62] Step 3: Return to Step 0 to prepare for the next fluid transfer
operation.
[63] As explained below, the fluid transfer rule may be executed by a
pressure sequencer
that provides the necessary sequence of pressures to accomplish any desired
fluid transfer
operation. Two examples show how the fluid transfer rule is used to perform
common fluid
transfer experiments. The first example demonstrates flow rate control when
fluid is
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transferred from one cavity to another; the second example demonstrates
automated
dilution of a fluid sample.
[64] Example 1: Flow rate control.
[65] Figs. 4A, 46 and 4C are diagrams illustrating operation of the device
of Fig. 1, seen in
plan view. In particular, Fig. 4A shows a plan view of reservoir A, node B and
reservoir C,
connected by channels 125 and 130. In Figs. 4B and 4C, labels 'A', 'B' and 'C'
are replaced by
'1', `L' and `L' (Fig. 4B) and 'H', 'L' and 'L' (Fig. 4C). Fig. 4A serves as a
key for Figs. 4B and 4C.
'H' and I' in Figs. 4B and 4C show which cavities have high and low pressure
applied to
them. Shading in Figs. 4B and 4C, and the arrow in Fig. 4C, shows that fluid
moves from
reservoir A to node B.
[66] The fluid transfer rule explains how the fluid transfer depicted in
Figs. 4B and 4C is
accomplished. Step 0 of the rule specifies that low pressure is applied to all
cavities. Fig. 4B
shows low pressure, 'L', applied to reservoir A, node B and reservoir C.
Shading of reservoir
A in Fig. 46 means that the reservoir has fluid in it, while node B and
reservoir C are empty.
Reservoir A is the origin.
[67] Step 1 of the fluid transfer rule specifies that high pressure is
applied to the origin
and any cavity connected to the origin by a channel, other than the
destination. Further,
low pressure is applied to the destination and any cavity connected to the
destination, other
than the origin. This is the situation depicted in Fig. 4C. The result is
fluid transfer from the
origin to the destination.
[68] All other conditions being equal, the volume of fluid transferred from
the origin to
the destination depends on the amount of time that pressure is applied during
Step 1 of the
fluid transfer rule_ An experiment was conducted to demonstrate flow rate
control in an
apparatus similar to that shown in Figs. 1 ¨4.
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[69] Fig. 5 is a graph of fluid volume transferred between a reservoir and
a node of a
device similar that of Fig. 1. The graph shows volume of fluid transferred in
microliters (u.L)
versus time (in seconds) that pressure was applied during Step 1 of the fluid
transfer rule.
The six black dots on the graph represent experimental data while the dashed
line is a linear
fit to the data. The observed flow rate is approximately 10 pi per second.
[70] During the experiment, there was no leakage of fluid to reservoir C,
even though
node B and reservoir C were held at the same low pressure compared to
reservoir A.
Leakage to reservoir C was prevented by the high flow resistance of channel
130 compared
to that of node B.
[71] Example 2: Automated dilution.
[72] Fig. 6 is a diagram illustrating operation of a reconfigurable
microfluidic device, seen
in plan view. In Fig. 6, the same device 605 is shown seven times under
headings 'STEP 0',
'STEP 1', ... , 'STEP 6'. Device 605 is similar in construction to the device
of Figs. 1 ¨4,
however device 605 has four reservoirs (610, 615, 620, 625) and one node
(630). To
improve visual clarity, reference numerals are not repeated for the device
when it is shown
under headings 'STEP 1' through 'STEP 6'. Each reservoir is connected to node
630 via its
own channel. For example, channel 635 connects reservoir 610 to node 630. The
other
channels do not have reference numerals. The reservoirs, the channels and the
node are
drawn in black, gray or white during various steps. Black and gray represent
two different
fluids, while white represents an absence of fluid.
[73] As discussed above, the fluid transfer rule in its basic form
alternates between two
states. The first state is an initial, rest condition where all cavities are
at low pressure. In
the second state, fluid is transferred from an origin to a destination. These
two states are
referred to as 'Step 0' and 'Step 1' above.
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[74] Fig. 6 uses "step" terminology. However, 'STEP 0' through 'STEP 6' in
Fig. 6 are not
intended to match the steps of the fluid transfer rule. Instead 'STEP 0'
through 'STEP 6' are
steps in an overall program during which the steps of the fluid transfer rule
are applied
repeatedly.
[75] The overall result of the program shown in Fig. 6 is that some fluid
from reservoir
610 is moved to reservoir 620 and some fluid from reservoir 615 is also moved
to reservoir
620. Thus, at the end of the program, in 'STEP 6', reservoir 620 contains a
mixture of fluids
from reservoirs 610 and 615. Equivalently, reservoir 620 contains a dilution
of fluid from
reservoir 610 by fluid from reservoir 615.
[76] A sequence of pressures is applied to the reservoirs and node of
device 605.
Pressures are indicated by labels 'H' for high pressure and 'L' for low
pressure in Fig. 6. STEP
0 shows the reservoirs and node all at low pressure. Reservoirs 620 and 625,
and node 630
do not contain fluid. Reservoirs 610 and 615 contain different fluids
indicated by black and
gray shading.
[77] In STEP 1, high pressure is applied to origin reservoir 610 and low
pressure is applied
to destination node 630 and to all cavities connected to the destination,
other than the
origin. Fluid flows from the origin to the destination. Although not
illustrated, after STEP 1,
system pressures are returned briefly to the initial condition, all cavities
at low pressure as
in STEP 0. A reset to all cavities at low pressure occurs before and after
each illustrated
STEP.
[78] In STEP 2, node 630 is the origin and reservoir 620 is the
destination. Therefore high
pressure is applied to the origin and all cavities connected to it, other than
the destination.
Low pressure is applied to the destination. Fluid flows from the origin to the
destination.
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[79] STEP 3 is an example of optional Step 2 of the fluid transfer rule.
The purpose of this
step is to clear the channels between node 630 and reservoirs 610 and 620. An
air gap must
exist in a channel in order for the channel to present a hydrophobic barrier
to fluid flow.
Without the operation shown in STEP 3, channel 635, and the channel connecting
node 630
to reservoir 620, could be left with fluid in them that would defeat their
hydrophobic
barriers.
[80] In STEP 3, reservoir 610 is switched briefly back to low pressure
while all other
pressures remain as in STEP 2. This causes a ny fluid left in channel 635 to
be sent back to
reservoir 610. There are alternative ways to accomplish this "channel
clearing" function as
discussed below. Channel clearing may be needed in cases where less than all
of the fluid at
the origin is moved to the destination in one cycle of the fluid transfer
rule.
[81] STEP 4, STEP 5 and STEP 6 are analogous to STEP 1, STEP 2 and STEP 3
except that
fluid is moved from reservoir 615 to reservoir 620 instead of from reservoir
610 to 620.
Since the amount of fluid moved from one cavity to another can be controlled
by the time
that pressures are applied, as demonstrated in Example 1, the ratio of fluid
moved to
reservoir 620 from reservoir 610 to fluid moved to reservoir 620 from
reservoir 615 can be
adjusted at the discretion of the experimenter. Thus automated dilution may be
performed
by selecting an appropriate sequence of pressures to be applied to the
cavities of device
605.
[82] An alternate means for clearing out channels when only some of the
fluid in an origin
cavity is transferred away involves dedicated gas tubes connected to the
channels. Fig. 7 is
a diagram of a reconfigurable microfluidic device, seen in cross section,
including ports for
clearing microfluidic channels. The device of Fig. 7 is nearly the same as
that of Fig. 1,
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except that gas tubes, pressure ports and gas pressure sources are provided to
enable
creation of air gaps in channels.
[83] In Fig. 7, microfluidic device 705 includes a substrate layer 710, a
hydrophobic fluidic
layer 715, and a pneumatic layer 720. Cavities in the hydrophobic fluidic
layer are labeled
'A', 'B' and 'C'. Reservoir A and node B are connected by channel 725 while
node B and
reservoir C are connected by channel 730.
[84] Pressure sources 735, 740 and 745 are connected to reservoir A, node B
and
reservoir C, respectively, via gas tubes 750, 755 and 760 respectively. Each
of the three
pressure sources is capable of providing at least two different pressures: a
high pressure and
a low pressure.
[85] Pressure sources 775 and 780 are connected to channels 725 and 730
respectively,
via gas tubes 785 and 790 respectively. The gas tubes present a higher barrier
to fluid flow
than the channels. In normal operation of device 705 only gas, never fluid,
flows in the gas
tubes.
[86] It is apparent that if device 605 of Fig. 6 were equipped with channel
clearing gas
tubes like gas tubes 785 and 790 of Fig. 7, then STEP 3 (optional Step 2 of
the fluid transfer
rule) could be replaced by a clearing STEP in which pressure is applied to
channel clearing
gas tubes while low pressure would be applied to all the cavities in the
system.
[87] An experiment was conducted to demonstrate automated dilution in an
apparatus
similar to that shown in Fig. 6. Fig. 8 is a graph of absorbance representing
results of an
automated dilution experiment. In the automated dilution experiment,
concentration of an
aqueous solution was inferred from optical absorbance measurements where
higher
absorbance corresponded to higher concentration of solute. (Optical absorbance
varies
linearly with concentration according to Beer's Law.) The graph in Fig. 8
therefore plots
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absorbance, representing measured concentration, versus target, or expected,
concentration. Target concentration is an expected result if the amounts of
fluid
transferred into the destination reservoir from the origin solute and solvent
reservoirs are
as expected.
[88] When no dilution is performed ("Zero dilution steps", "+" data point
marker),
absorbance 2.00 (in arbitrary units) corresponds to target concentration 1.00
in arbitrary
units). Target concentrations of 0.50 and 0.25 may be obtained in one dilution
step; i.e. one
time through STEPS 0 through 6 of Fig. 6. Data obtained in this way is labeled
"One dilution
step" and shown with "o" data point markers on the graph.
[89] Finally data obtained after two dilution steps ("Two dilution steps
(serial dilution)",
"x" data point markers) is shown for target concentrations of 0.25 and 0.0625.
In this case
the procedure of Fig. 6 was repeated twice. Target concentration 0.25 was
obtained in two
ways; using one dilution step or two dilution steps. The actual concentration,
as
represented by absorbance data, was nearly identical in the two cases.
[90] Examples 1 and 2 discussed above demonstrate that sequences of gas
pressures,
applied to reservoirs and nodes according to a fluid transfer rule, enable
fluid to be moved
from any reservoir to any other reservoir in a reconfigurable microfluidic
system. Fig. 9 is a
diagram of a reconfigurable microfluidic system 905, including a pressure
sequencer 915.
[91] In Fig. 9, microfluidic device 910 includes hydrophobic reservoirs,
nodes and
channels. These structures are formed in microfluidic layers of the device.
Each reservoir
and node is connected to pressure sequencer 915 via a gas tube, such as gas
tube 920.
Pressure sequencer 915 is connected to pressure sources 925 and 930. Pressure
sequencer
915 includes a set of programmable gas valves.
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[92] The sequencer receives pressure sequence data 940. This data includes
step by step
instructions specifying what pressure is to be applied to each reservoir and
node in device
910 in order to carry out a specific fluid transfer operation. As shown in
Example 2, fluid can
be moved from any reservoir to any other reservoir in a reconfigurable
microfluidic system
by repeating the steps of the fluid transfer rule.
[93] In a laboratory experiment, pressure sequencer 915 was implemented as
a set of
electronically controlled pneumatic valves that were programmed using LabVIEW
software
(National Instruments Corporation) running on a personal computer. For the
experiment,
pressure sequence data necessary to move fluid from one reservoir to another
in a
reconfigurable microfluidic device was worked out manually. However a
graphical software
program may be written that allows a user to select origin and destination
reservoirs, with
the program then generating appropriate pressure sequence data by repeated
application
of the fluid transfer rule. In this way an intuitive system may be created
that permits users
to perform arbitrary microfluidic experiments without needing to understand
the fluid
transfer rule or other system operation details.
[94] Reconfigurable microfluidic systems may have many reservoirs and
nodes, especially
those systems designed for parallel biochemical assays. One type of parallel
assay involves
performing many different biochemical experiments simultaneously on small
volumes of
fluid taken from one sample. A second type of parallel assay involves
processing many
different fluid samples simultaneously, in otherwise identical biochemical
experiments.
Both of these cases involve parallel operations in which groups of reservoirs
or nodes
change pressure together during the steps of a complex fluid transfer process.
[95] When a reconfigurable microfluidic device has reservoirs or nodes that
are operated
in a group, it is more convenient to integrate a gas flow manifold in the
pneumatic layer of
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the device than to dedicate a separate gas tube to each reservoir or node.
Figs. 10A (cross
sectional view) and 10B (plan view) are diagrams illustrating a gas flow
manifold in a
reconfigurable microfluidic device 1005.
[96] In Fig. 10A, the block arrow labeled 'B' indicates the perspective
from which Fig. 10B
is drawn. Device 1005 includes a substrate layer 1010, a hydrophobic
microfluidic layer
1015, and a pneumatic layer 1020. Dashed lines, e.g. 1030, designate channels
to
microfluidic cavities that are not shown in Fig. 10A because they are not in
the plane of the
page. Gas tube 1025 is connected via gas flow manifold 1035 to cavity 1040 and
cavity
1045. Any gas pressure supplied by the gas tube pressurizes both cavities at
once. The
layout of the gas flow manifold is shown in plan view in Fig. 10B. The gas
flow manifold acts
as a pressure port for groups of cavities that are operated in parallel.
[97] One application for reconfigurable microfluidic devices such as those
described
above is scalable, multiplexed immunoassays. The immunoassays considered
herein involve
surface interactions. At some point in each assay, molecules are linked to a
surface rather
than being free floating in solution. (Such surface-interaction assays are
sometimes called
inhomogeneous assays.) The surface to which molecules are linked is the wall
of a channel
in a reconfigurable microfluidic device.
[98] The most common immunoassays are various kinds of enzyme-linked
immunosorbent assays (ELISA); however the devices and techniques described
below are
not limited to ELISA. On the contrary, they are applicable to any assay in
which molecules
are linked to a surface. Furthermore, the devices and techniques described
below are
applicable to surface-interaction assays that are analogous to immunoassays
but do not
involve antibody ¨ antigen interactions. In these assays, a chemical species
that is bound to
a surface during an assay and captures another chemical species is referred to
as a capture
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analyte. The captured species is referred to as a sample analyte. A reagent
that is affected
by the presence of capture-analyte ¨ sample-analyte complexes is referred to
as a detection
reagent.
[99] An immunoassay is one that involves antigen ¨ antibody interactions.
In some kinds
of ELISA experiments an antigen is linked to a surface. In others, an antibody
is linked to the
surface. While the biochemical details of an ELISA, or other immunoassay
protocol, are
critically important to the scientific purpose of the particular experiment,
the devices and
techniques described below do not depend on these biochemical details. Thus,
whenever
the description mentions an antibody linked to a surface of a channel in a
microfluidic
device, it is understood that the same device could be employed in
biochemically different
kinds of experiments in which an antigen or other type of molecule is linked
to a surface.
[100] Single-channel, multichannel and multiplexed immunoassay devices are
described.
A single-channel assay is one that involves one kind of antibody linked to a
surface and one
sample. A multichannel assay is one in which many samples are processed in
parallel, but
with only one kind of antibody. In a multiplexed assay, experiments with many
different
kinds of antibodies are performed on one sample.
[1011 Multichannel and multiplexed assays may be scaled to implement assay
systems that
perform experiments with multiple samples and multiple antibodies. The
multiplexed assay
however, takes better advantage of the promise of microfluidics in terms of
optimum use of
small samples. In a multichannel assay, samples are loaded into each channel
from a
"macrofluidic" device, such as a pipette robot. In a multiplexed assay,
however, a single
sample is routed via microfluidic channels for testing with different kinds of
antibodies.
[102] The multiplexed assays described below depend on a microfluidic switched
interaction region which is implemented in a reconfigurable nnicrofluidic
device.
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Multiplexing is achieved by arranging multiple microfluidic switched
interaction regions in
series. A switched interaction region may also be implemented in a
microfluidic device
having conventional microvalves, albeit with increased complexity.
[103] Figs. 11 ¨ 16 are diagrams of a reconfigurable microfluidic device for
single channel
immunoassays, seen in plan view. Figs. 11 ¨ 16 outline steps in a single-
channel
immunoassay; i.e. an assay that involves one antibody linked to a surface and
one sample.
In Figs. 11¨ 16, reconfigurable microfluidic device 1105 includes: reservoirs
1110, 1115,
1120, 1125, 1130, 1145 and 1150; nodes 1135 and 1140; and channels 1155, 1160,
1165
and 1170. Other channels, such as the channel connecting reservoir 1115 to
node 1135, are
not labeled with reference numbers.
[104] Device 1105 may be constructed in layers exactly as described above; it
is only the
layout of reservoirs, nodes and channels that is different. The plan view
shown in Fig. 11 is
analogous to that of Fig. 4. A corresponding cross-sectional view of the
device of Fig. 11 is
not provided, but would essentially be a more complicated version of Fig. 1.
Channel 1170
is intentionally designed longer than the other channels as it serves as an
interaction region
where antigen ¨ antibody biochemical reactions take place.
[105] In an example ELISA experiment, reservoirs 1110, 1115, 1120, 1125, 1130
contained
wash buffer (e.g. phosphate buffered saline with Tween 20, "PBST"), horse
radish
peroxidase ("HRP") conjugate, 3,3',5,51-Tetramethylbenzidine substrate
("TMB"),
microcystin antibody, and blocking buffer (e.g. SuperBlockTM (Life
Technologies) or
equivalent), respectively. Of course, it does not matter which reservoir
contained what
solution, only that each solution had its own reservoir. Reservoir 1145
contained a sample
solution containing microcystin target antigen.
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[106] The example experiment involves coating the interaction region (channel
1170) with
antibody, followed by wash buffer, blocking buffer, wash buffer, sample
incubation, HRP
incubation, wash buffer, and TMB substrate incubation steps. Figs. 11 ¨ 14
show steps in
which a solution from one of reservoirs 1110, 1115, 1120, 1125, 1130 is
transferred to
reservoir 1150 via the interaction region, channel 1170. Figs. 15 and 16 show
steps in which
sample solution is transferred from reservoir 1145 to reservoir 1150 via
interaction region
1170.
[107] Figs. 11 ¨ 16 are labeled 'STEP 0', 'STEP 1' ... 'STEP 5'. The change in
configuration
from 'STEP 0' to 'STEP 1', and from 'STEP 1' to 'STEP 2', etc., is
accomplished by applying
pressures to the reservoirs and nodes of device 1105 according to the fluid
transfer rule. In
Figs. 11 ¨ 16, 'L' and 'H' indicate either low or high pressure, respectively,
applied to a
reservoir or node.
[108] Fig. 11, STEP 0, is the initial condition in which all reservoirs and
nodes are at low
pressure. Shading highlights the presence of fluid in reservoirs 1110 and
1145. In Fig. 12,
STEP 1, fluid from reservoir 1110 is transferred to node 1135. In Fig. 13,
STEP 2, fluid from
node 1135 is transferred to node 1140.
[109] In Fig. 14, STEP 3, fluid from node 1140 is transferred to reservoir
1150. In an actual
immunoassay experiment, this step is completed in two stages: first, fluid is
pushed from
node 1140 into channel 1170 and allowed to incubate there; second the fluid is
pushed into
reservoir 1150. This procedure permits, for example, coating the walls of
channel 1170 with
antibodies or incubation of a sample with antibodies that have been chemically
linked to the
walls of the channel in a previous step.
[110] In Fig. 15, STEP 4, sample solution from reservoir 1145 is transferred
to node 1140.
In Fig. 16, STEP 5, the sample solution is transferred from node 1140 to
reservoir 1150. As
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in STEP 3, this transfer is completed in two stages in an actual immunoassay,
including
incubation time in channel 1170.
[111] Results from the single-channel ELISA experiment outlined in Figs. 11-16
are
presented in Figs. 17 and 18 which are graphs of competitive ELISA absorbance
data. Figs.
17 and 18 compare ELISA results from the biochemical assay performed in the
device of
Figs. 11 ¨ 16 with results from the same biochemical assay performed in a
standard 96-well
plate.
[112] The assay performed in the reconfigurable microfluidic device used only
about 15%
of the sample, enzyme and substrate volumes that the 96-well plate version
required. Not
including antibody coating, the assay in the microfluidic format took 29
minutes versus 94
minutes for the 96-well plate assay. (The 96-well plate assay kit comes with
antibodies pre-
coated on the plate. Antibody coating took 23 minutes in the microfluidic
format.) A
competitive ELISA has been demonstrated and extensions to other kinds of
ELISA, such as
sandwich ELISA, are straightforward.
[113] Fig. 17 is a graph of optical absorbance (arbitrary units) versus
antigen concentration
(parts per billion) in a sample for a competitive ELISA experiment performed
in a 96-well
plate (darker shaded data bars) and for the same experiment performed in the
reconfigurable microfluidic device of Figs. 11 ¨ 16. There is good agreement
among the data
for the two assay formats.
[114] Fig. 18 is a graph of optical absorbance normalized to absorbance
measured for a
negative control; i.e. an experiment where the concentration of antigen was
zero. Diamond
and square data markers correspond to data obtained in a 96-well plate assay
and a
microfluidic device format, respectively. The dashed line is a logarithmic fit
to the 96-well
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plate data while the solid line is a logarithmic fit to the microfluidic
device data. There is
good agreement among the data for the two assay formats.
[115] The single channel immunoassay device just described may be extended for
multichannel operation. Figs. 19 ¨ 24 are diagrams of a reconfigurable
microfluidic device
for multichannel immunoassays, seen in plan view. Figs. 19 ¨ 24 outline steps
in a
multichannel immunoassay; i.e. an assay that involves many samples and one
surface-linked
antibody. In Figs. 19 ¨ 24, reconfigurable microfluidic device 1905 includes:
reservoirs 1910,
1915, 1920, 1925, 1930, 1945 and 1950; nodes 1935 and 1940; and channels 1955,
1960,
1965 and 1970. Other channels, such as the channel connecting reservoir 1915
to node
1935, are not labeled with reference numbers. Structures that are duplicated
from one
immunoassay experiment to the next are also not labeled with reference
numbers. The
volume of node 1935 is about eight times larger than the corresponding node
1135 in Fig.
11.
[116] Although it is clear in context, a distinction should be kept in mind
between a
"multichannel" immunoassay and a microfluidic device having two or more, i.e.
"multiple",
microfluidic channels. Every microfluidic device discussed herein has more
than one
microfluidic channel. (If a device had only one microfluidic channel, it would
also have only
two reservoirs and no nodes, and probably would not be very useful.) In the
context of
immunoassays, "multichannel" means that more than one immunoassay experiment
is
performed simultaneously. Each experiment is performed in an experimental
"channel" of a
multichannel device. Device 1905 has 29 microfluidic channels and eight
immunoassay
experimental channels. Dashed rectangle 1975 encloses one immunoassay channel,
for
example.
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[117] Device 1905 may be constructed in layers exactly as described above; it
is only the
layout of reservoirs, nodes and channels that is different. The plan view
shown in Fig. 19 is
analogous to that of Fig. 4. A corresponding cross-sectional view of the
device of Fig. 19 is
not provided, but would essentially be a more complicated version of Fig. 1.
Channel 1970
is intentionally designed longer than the other channels as it serves as an
interaction region
where antigen ¨ antibody biochemical reactions take place.
[118] As an example, a multichannel ELISA may be performed with reservoirs
1910, 1915,
1920, 1925, 1930 containing PBST, HRP conjugate, TMB, microcystin antibody,
and blocking
buffer, respectively. Of course, it does not matter which reservoir contains
what solution,
only that each solution has its own reservoir. Reservoir 1145 contains a
sample solution and
each corresponding reservoir in the eight immunoassay channels may contain its
own,
different sample solution.
[119] Multichannel [LISA involves coating the interaction region (channel
1970) with
antibody, followed by wash buffer, blocking buffer, wash buffer, sample
incubation, HRP
incubation, wash buffer, and TMB substrate incubation steps. Figs. 19 ¨ 22
show steps in
which, for example, a solution from one of reservoirs 1910, 1915, 1920, 1925,
1930 is
transferred to reservoir 1950 via the interaction region, channel 1970. These
steps are
performed simultaneously on all eight immunoassay channels. Figs. 23 and 24
show steps in
which, for example, sample solution is transferred from reservoir 1945 to
reservoir 1950 via
interaction region 1970. These steps are performed simultaneously on all eight
immunoassay channels, each with its own, possibly different, sample solution.
[120] Figs. 19-24 are labeled 'STEP 0', 'STEP 1' ... 'STEP 5'. The change in
configuration
from 'STEP 0' to 'STEP 1', and from 'STEP 1' to 'STEP 2', etc., is
accomplished by applying
pressures to the reservoirs and nodes of device 1905 according to the fluid
transfer rule. In
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Figs. 19¨ 24,1' and 'H' indicate either low or high pressure, respectively,
applied to a
reservoir or node.
[121] Fig. 19, STEP 0, is the initial condition in which all reservoirs and
nodes are at low
pressure. Shading highlights the presence of fluid in reservoirs 1910 and
1945. Fluid is also
present in the other, unnumbered reservoirs corresponding to reservoir 1945.
In Fig. 20,
STEP 1, fluid from reservoir 1910 is transferred to node 1935. In Fig. 21,
STEP 2, fluid from
node 1935 is transferred to node 1940 and to the other unnumbered nodes
corresponding
to node 1940.
[122] In Fig. 22, STEP 3, fluid from node 1940 is transferred to reservoir
1950. Similar fluid
transfer occurs simultaneously in each of the other immunoassay channels. In
an
immunoassay experiment, this step is completed in two stages: first, fluid is
pushed from
node 1940 into channel 1970 and allowed to incubate there; second the fluid is
pushed into
reservoir 1950. This procedure permits, for example, coating the walls of
channel 1970 with
antibodies or incubation of a sample with antibodies that have been chemically
linked to the
walls of the channel in a previous step.
[123] In Fig. 23, STEP 4, sample solution from reservoir 2945 is transferred
to node 1940.
This same fluid movement occurs in each of the eight immunoassay channels, but
the
composition of the sample may be different in each one. In Fig. 24, STEP 5,
the sample
solution is transferred from node 1940 to reservoir 1950. As in STEP 3, this
transfer is
completed in two stages in an actual immunoassay, including incubation time in
channel
1970. This same fluid movement occurs in each of the eight immunoassay
channels. After
STEP 5, fluid in reservoir 1950 and corresponding reservoirs may be tested,
e.g., for optical
absorption. Absorption may be measured while fluid is in device 1905 or fluid
may be
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unloaded to an external container (a 96-well plate, for example) as discussed
above in
connection with Fig. 3.
[124] Multichannel immunoassay device 1905 is a generalization of single-
channel device
1105. It permits a particular immunoassay chemistry to be applied to many
samples at
once. Although device 1905 processes eight samples simultaneously, additional
immunoassay channels may be included in a design to process even more samples.
[1251 For example, Fig. 25 is a conceptual diagram of a reconfigurable
rnicrofluidic device
2505 for 96-channel immunoassays. The 96-fluid-sample output of device 2505
may be
loaded into a 96-well plate for analysis with a standard plate reader. Fig. 25
is schematic. In
the figure, circles 2510 represent reservoirs that may contain wash buffers,
enzymes,
substrates, antibodies and blocking buffers, for example. These reservoirs are
analogous to
reservoirs 1910¨ 1930 in Figs. 19 ¨ 24. Oval 2515 represents a reservoir that
is analogous to
reservoir 1935 in Figs. 19-24. Braces 2520 denote groups of immunoassay
channels such
as 2525. These immunoassay channels are analogous to immunoassay channel 1975
in Figs.
19 ¨24. Each channel may be loaded with a unique sample. The large number "24"
in Fig.
25 indicates that there are 24 immunoassay channels arranged in a group. Four
such groups
of 24 make 96 immunoassay channels in total. Of course, devices like 2505 may
be
designed with different numbers of immunoassay channels. Devices with 384 or
1536
channels may be constructed to be compatible with popular well plate
configurations, for
example.
[126] One of the limitations of the multichannel immunoassay devices of Figs.
19 ¨25 is
that each sample is loaded into the microfluidic device from an external
"macrofluidic"
system such as a pipette robot. This means that macroscopic sample volumes are
required
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for each type of immunoassay. The multiplexed assay devices described below
remove this
limitation.
[127] Once a sample is loaded into a multiplexed assay device, it can be
tested in many,
biochemically different immunoassays all on the same device. This means that a
smaller
starting sample volume is required when compared to the multichannel assays
discussed
above.
[128] Multiplexed immunoassay devices include a microfluidic structure defined
here as a
"microfluidic switched interaction region". A microfluidic switched
interaction region is a
microfluidic channel connected at one of its ends to two input channels via a
node. The
interaction region is connected at its other end to two output channels via
another node.
The interaction region is "switched" because the action of nodes described
above allows
operation of the device such that fluid travels from one (but not the other)
of the input
channels to one (but not the other) of the output channels. The switching
action of a node
cannot be replicated with a single microfluidic valve. However a switched
interaction region
may be implemented with a more complicated arrangement of microvalves as
discussed
below.
[129] Fig. 26 illustrates a microfluidic switched interaction region 2605 in
two operational
modes. At "A" an interaction region is operated such that fluid travels from
one of its inputs
to one of its outputs. At "B" the interaction region is operated such that
fluid travels from
the other input to the other output. Shading in the figure highlights these
two modes. Fluid
may also travel in the reverse direction, so "input" and "output" serve only
as channel
labels, not as indicators of flow direction.
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[130] In Fig. 26, input microfluidic channels 2610 and 2620 are connected to
microfluidic
channel 2630 via node 2635. Microfluidic channel 2635 is connected to output
rnicrofluidic
channels 2615 and 2625 via node 2640.
[131] Operation of the switched interaction region at "A" is as follows. Fluid
from channel
2610 is accumulated in node 2635. Then the fluid is sent from node 2635 to
node 2640.
Finally the fluid is sent out via channel 2615. Fluid does not leak into
channels 2620 or 2625,
just as fluid did not leak into reservoir "C" in the fluid transfer experiment
of Figs. 4 and 5
discussed above. Operation of the switched interaction region at "B" is
analogous, except
that fluid arrives at node 2635 from channel 2620 and departs node 2640 via
channel 2615.
[132] Two other modes of operation are possible but not illustrated. Fluid may
be
switched from channel 2610 to 2625, or fluid may be switched from channel 2620
to 7615.
These additional modes are not illustrated because they are analogous to modes
"A" and
"B", and they are not necessary to the discussion of multiplexed assays.
[133] Multiplexed immunoassays are performed with reconfigurable microfluidic
devices
having multiple microfluidic switched interaction regions connected in series
as illustrated in
Fig. 27. Fig. 27 shows an example in which two switched interaction regions
are connected
in series. A system with three switched interaction regions in series is
illustrated in Figs. 28
¨33. In fact, systems may be designed with any number of switched interaction
regions
connected in series. When switched interaction regions are connected in
series, an output
channel of one interaction region is connected to an input channel of the next
interaction
region. All of the interaction regions may also be connected to a common input
channel.
[134] In a system like that of Fig. 27, one sample is processed in multiple
interaction
regions. Each interaction region supports an immunoassay with a different
antibody. Thus
one sample can be tested for the presence of many different antigens. The high
specificity
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of antigen-antibody interactions permits the same sample to be processed in
different
immunoassays, simultaneously or one after the other. The same enzyme linked
detection
chemistry may be used in each interaction region because each interaction
region has its
own substrate output.
[135] In Fig. 27, substrate input channel 2705 is split into two branches 2710
and 2715
which lead to interaction regions 2720 and 2725, respectively. Interaction
regions 2720 and
2725 have the same structure as interaction region 2605 in Fig. 26. During
normal
immunoassay operations, interaction region 2720 either takes fluid input from
channel 2710
and sends it out via channel 2730 or it takes fluid input from channel 2745
and sends it out
via channel 2750. Similarly, interaction region 2725 either takes fluid input
from channel
2715 and sends it out via channel 2735 or it takes fluid input from channel
2740 and sends it
out via channel 2745.
[136] The overall steps (ignoring rinses, buffers, etc.) for performing
multiplexed
immunoassays with a system like that of Fig. 27 are: coat each interaction
region with a
(possibly different) kind of antibodies; load a sample into the interaction
regions and
incubate; load substrate into the interaction regions; collect substrate from
each interaction
region separately; analyze collected substrates, e.g. by optical absorption.
Alternatively, the
sample can be loaded into one interaction region for incubation and then sent
to
subsequent interaction regions later. These steps are illustrated in Figs. 28-
33 which are
diagrams of a reconfigurable microfluidic device for multiplexed immunoassays.
[137] Figs. 28 ¨ 33 show a reconfigurable microfluidic device in plan view,
like Figs. 11 ¨ 16
and Figs. 19 ¨24. The device includes reservoirs, nodes and channels, and it
moves fluid via
application of the fluid transfer rule. Since the fluid transfer rule has been
explained and
demonstrated in several examples above, Figs. 28-33 do not include pressure
labels such
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as "H" and "L". Figs. 28 - 33 also omit some optional nodes which are
described later in
connection with Fig. 34. Furthermore, Figs. 28 - 33 and associated description
omit various
rinsing, buffer and enzyme conjugate steps. Rather, the discussion of Figs. 28
- 33 is
directed to describing fluid movements that permit multiplexed immunoassays in
a
multiplexed immunoassay device. All such fluid movements may be accomplished
via
application of the fluid transfer rule and pressures may be applied at nodes
and reservoirs
automatically with a pressure sequencer such as that discussed above and shown
in Fig. 9.
[138] In Fig. 28, reconfigurable microfluidic device 2805 includes reservoirs
2807, 2820,
2832, 2847, 2862, 2870, nodes 2825, 2831, 2840, 2846, 2855, 2861, 2877, and
channels
2810, 2812, 2815, 2817, 2822, 2830, 2835, 2837, 2845, 2850, 2852, 2860, 2865,
and 2867.
Antibody supplies Ab1, Ab2 and Ab3 are connected to nodes 2831, 2846 and 2861
via
supply tubes 2827, 2842 and 2857, respectively.
[139] In Fig. 28, antibody solution from Abl is loaded into node 2831,
antibody solution
from Ab2 is loaded into node 2846, and antibody solution from Ab3 is loaded
into node
2861. Antibody loading from the antibody supplies to the nodes may
accomplished in the
manner described above in connection with Fig. 2. This is an example of nodes
temporarily
storing fluid.
[140] In Fig. 29, antibodies from Ab1 are chemically linked to the walls of
channel 2830,
antibodies from Ab2 are chemically linked to the walls of channel 2845, and
antibodies from
Ab3 are chemically linked to the walls of channel 2860.
[141] In Fig. 30, sample solution from Sample supply is loaded into reservoir
2870 via
supply tube 2872. After the sample solution is loaded in the reservoir it is
sent through
channels 2867, 2860, 2850, 2845, 2835, 2830 and 2822. The sample solution
interacts with
antibodies from antibody solutions ,4h1, Ah2 and Ab3 in channels 2830, 2845
and 2860,
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respectively. Sample solution may be distributed in all three interaction
regions (2830, 2845
and 2860) at once for simultaneous immunoassays, or it may be first kept in
one interaction
region and later sent to other interaction regions for sequential
immunoassays. Antibodies
do not move from one interaction region to another because they are chemically
linked to
the walls of channels 2830, 2845 and 2860.
[142] In Fig. 31, substrate solution from Substrate supply is loaded into
reservoir 2807 via
supply tube 2875. Substrate solution is then moved to node 2877 and on to
nodes 2825,
2840 and 2855 via channels 2812, 2815 and 2817, respectively. Channels 2812,
2815 and
2817 may be designed to have the same length. Alternatively, substrate
solution may
loaded into node 2877; the substrate solution may then be sent to nodes 2825,
2840 and
2855 sequentially, if desired.
[143] In Fig. 32, substrate solution is moved from node 2825 to node 2831,
from node
2840 to node 2846, and from node 2855 to node 2861, after interacting with
antigen-
antibody complexes in channels 2830, 2845 and 2860 respectively.
[144] Finally in Fig. 33, substrate solution is moved from nodes 2831, 2846
and 2861 to
reservoirs 2832, 2847 and 2862 respectively. Here the solution may be unloaded
(as in Fig.
3, for example) for optical absorption analysis.
[145] Device 2805, based on multiple microfluidic switched interaction regions
connected
in series, permits one sample solution to interact with different kinds of
antibodies that are
linked to the walls of different microfluidic channels. Detection of antigen-
antibody
interactions is then performed separately in each of those channels. This is
helpful for
immunoassays because only a limited number of different enzyme-linked
detection
protocols are known, with one based on HRP cleaving TMB being the most common.
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[146] Device 2805 has three interaction regions for testing a sample with as
many as three
different kinds of antibodies. However, the device can be extended for
operation with more
different kinds of antibodies by adding more microfluidic switched interaction
regions in
series.
[147] Fig. 34 shows the reconfigurable microfluidic device of Figs. 28 ¨ 33
with the addition
of optional nodes 2880, 2882, 2884, 2886, 2888 and 2890. These optional nodes,
or "buffer
nodes", permit fully parallel operation of device 2805 for antibody and
substrate loading
into interaction regions. Figs. 29 and 32 discussed above illustrate
operations in which fluid
flows in the three interaction regions of device 2805. However, when only the
nodes shown
in those figures are present, the fluid flows in the interaction regions must
occur
sequentially, not at the same time. (Other fluid flows, outside the
interaction regions, may
occur simultaneously.)
[148] As an example, consider coating channel 2830 with Ab1 in Fig. 29. This
is
accomplished by setting node 2831 to high pressure and node 2825 to low
pressure. If the
same operations were performed at the same time with channel 2845 and nodes
2846 and
2840, then fluid would also travel from node 2831 to node 2840 (low pressure).
In Fig. 34,
buffer node 2880, set to high pressure, and buffer node 2882, set to low
pressure, prevent
the undesired flow of fluid from node 2831 to node 2840 during this operation.
[149] A similar situation exists in the scenario of Fig. 32 when substrate
solution is moved
from node 2825 to node 2831 (and from 2840 to 2846, and from 2855 to 2861). In
Fig. 34,
buffer node 2880 is set to low pressure and buffer node 2882 is set to high
pressure to
prevent undesired flow of fluid from node 2840 to node 2831. In all cases,
buffer node 2880
is set to the same pressure as node 2831 and buffer node 2882 is set to the
same pressure
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as node 2840. Similarly, buffer node 2884 may always be set to the same
pressure as node
2846 and buffer node 2886 may always be set to the same pressure as node 2855,
etc.
[150] Optional buffer nodes 2880, 2882, 2884, 2886, 2888 and 2890 therefore
prevent
simultaneous fluid flows in the series-connected interaction regions from
contaminating
each other. This is not a required capability for multiplexed immunoassays, as
fluid flows in
the interaction regions may be performed sequentially. However, simultaneous
operation
also reduces the complexity of node pressure sequencing. When the optional
buffer nodes
are present, the pressures at, for example, nodes 2825 and 2840, may always be
set equal
to each other, both high or both low, and therefore they may be supplied from
a common
pressure tube or pressure manifold. This reduces the number of pressure tubes
and
external pressure sources needed.
[151] Immunoassay devices with microfluidic switched interaction regions may
also be
implemented with microfluidic valves as shown in Fig. 35. Fig. 35 may be
compared to Figs.
26 and 27. Figs. 27 and 35 show microfluidic devices having multiple
microfluidic switched
interaction regions connected in series. However, the device of Fig. 35 is
implemented with
conventional microvalves while the device of Fig. 27 is implemented with
reservoirs and
nodes. A microva lye is a microfluidic device that opens and closes to allow
or prevent fluid
flow past the microvalve in a microfluidic channel. Microvalves considered
here may be of
any conventional design, such as normally-open microvalves or normally-closed
microvalves.
[152] In Fig. 35, substrate input channel 3505 is split into two branches 3510
and 3515
which lead to interaction regions 3520 and 3525, respectively. Interaction
regions 3520 and
3525 include the same structure as interaction region 2605 in Fig. 26.
However, in Fig. 35
the interaction regions are based on microvalves and further include an extra
dump port.
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[153] During normal immunoassay operations, interaction region 3520 may route
fluid
input from channel 3510 to SUBSTRATE OUTPUT 1; or it may route fluid input
from PUMP 1
to SAMPLE OUTPUT; or it may route fluid from PUMP 1 to Ab DUMP 1. Similarly,
interaction
region 3525 either routes fluid input from channel 3515 to SUBSTRATE OUTPUT 2;
or it
routes fluid input from PUMP 2 to SAMPLE OUTPUT (via interaction region 3520);
or it
routes fluid from PUMP 2 to Ab DUMP 2.
[154] Dump ports Ab DUMP 1 and Ab DUMP 2 are needed because a microvalve-based
system does not include nodes that can temporarily store fluid. When PUMP 1
operates to
coat antibodies supplied at Ab LOAD PORT 1 on the walls of channel 3560, the
fluid already
in that channel must be provided with somewhere to go ¨ dump port Ab DUMP 1,
in this
case.
[155] Interaction region 3520 serves as an example of a microvalve
implementation of a
nnicrofluidic switched interaction region. Interaction region 3520 includes
microvalves 3530,
3535, 3540, 3545, 3550 and channel 3560.
[156] To route fluid from channel 3510 to SUBSTRATE OUTPUT 1, microvalves 3540
and
3545 are opened and microvalves 3530, 3535 and 3550 are closed. To route fluid
from
PUMP 1 to SAMPLE OUTPUT, microvalves 3530 and 3550 are opened and microvalves
3535,
3540 and 3545 are closed. To route fluid from PUMP 1 to Ab DUMP 1, microvalves
3535
and 3550 are opened and microvalves 3530, 3540 and 3545 are closed.
[157] Interaction regions based on microvalves, connected in series, can
perform the
functions of a node-based device, such as shown in Fig. 27. In particular, the
devices of Figs.
27 and 35 both: (a) permit a single sample solution to interact with multiple
interaction
regions; and (b) permit antigen detection (via substrate interactions) in each
interaction
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region separately. The devices of Figs. 34 and 35 permit simultaneous fluid
flows in all of
their interaction regions.
[158] PUMP 1 and PUMP 2 in Fig. 35 are microfluidic pumps which may be
implemented as
a series of three microfluidic valves each. Ab LOAD PORT 1 and Ab LOAD PORT 2
are ports
via which antibodies may be loaded into the microfluidic system. SAMPLE INPUT
is a port
via which a sample may be loaded into the microfluidic system.
[159] Multiplexed immunoassay devices based on multiple microfluidic switched
interaction regions permit a single small-volume sample to be tested in many
different
immunoassays. Detection of different antigens in the sample is performed in
different
interaction regions; hence, the detection mechanism may be the same in each
interaction
region. Multiplexed assays may be scaled to analyze multiple samples across
multiple
immunoassays in systems containing many copies of a devices such as those
illustrated in
Figs. 27-35.
[160] The above description of the disclosed embodiments is provided to enable
any
person skilled in the art to make or use the invention. Various modifications
to these
embodiments will be readily apparent to those skilled in the art, and the
principles defined
herein may be applied to other embodiments without departing from the scope of
the
disclosure. Thus, the disclosure is not intended to be limited to the
embodiments shown
herein but is to be accorded the widest scope consistent with the principles
and novel
features disclosed herein.
[161] All elements, parts and steps described herein are preferably included.
It is to be
understood that any of these elements, parts and steps may be replaced by
other elements,
parts and steps or deleted altogether as will be obvious to those skilled in
the art.
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Concepts
In greater detail, this writing also discloses at least the following
concepts.
Concept 1. A reconfigurable microfluidic system comprising:
two or more microfluidic switched interaction regions, a plurality of the
interaction regions
having at least two microfluidic input channels and two microfluidic output
channels, and a
plurality of the interaction regions being connected in series such that an
output channel of
one interaction region is connected to an input channel of the next
interaction region.
Concept 2. The reconfigurable microfluidic system of Concept 1, a plurality of
the interaction
regions also connected to a common microfluidic input channel.
Concept 3. The reconfigurable microfluidic system of Concept 1 wherein a
plurality of the
microfluidic switched interaction regions include a hydrophobic microfluidic
channel having
a first end connected to two hydrophobic microfluidic input channels via a
first microfluidic
cavity and a second end connected to two hydrophobic microfluidic output
channels via a
second microfluidic cavity, the hydrophobic microfluidic channels having a
higher resistance
to fluid flow than that of the cavities, and a plurality of the cavities
including a gas pressure
port.
Concept 4. The reconfigurable microfluidic system of Concept 3, where a
hydrophobic
microfluidic output channel of one interaction region is connected to a
hydrophobic
microfluidic input channel of the next interaction region via: (1) a first
microfluidic cavity, (2)
a hydrophobic microfluidic channel, and (3) a second microfluidic cavity,
connected in
series.
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Concept 5. The reconfigurable microfluidic system of Concept 3, a plurality of
the
hydrophobic microfluidic channels having a resistance to fluid flow at least
100 times
greater than that of the cavities.
Concept 6. The reconfigurable microfluidic system of Concept 3, a plurality of
the
hydrophobic microfluidic channels having a resistance to fluid flow at least
1,000 times
greater than that of the cavities.
Concept 7. The reconfigurable microfluidic system of Concept 3, a plurality of
the
hydrophobic microfluidic channels having a resistance to fluid flow at least
10,000 times
greater than that of the cavities.
Concept 8. The reconfigurable microfluidic system of Concept 3, the cavities
being formed in
a hydrophobic microfluidic layer that is bonded to a substrate layer, and the
cavities being
sealed by a pneumatic layer that is bonded to the microfluidic layer.
Concept 9. The reconfigurable microfluidic system of Concept 8, the
microfluidic layer being
made from polydimethylsiloxane.
Concept 10. The reconfigurable microfluidic system of Concept 8, the
microfluidic layer
being made from fluorinated ethylene propylene.
Concept 11. The reconfigurable microfluidic system of Concept 8, the
microfluidic layer
being made from polytetrafluoroethylene.
Concept 12. The reconfigurable microfluidic system of Concept 8; the pneumatic
layer
including a gas manifold that acts as a pressure port for two or more
cavities.
Concept 13. The reconfigurable microfluidic system of Concept 3 further
comprising fluid
tubing connecting a cavity to an external fluid store maintained at
atmospheric pressure.
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Concept 14. The reconfigurable microfluidic system of Concept 3 further
comprising gas
tubing connecting one or more cavities to gas pressure sources via the gas
pressure ports.
Concept 15. The reconfigurable microfluidic system of Concept 3, at least one
microfluidic
channel having a gas pressure port.
Concept 16. The reconfigurable microfluidic system of Concept 3 further
comprising a
pressure sequencer including a set of gas valves, the pressure sequencer
connected by gas
tubing to: a high pressure gas source, a low pressure gas source, and to at
least one cavity.
Concept 17. The reconfigurable microfluidic system of Concept 16, the pressure
sequencer
applying high gas pressure and low gas pressure to the at least one cavity
according to
pressure sequence data.
Concept 18. The reconfigurable microfluidic system of Concept 17, a plurality
of the
hydrophobic microfluidic channels having a hydrophobic pressure barrier to
fluid flow that is
less than the pressure difference between the high gas pressure and the low
gas pressure.
Concept 19. The reconfigurable microfluidic system of Concept 18, the pressure
sequence
data following a fluid transfer rule in which high gas pressure is applied to
an origin cavity
from which a fluid is transferred and low gas pressure is applied to a
destination cavity to
which the fluid is transferred, and high gas pressure is applied to any cavity
(other than the
destination cavity) connected to the origin cavity by a channel and low gas
pressure is
applied to any cavity (other than the origin cavity) connected to the
destination cavity by a
channel, and where high gas pressure is a pressure greater than low gas
pressure.
Concept 20. The reconfigurable microfluidic system of Concept 1 where each
microfluidic
switched interaction region includes a microfluidic channel having a first end
connected to
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three input microfluidic channels and a second end connected to two output
microfluidic
channels, each input or output microfluidic channel including a microfluidic
valve.
Concept 21. A method for performing a multiplexed immunoassay, the method
comprising
operating the reconfigurable microfluidic system of Concept 19 according to
pressure
sequence data such that the pressure sequencer directs fluid flows in the
system that cause
different kinds of sample-analyte ¨ capture-analyte reactions to occur in
different
interaction regions, but the same kind of detection reagent reaction to occur
in a plurality of
interaction regions.
Concept 22. The method of Concept 21 wherein the sample-analyte ¨ capture-
analyte
reactions are antibody ¨ antigen reactions.
Concept 23. The method of Concept 21 wherein the detection reagent reaction is
an
enzyme-linked detection reaction.
Concept 24. The method of Concept 21 wherein the pressure sequencer directs
fluid flows
via which capture analyte solutions are loaded into a plurality of interaction
regions
simultaneously.
Concept 25. The method of Concept 21 wherein the pressure sequencer directs
fluid flows
via which detection reagent solution in loaded into a plurality of interaction
regions
simultaneously.
Concept 26. The method of Concept 21 wherein the pressure sequencer directs
fluid flows
via which sample analyte solution is incubated a plurality of interaction
regions
simultaneously.
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Concept 27. The method of Concept 21 wherein the pressure sequencer directs
fluid flows
via which sample analyte solution is incubated a plurality of interaction
regions sequentially.
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