U.S. patent application number 13/024206 was filed with the patent office on 2011-07-21 for device and method for pressure-driven plug transport.
This patent application is currently assigned to The University of Chicago. Invention is credited to Cory John Gerdts, Rustem F. Ismagilov, Joshua David Tice, Bo Zheng.
Application Number | 20110176966 13/024206 |
Document ID | / |
Family ID | 43706227 |
Filed Date | 2011-07-21 |
United States Patent
Application |
20110176966 |
Kind Code |
A1 |
Ismagilov; Rustem F. ; et
al. |
July 21, 2011 |
DEVICE AND METHOD FOR PRESSURE-DRIVEN PLUG TRANSPORT
Abstract
The present invention provides microfabricated substrates and
methods of conducting reactions within these substrates. The
reactions occur in plugs transported in the flow of a
carrier-fluid.
Inventors: |
Ismagilov; Rustem F.;
(Chicago, IL) ; Tice; Joshua David; (Webster,
NY) ; Gerdts; Cory John; (Chicago, IL) ;
Zheng; Bo; (Chicago, IL) |
Assignee: |
The University of Chicago
Chicago
IL
|
Family ID: |
43706227 |
Appl. No.: |
13/024206 |
Filed: |
February 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12777099 |
May 10, 2010 |
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13024206 |
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10765718 |
Jan 26, 2004 |
7901939 |
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12777099 |
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10434970 |
May 9, 2003 |
7129091 |
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10765718 |
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60394544 |
Jul 8, 2002 |
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60379927 |
May 9, 2002 |
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Current U.S.
Class: |
422/82.05 ;
250/288 |
Current CPC
Class: |
B01J 2219/00585
20130101; B82Y 30/00 20130101; B01J 2219/00891 20130101; B01L
3/502715 20130101; C30B 7/14 20130101; C30B 29/54 20130101; B01F
13/0071 20130101; B01J 19/0046 20130101; B01J 2219/00756 20130101;
B01J 2219/00725 20130101; C07K 14/43 20130101; C12Q 1/6806
20130101; B01F 5/0646 20130101; Y10T 436/25 20150115; B01J
2219/00975 20130101; B01L 3/502784 20130101; B01J 2219/00894
20130101; B01J 2219/00977 20130101; B01J 2219/00722 20130101; B01D
9/0072 20130101; B01F 5/0647 20130101; B01L 2200/12 20130101; B01L
2400/0487 20130101; G01N 1/28 20130101; Y10T 137/0318 20150401;
B01J 2219/0086 20130101; B01J 2219/00889 20130101; B01J 2219/00286
20130101; B01J 2219/00599 20130101; B01J 2219/00783 20130101; B01L
2200/0673 20130101; B01D 2009/0086 20130101; Y10T 436/117497
20150115; B01L 2300/0867 20130101; G01N 35/08 20130101; B01J 14/00
20130101; B01J 2219/00869 20130101; C12N 9/2462 20130101; Y10T
117/10 20150115; B01J 19/0093 20130101; C30B 29/58 20130101; B01J
2219/00576 20130101; B01J 2219/00736 20130101; B01J 2219/00837
20130101; B01J 2219/00903 20130101; C12Y 302/01017 20130101; B01L
3/50273 20130101; Y10T 436/143333 20150115 |
Class at
Publication: |
422/82.05 ;
250/288 |
International
Class: |
G01N 21/00 20060101
G01N021/00; H01J 49/26 20060101 H01J049/26 |
Claims
1. A system comprising: a mass spectrometer; a microfluidic device
comprising a microchannel having an exit point leading to the mass
spectrometer; and a first plurality of plugs comprising a first
plug fluid flowing in the microchannel toward the exit point,
wherein at least a first plug of the first plurality of plugs is
detected by the mass spectrometer following its exit through the
exit point.
2. The system of claim 1, wherein the mass spectrometer is an
electrospray mass spectrometer.
3. The system of claim 1, wherein the first plug is driven by
pressure through the microchannel toward the exit point.
4. The system of claim 1, wherein the first plug is substantially
surrounded by a carrier fluid in the microchannel.
5. The system of claim 4, wherein the carrier fluid comprises a
fluorinated oil.
6. The system of claim 4, wherein the carrier fluid and/or the
first fluid comprises a surfactant.
7. The system of claim 6, wherein the surfactant is a fluorinated
surfactant.
8. The system of claim 1, wherein the first plug comprises one or
more components and wherein the detection of the first plug by the
mass spectrometer includes detecting one or more mass spectra of
the components.
9. The system of claim 1, wherein the first plug comprises a
plurality of components including one or more reactants and/or
reagents and wherein the detection of the first plug by the mass
spectrometer includes detecting one or more mass spectra of the
reactants and/or reagents, the components, and/or one or more
reaction products or intermediates or any combination thereof.
10. The system of claim 9, wherein the first plug is a merged
plug.
11. The system of claim 10, wherein the first plug is formed by the
merger of a second plug and a third plug, wherein the second plug
comprises a second plug fluid comprising at least a first reactant
and/or first reagent and the third plug comprises a third plug
fluid comprising at least a first component other than the first
reactant and/or first reagent, and further wherein, at or after the
merger, the first reactant and/or first reagent undergoes or
participates in a reaction involving the first component.
12. The system of claim 8, wherein the first plug flows through the
microchannel at a known first flow rate and wherein a first
distance traversed by the first plug from a first point in the
microchannel upstream of the exit point, through the exit point, to
the mass spectrometer is known.
13. The system of claim 9, wherein the first plug flows through the
microchannel at a known first flow rate and wherein a first
distance traversed by the first plug from a first point in the
microchannel upstream of the exit point, through the exit point, to
the mass spectrometer is known.
14. The system of claim 12, wherein at least one component, and/or
at least one reactant and/or reagent if present, undergoes or
participates in a reaction initiated or occurring when the first
plug is at the first point.
15. The system of claim 13, wherein at least one component, and/or
at least one reactant and/or reagent if present, undergoes or
participates in a reaction initiated or occurring when the first
plug is at the first point.
16. The system of claim 14, wherein the reaction is induced by
radiation, heat, temperature change, pressure change, ultrasonic
wave, and/or a catalyst.
17. The system of claim 15, wherein the reaction is induced by
radiation, heat, temperature change, pressure change, ultrasonic
wave, and/or a catalyst.
18. The system of claim 1, wherein an additional second plug fluid
immiscible with the first plug fluid is disposed directly between
and separates the first plug and a second plug of the first
plurality of plugs.
19. The system of claim 1, further comprising a second plurality of
plugs comprising a second plug fluid flowing in the microchannel
toward the exit point, and wherein the second plug fluid is
immiscible with the first plug fluid and the second plurality of
plugs separate the first plurality of plugs from each other.
20. The system of claim 19, wherein the first plurality of plugs
and the second plurality of plugs are substantially surrounded by a
carrier fluid in the microchannel.
Description
[0001] This application is a continuation of application Ser. No.
12/777,099, filed May 5, 2010, which is a continuation of
application Ser. No. 10/765,718, filed Jan. 26, 2004, which is a
continuation-in-part of application Ser. No. 10/434,970, filed May
9, 2003, which claims the benefit of U.S. Provisional Application
No. 60/394,544, filed Jul. 8, 2002, and U.S. Provisional
Application No. 60/379,927, filed May 9, 2002, all of which are
incorporated herein by reference.
BACKGROUND
[0002] Nonlinear dynamics, in conjunction with microfluidics, play
a central role in the design of the devices and the methods
according to the invention. Microfluidics deals with the transport
of fluids through networks of channels, typically having micrometer
dimensions. Microfluidic systems (sometimes called labs-on-a-chip)
find applications in microscale chemical and biological analysis
(micro-total-analysis systems). The main advantages of microfluidic
systems are high speed and low consumption of reagents. They are
thus very promising for medical diagnostics and high-throughput
screening. Highly parallel arrays of microfluidic systems are used
for the synthesis of macroscopic quantities of chemical and
biological compounds, e.g., the destruction of chemical warfare
agents and pharmaceuticals synthesis. Their advantage is improved
control over mass and heat transport.
[0003] Microfluidic systems generally require means of pumping
fluids through the channels. In the two most common methods, the
fluids are either driven by pressure or driven by electroosmotic
flow (EOF). Flows driven by EOF are attractive because they can be
easily controlled even in complicated networks. EOF-driven flows
have flat, plug-like velocity profile, that is, the velocity of the
fluid is the same near the walls and in the middle of the channel.
Thus, if small volumes of multiple analytes are injected
sequentially into a channel, these plugs are transported as
non-overlapping plugs (low dispersion), in which case the
dispersion comes mostly from the diffusion between plugs. A main
disadvantage of EOF is that it is generated by the motion of the
double layer at the charged surfaces of the channel walls. EOF can
therefore be highly sensitive to surface contamination by charged
impurities. This may not be an issue when using channels with
negative surface charges in DNA analysis and manipulation because
DNA is uniformly negatively charged and does not adsorb to the
walls. However, this can be a serious limitation in applications
that involve proteins that are often charged and tend to adsorb on
charged surfaces. In addition, high voltages are often undesirable,
or sources of high voltages such as portable analyzers may not be
available.
[0004] Flows driven by pressure are typically significantly less
sensitive to surface chemistry than EOF. The main disadvantage of
pressure-driven flows is that they normally have a parabolic flow
profile instead of the flat profile of EOF. Solutes in the middle
of the channel move much faster (about twice the average velocity
of the flow) than solutes near the walls of the channels. A
parabolic velocity profile normally leads to high dispersion in
pressure-driven flows; a plug of solute injected into a channel is
immediately distorted and stretched along the channel. This
distortion is somewhat reduced by solute transport via diffusion
from the middle of the channel towards the walls and back. But the
distortion is made worse by diffusion along the channel (the
overall dispersion is known as Taylor dispersion).
[0005] Taylor dispersion broadens and dilutes sample plugs. Some of
the sample is frequently left behind the plug as a tail. Overlap of
these tails usually leads to cross-contamination of samples in
different plugs. Thus, samples are often introduced into the
channels individually, separated by buffer washes. On the other
hand, interleaving samples with long buffer plugs, or washing the
system with buffer between samples, reduces the throughput of the
system.
[0006] In EOF, flow transport is essentially linear, that is, if
two reactants are introduced into a plug and transported by EOF,
their residence time (and reaction time) can be calculated simply
by dividing the distance traveled in the channel by the velocity.
This linear transport allows precise control of residence times
through a proper adjustment of the channel lengths and flow rates.
In contrast, dispersion in pressure-driven flow typically creates a
broad range of residence times for a plug traveling in such flows,
and this diminishes time control.
[0007] The issue of time control is important. Many chemical and
biochemical processes occur on particular time scales, and
measurement of reaction times can be indicative of concentrations
of reagents or their reactivity. Stopped-flow type instruments are
typically used to perform these measurements. These instruments
rely on turbulent flow to mix the reagents and transport them with
minimal dispersion. Turbulent flow normally occurs in tubes with
large diameter and at high flow rates. Thus stopped-flow
instruments tend to use large volumes of reagents (on the order of
ml/s). A microfluidic analog of stopped-flow, which consumes
smaller volumes of reagents (typically .mu.L/min), could be useful
as a scientific instrument, e.g., as a diagnostic instrument. So
far, microfluidic devices have not be able to compete with
stopped-flow type instruments because EOF is usually very slow
(although with less dispersion) while pressure-driven flows suffer
from dispersion.
[0008] In addition, mixing in microfluidic systems is often slow
regardless of the method used to drive the fluid because flow is
laminar in these systems (as opposed to turbulent in larger
systems). Mixing in laminar flows relies on diffusion and is
especially slow for larger molecules such as DNA and proteins.
[0009] In addition, particulates present handling difficulty in
microfluidic systems. While suspensions of cells in aqueous buffers
can be relatively easy to handle because cells are isodense with
these buffers, particulates that are not isodense with the fluid
tend to settle at the bottom of the channel, thus eventually
blocking the channel. Therefore, samples for analysis often require
filtration to remove particulates.
SUMMARY ACCORDING TO THE INVENTION
[0010] In one aspect, a system includes a mass spectrometer, a
microfluidic device comprising a microchannel having an exit point
leading to the mass spectrometer, and a first plurality of plugs
comprising a first plug fluid flowing in the microchannel toward
the exit point. At least a first plug of the first plurality of
plugs is detected by the mass spectrometer following its exit
through the exit point.
[0011] In another aspect, the mass spectrometer may be an
electrospray mass spectrometer.
[0012] In another aspect, the first plug may be driven by pressure
through the microchannel toward the exit point.
[0013] In yet another aspect, the first plug may be substantially
surrounded by a carrier fluid in the microchannel. The carrier
fluid may include a fluorinated oil. Furthermore, the carrier fluid
and/or the first fluid may include a surfactant. The surfactant may
be a fluorinated surfactant.
[0014] In a further aspect, the first plug includes one or more
components and the detection of the first plug by the mass
spectrometer includes detecting one or more mass spectra of the
components.
[0015] In another aspect, the first plug includes a plurality of
components including one or more reactants and/or reagents, and the
detection of the first plug by the mass spectrometer includes
detecting one or more mass spectra of the reactants and/or
reagents, the components, and/or one or more reaction products or
intermediates or any combination thereof.
[0016] In yet another aspect, the first plug may be a merged plug.
The first plug may be formed by the merger of a second plug and a
third plug, wherein the second plug includes a second plug fluid
comprising at least a first reactant and/or first reagent and the
third plug includes a third plug fluid comprising at least a first
component other than the first reactant and/or first reagent. At or
after the merger, the first reactant and/or first reagent may
undergo or participate in a reaction involving the first
component.
[0017] In a further aspect, the first plug flows through the
microchannel at a known first flow rate, wherein a first distance
traversed by the first plug from a first point in the microchannel
upstream of the exit point, through the exit point, to the mass
spectrometer is known.
[0018] In another aspect, at least one component, and/or at least
one reactant and/or reagent if present, undergoes or participates
in a reaction initiated or occurring when the first plug is at the
first point. The reaction may be induced by radiation, heat,
temperature change, pressure change, ultrasonic wave, and/or a
catalyst.
[0019] In another aspect, an additional second plug fluid
immiscible with the first plug fluid is disposed directly between
and separates the first plug and a second plug of the first
plurality of plugs.
[0020] In yet another aspect, the system further includes a second
plurality of plugs comprising a second plug fluid flowing in the
microchannel toward the exit point, wherein the second plug fluid
is immiscible with the first plug fluid and the second plurality of
plugs separate the first plurality of plugs from each other. In
addition, the first plurality of plugs and the second plurality of
plugs may be substantially surrounded by a carrier fluid in the
microchannel.
BRIEF DESCRIPTION OF THE DRAWINGS AND PHOTOGRAPHS
[0021] FIG. 1A is a schematic diagram of a basic channel design
that may be used to induce rapid mixing in plugs. FIG. 1B(1)-(4)
are schematic diagrams depicting a series of periodic variations of
the basic channel design. FIG. 1C(1)-(4) are schematic diagrams
depicting a series of aperiodic combinations resulting from a
sequence of alternating elements taken from a basic design element
shown in FIG. 1A and an element from the periodic variation series
shown in FIGS. 10B(1)-(4).
[0022] FIG. 2A is a schematic diagram contrasting laminar flow
transport and plug transport in a channel. FIG. 2B(1) shows a
photograph (right side, top portion) illustrating rapid mixing
inside plugs moving through winding channels. FIG. 2B(2) shows a
photograph (right side, lower portion) showing that winding
channels do not accelerate mixing in a laminar flow in the absence
of PFD.
[0023] FIG. 3 shows photographs (right side) and schematic diagrams
(left side) that depict a stream of plugs from an aqueous
plug-fluid and an oil (carrier-fluid) in curved channels at flow
rates of 0.5 .mu.L/min and 1.0 .mu.L/min.
[0024] FIG. 4 shows a photograph (lower portion) and a schematic
diagram (upper portion) that illustrate plug formation through the
injection of oil and multiple plug-fluids.
[0025] FIG. 5 is a schematic diagram that illustrates a two-step
reaction in which plugs are formed through the injection of oil and
multiple plug-fluids using a combination of different geometries
for controlling reactions and mixing.
[0026] FIG. 6 is a schematic representation of part of a
microfluidic network that uses multiple inlets and that allows for
both splitting and merging of plugs. This schematic diagram shows
two reactions that are conducted simultaneously. A third reaction
(between the first two reaction mixtures) is conducted using
precise time delay.
[0027] FIG. 7(a)-(b) show microphotographs (10 .mu.s exposure)
illustrating rapid mixing inside plugs (a) and negligible mixing in
a laminar flow (b) moving through winding channels at the same
total flow velocity. FIG. 7(c) shows a false-color microphotograph
(2 s exposure, individual plugs are invisible) showing
time-averaged fluorescence arising from rapid mixing inside plugs
of solutions of Fluo-4 and CaCl.sub.2. FIG. 7(d) shows a plot of
the relative normalized intensity (I) of fluorescence obtained from
images such as shown in (c) as a function of distance (left)
traveled by the plugs and of time required to travel that distance
(right) at a given flow rate. FIG. 7(e) shows a false-color
microphotograph (2 s exposure) of the weak fluorescence arising
from negligible mixing in a laminar flow of the solutions used in
(c).
[0028] FIG. 8 shows photographs (right side) and schematics (left
side) that illustrate fast mixing at flow rates of about 0.5
.mu.L/min and about 1.0 .mu.L/min using 90.degree.-step
channels.
[0029] FIG. 9 shows schematics (left side) and photographs (right
side) illustrates fast mixing at flow rates of about 1.0 .mu.L/min
and about 0.5 .mu.L/min using 135.degree.-step channels.
[0030] FIG. 10a) is a schematic diagram depicting three-dimensional
confocal visualization of chaotic flows in plugs. FIG. 10b) is a
plot showing a sequence preferably used for visualization of a
three-dimensional flow.
[0031] FIG. 11 shows a schematic diagram of a channel geometry
designed to implement and visualize the baker's transformation of
plugs flowing through microfluidic channels.
[0032] FIG. 12 shows photographs depicting the merging of plugs
(top) and splitting of plugs (bottom) that flow in separate
channels or channel branches that are perpendicular.
[0033] FIG. 13 shows UV-VIS spectra of CdS nanoparticles formed by
rapid mixing in plugs (spectrum with a sharp absorption peak) and
by conventional mixing of solutions.
[0034] FIG. 14 shows schematic diagrams (left side) and photographs
(right side) that illustrate the synthesis of CdS nanoparticles in
PDMS microfluidic channels in single-phase aqueous laminar flow
(FIG. 14A) and in aqueous plugs that are surrounded by
water-immiscible perfluorodecaline (FIG. 14B).
[0035] FIG. 15 shows schematic representations of the synthesis of
CdS nanoparticles inside plugs.
[0036] FIG. 16 is a schematic illustration of a microfluidic device
according to the invention that illustrates the trapping of
plugs.
[0037] FIG. 17 is a schematic of a microfluidic method for forming
plugs with variable compositions for protein crystallization.
[0038] FIG. 18 is a schematic illustration of a method for
controlling heterogeneous nucleation by varying the surface
chemistry at the interface of an aqueous plug-fluid and a
carrier-fluid.
[0039] FIG. 19 is a schematic diagram that illustrates a method of
separating nucleation and growth using a microfluidic network
according to the present invention.
[0040] FIG. 20 show schematic diagrams that illustrate two methods
that provide a precise and reproducible degree of control over
mixing and that can be used to determine the effect of mixing on
protein crystallization.
[0041] FIG. 21 is a reaction diagram illustrating an unstable point
in the chlorite-thiosulfate reaction.
[0042] FIG. 22A-D are schematic diagrams that show various examples
of geometries of microfluidic channels according to the invention
for obtaining kinetic information from single optical images.
[0043] FIG. 23 shows a schematic of a microfluidic network (left
side) and a table of parameters for a network having channel
heights of 15 and 2 .mu.m.
[0044] FIG. 24 shows a reaction scheme that depicts examples of
fluorinated surfactants that form monolayers that are: (a)
resistant to protein adsorption; (b) positively charged; and (c)
negatively charged. FIG. 24b shows a chemical structure of neutral
surfactants charged by interactions with water by protonation of an
amine or a guanidinium group. FIG. 24c shows a chemical structure
of neutral surfactants charged by interactions with water
deprotonation of a carboxylic acid group.
[0045] FIG. 25 are schematic diagrams of microfluidic network (left
side of a), b), and c)) that can be used for controlling the
concentrations of aqueous solutions inside the plugs, as well as
photographs (right side of a), b), and c)) showing the formation of
plugs with different concentrations of the aqueous streams.
[0046] FIG. 26 are schematic diagrams of microfluidic network (left
side of a) and b)) and photographs (right side of a) and b)) of the
plug-forming region of the network in which the aqueous streams
were dyed with red and green food dyes to show their flow
patterns.
[0047] FIG. 27 are photographs and plots showing the effects of
initial conditions on mixing by recirculating flow inside plugs
moving through straight microchannels. FIG. 27a1) is a schematic
diagram showing that recirculating flow (shown by black arrows)
efficiently mixed solutions of reagents that were initially
localized in the front and back halves of the plug. FIG. 27a2) is a
schematic diagram showing that recirculating flow (shown by black
arrows) did not efficiently mix solutions of reagents that were
initially localized in the left and right halves of the plugs. FIG.
27b) shows a schematic diagram showing the inlet portions (left
side) and photographs of images showing measurements of various
periods and lengths of plugs. FIG. 27c1) shows a graph of the
relative optical intensity of Fe(SCN).sub.x.sup.(3-x)+ complexes in
plugs of varying lengths. FIG. 27c2) is the same as FIG. 7c1)
except that each plug traverses a distance of 1.3 mm.
[0048] FIG. 28 is a schematic illustration of a plug showing the
notation used to identify different regions of the plugs relative
to the direction of motion.
[0049] FIG. 29a)-b) are plots of the periods and the lengths of
plugs as a function of total flow velocity (FIG. 29a)) and water
fraction (FIG. 29b)).
[0050] FIG. 30 shows photographs illustrating weak dependence of
periods, length of plugs, and flow patterns inside plugs on total
flow velocity.
[0051] FIG. 31 are plots showing the distribution of periods and
lengths of plugs where the water fractions were 0.20, 0.40, and
0.73, respectively.
[0052] FIG. 32 shows photographs (middle and right side) that show
that plug traps are not required for crystal formation in a
microfluidic network, as well as a diagram of the microfluidic
network (left side).
[0053] FIG. 33a-d (left side) are top views of microfluidic
networks (left side) and photographs (right side) that comprise
channels having either uniform or nonuniform dimension. FIG. 33a
shows that merging of the plugs occurs infrequently in the T-shaped
channel shown in the photographs. FIG. 33b illustrates plug merging
occurring between plugs arriving at different times at the Y-shaped
junction (magnified view shown). FIG. 33c depicts in-phase merging,
i.e., plug merging upon simultaneous arrival of at least two plugs
at a junction, of plugs of different sizes generated using
different oil/water ratios at the two pairs of inlets. FIG. 33d
illustrates defects (i.e., plugs that fail to undergo merging when
they would normally merge under typical or ideal conditions)
produced by fluctuations in the relative velocity of the two
incoming streams of plugs.
[0054] FIG. 34a-c show a schematic diagram (a, left side) and
photographs (b, c) each of which depicts a channel network viewed
from the top. FIG. 34a is a schematic diagram of the channel
network used in the experiment. FIG. 34b is a photograph showing
the splitting of plugs into plugs of approximately one-half the
size of the initial plugs. FIG. 34c is a photograph showing the
asymmetric splitting of plugs which occurred when
P.sub.1<P.sub.2.
[0055] FIG. 35 shows a schematic diagram (a, left side) and
photographs (b, c) that depicts the splitting of plugs using
microfluidic networks without constrictions near the junction.
[0056] FIG. 36 shows a photograph (right side) of lysozyme crystals
grown in water plugs in the wells of the microfluidic channel, as
well as a diagram (left side) of the microfluidic network used in
the crystallization.
[0057] FIG. 37 is a schematic diagram that depicts a microfluidic
device according to the invention that can be used to amplify a
small chemical signal using an autocatalytic (and possibly
unstable) reaction mixture.
[0058] FIG. 38 is a schematic diagram that illustrates a method for
a multi-stage chemical amplification which can be used to detect as
few as a single molecule.
[0059] FIG. 39 shows a diagram (left side) of the microfluidic
network and a photograph (right side) of water plugs attached to
the PDMS wall.
[0060] FIG. 40 is a schematic representation (left side) of a
microfluidic network used to measure kinetics data for the reaction
of RNase A using a fluorogenic substrate (on-chip enzyme kinetics),
and plots that shows the kinetic data for the reaction between
RNase A and a fluorogenic substrate.
[0061] FIG. 41 shows a photograph (middle and right side) of the
water droplet region of the microfluidic network (T stands for
time), as well as a diagram of the microfluidic network (left
side).
[0062] FIG. 42 shows a schematic diagram (left side) of a
microfluidic network and a photograph (right side) of the ink plug
region of the microfluidic network in which the gradients were
formed by varying the flow rates.
[0063] FIG. 43 shows a schematic diagram (left side) of a
microfluidic network and a photograph (right side) of lysozyme
crystals formed in the microfluidic network using gradients.
[0064] FIG. 44 are schematic illustrations showing how an initial
gradient may be created by injecting a discrete aqueous sample of a
reagent B into a flowing stream of water.
[0065] FIG. 45a) shows a schematic of the microfluidic network used
to demonstrate that on-chip dilutions can be accomplished by
varying the flow rates of the reagents. The blue rectangle outlines
the field of view for images shown in FIG. 45c)-d). FIG. 45b) shows
a graph quantifying this dilution method by measuring fluorescence
of a solution of fluorescein diluted in plugs in the
microchannel.
[0066] FIG. 46 shows a microbatch protein crystallization analogue
scheme using a with a substrate that includes capillary tubing.
[0067] FIG. 47a) shows a lysozyme crystal grown attached to a
capillary tube wall.
[0068] FIG. 47b) shows a thaumatin crystal grown at the interface
of protein solution and oil.
[0069] FIG. 48a) shows a schematic illustration of a process for
direct screening of crystals in a capillary tube by x-ray
diffraction.
[0070] FIG. 48b) shows an x-ray diffraction pattern from a
thaumatin crystal grown inside a capillary tube using a microbatch
analogue method (no evaporation).
[0071] FIG. 49 shows a vapor-diffusion protein crystallization
analogue scheme with a substrate that includes capillary
tubing.
[0072] FIG. 50a) shows vapor diffusion in droplets surrounded by
FMS-121 inside a capillary right after the flow was stopped and the
capillary was sealed.
[0073] FIG. 50b) shows vapor diffusion in droplets surrounded by
FMS-121 inside a capillary 5 days after the flow was stopped and
the capillary was sealed.
[0074] FIG. 51a) shows a schematic drawing of an experimental setup
to form alternating droplets.
[0075] FIG. 51b) shows a schematic drawing of an experimental setup
to form alternating droplets where instead of single solutions 1
and 2, a set of multiple solutions A and B can be used in a similar
system.
[0076] FIG. 51c) shows a microphotograph illustrating the formation
of alternating NaCl--Fe(SCN).sub.3--NaCl droplets.
[0077] FIG. 52a) shows another example of generating alternating
droplets from two different aqueous solutions.
[0078] FIG. 52b) shows a microphotograph illustrating the formation
of alternating NaCl--Fe(SCN).sub.3--NaCl droplets.
[0079] FIG. 53a-c) shows several representative geometries in which
alternating plugs may be formed.
[0080] FIG. 54a-b) illustrates two representative geometries for
indexing a component in a plug using markers.
DETAILED DESCRIPTION ACCORDING TO THE INVENTION
[0081] The term "analysis" generally refers to a process or step
involving physical, chemical, biochemical, or biological analysis
that includes characterization, testing, measurement, optimization,
separation, synthesis, addition, filtration, dissolution, or
mixing.
[0082] The term "analysis unit" refers to a part of or a location
in a substrate or channel wherein a chemical undergoes one or more
types of analyses.
[0083] The term "capillary tube" refers to a hollow, tube-shaped
structure with a bore. The cross-sections of the tube and bore can
be round, square or rectangular. The corners of the tube or bore
can also be rounded. The bore diameters can range in size from
1.mu. to several millimeters; the outer diameters can be between
about 60 .mu.m up to several millimeters. The tube can be made
using any material suitable for x-ray diffraction analysis (e.g.,
silica, plastic, etc.), and can additionally include coatings (e.g.
polyimide) suitable for use under variable (e.g, high) temperatures
or for UV transparency.
[0084] The term "carrier-fluid" refers to a fluid that is
immiscible with a plug-fluid. The carrier-fluid may comprise a
substance having both polar and non-polar groups or moieties.
[0085] The term "channel" refers to a conduit that is typically
enclosed, although it may be at least partially open, and that
allows the passage through it of one or more types of substances or
mixtures, which may be homogeneous or heterogeneous, including
compounds, solvents, solutions, emulsions, or dispersions, any one
of which may be in the solid, liquid, or gaseous phase. A channel
can assume any form or shape such as tubular or cylindrical, a
uniform or variable (e.g., tapered) diameter along its length, and
one or more cross-sectional shapes along its length such as
rectangular, circular, or triangular. A channel is typically made
of a suitable material such as a polymer, metal, glass, composite,
or other relatively inert materials. As used herein, the term
"channel" includes microchannels that are of dimensions suitable
for use in devices. A network of channels refers to a multiplicity
of channels that are typically connected or in communication with
each other. A channel may be connected to at least one other
channel through another type of conduit such as a valve.
[0086] The term "chemical" refers to a substance, compound,
mixture, solution, emulsion, dispersion, molecule, ion, dimer,
macromolecule such as a polymer or protein, biomolecule,
precipitate, crystal, chemical moiety or group, particle,
nanoparticle, reagent, reaction product, solvent, or fluid any one
of which may exist in the solid, liquid, or gaseous state, and
which is typically the subject of an analysis.
[0087] The term "detection region" refers to a part of or a
location in a substrate or channel wherein a chemical is
identified, measured, or sorted based on a predetermined property
or characteristic.
[0088] The term "device" refers to a device fabricated or
manufactured using techniques such as wet or dry etching and/or
conventional lithographic techniques or a micromachining technology
such as soft lithography. As used herein, the term "devices"
includes those that are called, known, or classified as
microfabricated devices. A device according to the invention may
have dimensions between about 0.3 cm to about 15 (for 6 inch wafer)
cm per side and between about 1 micrometer to about 1 cm thick, but
the dimensions of the device may also lie outside these ranges.
[0089] The term "discrimination region" refers to a part of or a
location in a substrate or channel wherein the flow of a fluid can
change direction to enter at least one other channel such as a
branch channel.
[0090] The term "downstream" refers to a position relative to an
initial position which is reached after the fluid flows past the
initial point. In a circulating flow device, downstream refers to a
position farther along the flow path of the fluid before it crosses
the initial point again. "Upstream" refers to a point in the flow
path of a fluid that the fluid reaches or passes before it reaches
or passes a given initial point in a substrate or device.
[0091] The term "flow" means any movement of a solid or a fluid
such as a liquid. For example, the movement of plug-fluid,
carrier-fluid, or a plug in a substrate, or component of a
substrate according to the invention, or in a substrate or
component of a substrate involving a method according to the
invention, e.g., through channels of a microfluidic substrate
according to the invention, comprises a flow. The application of
any force may be used to provide a flow, including without
limitation: pressure, capillary action, electro-osmosis,
electrophoresis, dielectrophoresis, optical tweezers, and
combinations thereof, without regard for any particular theory or
mechanism of action.
[0092] The term "immiscible" refers to the resistance to mixing of
at least two phases or fluids under a given condition or set of
conditions (e.g., temperature and/or pressure) such that the at
least two phases or fluids persist or remain at least partially
separated even after the phases have undergone some type of
mechanical or physical agitation. Phases or fluids that are
immiscible are typically physically and/or chemically discernible,
or they may be separated at least to a certain extent.
[0093] The term "inlet port" refers to an area of a substrate that
receives plug-fluids. The inlet port may contain an inlet channel,
a well or reservoir, an opening, and other features that facilitate
the entry of chemicals into the substrate. A substrate may contain
more than one inlet port if desired. The inlet port can be in fluid
communication with a channel or separated from the channel by a
valve.
[0094] The term "nanoparticles" refers to atomic, molecular or
macromolecular particles typically in the length scale of
approximately 1-100 nanometer range. Typically, the novel and
differentiating properties and functions of nanoparticles are
observed or developed at a critical length scale of matter
typically under 100 nm. Nanoparticles may be used in constructing
nanoscale structures and they may be integrated into larger
material components, systems and architectures. In some particular
cases, the critical length scale for novel properties and phenomena
involving nanoparticles may be under 1 nm (e.g., manipulation of
atoms at approximately 0.1 nm) or it may be larger than 100 nm
(e.g., nanoparticle reinforced polymers have the unique feature at
approximately 200-300 nm as a function of the local bridges or
bonds between the nanoparticles and the polymer).
[0095] The term "nucleation composition" refers to a substance or
mixture that includes one or more nuclei capable of growing into a
crystal under conditions suitable for crystal formation. A
nucleation composition may, for example, be induced to undergo
crystallization by evaporation, changes in reagent concentration,
adding a substance such as a precipitant, seeding with a solid
material, mechanical agitation, or scratching of a surface in
contact with the nucleation composition.
[0096] The term "outlet port" refers to an area of a substrate that
collects or dispenses the plug-fluid, carrier-fluid, plugs or
reaction product. A substrate may contain more than one outlet port
if desired.
[0097] The term "particles" means any discrete form or unit of
matter. The term "particle" or "particles" includes atoms,
molecules, ions, dimers, polymers, or biomolecules.
[0098] The term "particulate" refers to a cluster or agglomeration
of particles such as atoms, molecules, ions, dimers, polymers, or
biomolecules. Particulates may comprise solid matter or be
substantially solid, but they may also be porous or partially
hollow. They may contain a liquid or gas. In addition, particulates
may be homogeneous or heterogeneous, that is, they may comprise one
or more substances or materials.
[0099] "Plugs" in accordance with the present invention are formed
in a substrate when a stream of at least one plug-fluid is
introduced into the flow of a carrier-fluid in which it is
substantially immiscible. The flow of the fluids in the device is
induced by a driving force or stimulus that arises, directly or
indirectly, from the presence or application of, for example,
pressure, radiation, heat, vibration, sound waves, an electric
field, or a magnetic field. Plugs in accordance with the present
invention may vary in size but when formed, their cross-section
should be substantially similar to the cross-section of the
channels in which they are formed. When plugs merge or get trapped
inside plug traps, the cross-section of the plugs may change. For
example, when a plug enters a wider channel, its cross-section
typically increases.
[0100] Further, plugs in accordance with the present invention may
vary in shape, and for example may be spherical or non-spherical.
The shape of the plug may be independent of the shape of the
channel (e.g., a plug may be a deformed sphere traveling in a
rectangular channel). The plugs may be in the form of plugs
comprising an aqueous plug-fluid containing one or more reagents
and/or one or more products formed from a reaction of the reagents,
wherein the aqueous plug-fluid is surrounded by a non-polar or
hydrophobic fluid such as an oil. The plugs may also be in the form
of plugs comprising mainly a non-polar or hydrophobic fluid which
is surrounded by an aqueous fluid. The plugs may be encased by one
or more layers of molecules that comprise both hydrophobic and
hydrophilic groups or moieties. The term "plugs" also includes
plugs comprising one or more smaller plugs, that is,
plugs-within-plugs. The relative amounts of reagents and reaction
products contained in the plugs at any given time depend on factors
such as the extent of a reaction occurring within the plugs.
Preferably, plugs contain a mixture of at least two plug
fluids.
[0101] The term "plug-forming region" refers to a junction between
an inlet port and the first channel of a substrate according to the
invention. Preferably, the fluid introduced into the inlet port is
"incompatible" (i.e., immiscible) with the fluid in the first
channel so that plugs of the fluid formed in the plug-forming
region are entrained into the stream of fluid from the first
channel.
[0102] The term "plug-fluid" refers to a fluid wherein or using
which a reaction or precipitation can occur. Typically, the
plug-fluid contains a solvent and a reagent although in some
embodiments at least one plug-fluid may not contain a reagent. The
reagent may be soluble or insoluble in the solvent. The plug-fluid
may contain a surfactant. At least two different plug-fluids are
used in the present invention. When both plug-fluids contain
reagents, the fluids are typically miscible, but can also be
partially immiscible, so long as the reagents within each
plug-fluid can react to form at least one product or
intermediate.
[0103] The term "polymer" means any substance or compound that is
composed of two or more building blocks (`mers`) that are
repetitively linked to each other. For example, a "dimer" is a
compound in which two building blocks have been joined together.
Polymers include both condensation and addition polymers. Typical
examples of condensation polymers include polyamide, polyester,
protein, wool, silk, polyurethane, cellulose, and polysiloxane.
Examples of addition polymers are polyethylene, polyisobutylene,
polyacrylonitrile, poly(vinyl chloride), and polystyrene. Other
examples include polymers having enhanced electrical or optical
properties (e.g., a nonlinear optical property) such as
electroconductive or photorefractive polymers. Polymers include
both linear and branched polymers.
[0104] The term "protein" generally refers to a set of amino acids
linked together usually in a specific sequence. A protein can be
either naturally-occurring or man-made. As used herein, the term
"protein" includes amino acid sequences that have been modified to
contain moieties or groups such as sugars, polymers, metalloorganic
groups, fluorescent or light-emitting groups, moieties or groups
that enhance or participate in a process such as intramolecular or
intermolecular electron transfer, moieties or groups that
facilitate or induce a protein into assuming a particular
conformation or series of conformations, moieties or groups that
hinder or inhibit a protein from assuming a particular conformation
or series of conformations, moieties or groups that induce,
enhance, or inhibit protein folding, or other moieties or groups
that are incorporated into the amino acid sequence and that are
intended to modify the sequence's chemical, biochemical, or
biological properties. As used herein, a protein includes, but is
not limited to, enzymes, structural elements, antibodies, hormones,
electron carriers, and other macromolecules that are involved in
processes such as cellular processes or activities. Proteins
typically have up to four structural levels that include primary,
secondary, tertiary, and quaternary structures.
[0105] The term "reaction" refers to a physical, chemical,
biochemical, or biological transformation that involves at least
one chemical, e.g., reactant, reagent, phase, carrier-fluid, or
plug-fluid and that generally involves (in the case of chemical,
biochemical, and biological transformations) the breaking or
formation of one or more bonds such as covalent, noncovalent, van
der Waals, hydrogen, or ionic bonds. The term includes typical
chemical reactions such as synthesis reactions, neutralization
reactions, decomposition reactions, displacement reactions,
reduction-oxidation reactions, precipitation, crystallization,
combustion reactions, and polymerization reactions, as well as
covalent and noncovalent binding, phase change, color change, phase
formation, crystallization, dissolution, light emission, changes of
light absorption or emissive properties, temperature change or heat
absorption or emission, conformational change, and folding or
unfolding of a macromolecule such as a protein.
[0106] The term "reagent" refers to a component of a plug-fluid
that undergoes or participates (e.g., by influencing the rate of a
reaction or position of equilibrium) in at least one type of
reaction with one or more components of other plug-fluids or a
reagent-containing carrier-fluid in the substrate to produce one or
more reaction products or intermediates which may undergo a further
reaction or series of reactions. A reagent contained in a
plug-fluid may undergo a reaction in which a stimulus such as
radiation, heat, temperature or pressure change, ultrasonic wave,
or a catalyst induces a reaction to give rise to a transformation
of the reagent to another reagent, intermediate, or product. A
reagent may also undergo a reaction such as a phase change (e.g.,
precipitation) upon interaction with one or more components of
other plug-fluids or a reagent-containing carrier-fluid.
[0107] The term "substrate" refers to a layer or piece of material
from which devices or chips are prepared or manufactured. As used
herein, the term "substrate" includes any substrate fabricated
using any traditional or known microfabrication techniques. The
term "substrate" also refers either to an entire device or chip or
to a portion, area, or section of a device or chip which may or may
not be removable or detachable from the main body of the device or
chip. The substrate may be prepared from one or more materials such
as glass, silicon, silicone elastomer, and polymers including, but
not limited to, polypropylene or polyethylene.
[0108] The discussion below provides a detailed description of
various devices and methods according to the invention for forming
plugs, generating gradients in a series of plugs, varying the
concentration of reagents inside plugs, rapid mixing in plugs, and
scaling of mixing times. In particular, a detailed description of
methods for merging, splitting and/or sorting plugs using channels,
which form the bases for various applications ranging from the
manufacture and analysis of various products to applications in
electronics, medicine, diagnostics, and pharmaceuticals, to name a
few, is discussed. Methods of detection and measurement of, among
others, plugs and processes occurring within plugs are also
described.
[0109] Among the various applications involving the devices and
methods according to the invention are particle separation/sorting,
synthesis, investigation of nonlinear and stochastic systems,
nonlinear amplification using unstable autocatalytic mixtures, use
of stochastic chemical systems for chemical amplification, kinetic
measurements, time control of processes, increasing the dynamic
range of kinetic measurements, ultrafast measurements,
crystallization of proteins, and dynamic control of surface
chemistry.
[0110] In addition, the devices and methods according to the
invention offer a wide-range of other applications. For example,
the devices and methods according to the invention provide for
effective, rapid, and precise manipulation and monitoring of
solutions or reactions over a range of time scales (e.g., from tens
of microseconds, to hours or weeks in case of, for example,
crystallization) and over a range of solution volumes (e.g., from
femtoliters to hundreds of nanoliters).
[0111] In one aspect of the invention, the various devices and
methods according to the invention are used to overcome one or more
of the following problems involving microfluidics. First, the
substantial dispersion of solutes in microfluidic channels
increases reagent consumption and makes experiments or measurements
over long time scales (e.g., minutes to hours) difficult to
perform. Various devices and methods according to the invention are
intended to overcome this problem by localizing reagents inside
plugs that are encapsulated by an immiscible carrier-fluid.
[0112] Second, slow mixing of solutions renders experiments, tests,
or reactions involving very short time scales (e.g., tens of
milliseconds and below) either difficult or impossible to perform
with existing technologies. In addition, turbulence-based mixing
techniques prohibitively increase sample consumption. In accordance
with the present invention, this problem is preferably addressed by
conducting the mixing process inside plugs. Rather than relying on
turbulence, the various devices and methods according to the
invention preferably rely on chaotic advection to accelerate the
mixing process. An advantage provided by chaotic advection is that
it is expected to operate efficiently in both small and large
channels.
[0113] Third, achieving control over the chemistry of internal
surfaces of devices can be very important at small scales. Thus,
being able to control surface chemistry in small devices for
example is highly desirable. In accordance with the devices and
methods according to the invention, the surface chemistry to which
solutions are exposed is preferably controlled through a careful
selection of surfactants that are preferably designed to assemble
at the interface between the plugs and the immiscible fluid that
surrounds them.
[0114] Devices and methods of the invention are also provided for
use in traditional areas of microfluidics where, for example,
miniaturization and speed are important. Thus, the devices and
methods according to the invention may be used to develop various
tools such as those for high-throughput chemical or biophysical
measurements, chemical synthesis, particle formation, and protein
crystallization. They may also be used in high-throughput
screening, combinatorial synthesis, analysis, and diagnostics,
either as a self-contained platform, or in combination with
existing technologies particularly those that rely on the use of
immiscible fluid flows.
[0115] Importantly, the devices of the invention can be adapted to
work with automation and robotic technology. They may be used, for
example, as a basis for ultra-high throughput automated systems for
structural and functional characterization of biological molecules.
Thus, the various devices and methods according to the invention
provide rapid, economical, and accessible means of synthesis,
analysis, and measurements in the fields of biology, chemistry,
biophysics, bioengineering, and medicine (e.g., for
diagnostics).
[0116] The devices and methods of the invention have numerous other
possible applications. For example, chaotic mixing at low values of
Reynolds number can be exploited as an important tool for
controlling unstable chemical reactions. In addition, the systems
and devices of the invention may be used for controlling and/or
monitoring reactions that generate highly unstable (or explosive)
intermediates. They can also be valuable for controlling or
monitoring reactions or processes involving autocatalytic
reactions. For example, pure hydrogen peroxide (H.sub.2O.sub.2) is
an inexpensive and highly effective oxidant, but its autocatalytic
decomposition often leads to explosions upon storage and handling.
In the microfluidic systems of the invention, H.sub.2O.sub.2 is
preferably generated in-situ, stabilized by the chaotic flow, and
used to destroy chemical and biological warfare agents. Because the
unstable mixtures in these systems are localized inside plugs
formed in accordance with the invention, occasional autocatalytic
decomposition in one or more plugs is kept localized within those
plugs thereby preventing a catastrophic reaction involving the
whole system. In addition, large arrays of microfluidic reactors
may be operated in parallel to provide substantial throughput.
[0117] It is also possible to couple multiple autocatalytic
reactions in a single network using the devices and methods
according to the invention. For example, a sample plug could be
split into many smaller plugs and forwarded to individual
amplification cascades. Because the contents of the cascades'
outflows exhibit patterns that correspond to the patterns of
analytes present in these systems, these patterns could be analyzed
using artificial neural network (ANN) (Jackson, R. B. a. T. Neural
Computing: An Introduction, Hilger, N.Y., 1991; Zornetzer et al.,
An Introduction to Neural and Electronic Networks, Academic Press,
San Diego, Calif., 1990) algorithms. For example, patterns that
arise in blood or saliva analysis may correspond to certain normal
or abnormal (e.g., disease, fatigue, infection, poisoning)
conditions involving, for example, human and animals.
[0118] Moreover, it may be possible to create intelligent
microfluidic systems in accordance with the invention, where the
nonlinear chemical reactions perform not only detection, but also
analysis using ANN algorithms. For example, after amplification,
the channels of the present invention typically will contain
sufficient amounts of material to operate hydrogel-based valves
(Liu et al., "Fabrication and characterization of hydrogel-based
microvalves," J. Microelectromech. Syst. 2002, vol. 11, pp. 45-53;
Yu et al., "Responsive biomimetic hydrogel valve for
microfluidics," Appl. Phys. Lett. 2001, vol. 78, pp. 2589-2591;
Beebe et al., "Functional hydrogel structures for autonomous flow
control inside microfluidic channels," Nature, 2000, vol. 404,
588). These valves can be used to control flows inside the system
as a function of the sample plug composition. Feedforward and even
feedback (e.g., by using the hydrogel valves to control the flow of
the input streams) networks may thus be created and used for
analysis. Such nonlinear networks may be used not only to recognize
patterns pre-programmed by the connectivity of the channels
(Hjelmfelt et al., "Pattern-Recognition in Coupled Chemical Kinetic
Systems," Science, 1993, 260, 335-337) but also to learn patterns
by reconfiguring themselves (Jackson, R. B. a. T. Neural Computing:
An Introduction, Hilger, N.Y., 1991; Zornetzer et al., An
Introduction to Neural and Electronic Networks, Academic Press, San
Diego, Calif., 1990). Such intelligent microfluidic devices could
have unprecedented capabilities for fully autonomous detection,
analysis, and signal processing, perhaps surpassing those of
biological and current man-made systems.
[0119] The devices and methods of the invention are also useful in
genomics and proteomics, which are used to identify thousands of
new biomolecules that need to be characterized, or are available
only in minute quantities. In particular, the success of genomics
and proteomics has increased the demand for efficient,
high-throughput mechanisms for protein crystallization. X-ray
structure determination remains the predominant method of
structural characterization of proteins. However, despite
significant efforts to understand the process of crystallization,
macromolecular crystallization largely remains an empirical field,
with no general theory to guide a rational approach. As a result,
empirical screening has remained the most widely used method for
crystallizing proteins.
[0120] The following areas also provide applications of the devices
and methods according to the invention. For example, a number of
problems still beset high-throughput kinetics and protein
crystallization. When it comes to determining protein structure and
quantitatively ascertaining protein interactions, there are at
least two technological challenges: (1) most robotic technology
still only automate existing methods and are often too expensive
for a small research laboratory; and (2) there remains the need for
conceptually new methods that provide greater degree of control
over the crystallization process. In addition, setting up and
monitoring crystallization trials typically involve handling of
sub-microliter volumes of fluids over periods ranging from seconds
to days.
[0121] Thus, various devices and methods according to the present
invention are designed to provide novel and efficient means for
high-throughput crystallization of soluble and membrane proteins.
In addition to being a simple and economical method of setting up
thousands of crystallization trials in a matter of minutes, a
system according to the invention will enable unique time control
of processes such as the mixing and nucleation steps leading to
crystallization. A system according to the present invention may
also be used to control protein crystallization by controlling not
only short time-scale events such as nucleation but also long
time-scale events such as crystal growth.
[0122] Further, the devices and methods of the present invention
may be used in high-throughput, kinetic, and biophysical
measurements spanning the 10.sup.-5-10.sup.7 second time regime.
Preferably, the various devices and methods according to the
present invention require only between about a few nanoliters to
about a few microliters of each solution. Applications of such
devices and methods include studies of enzyme kinetics and RNA
folding, and nanoparticle characterization and synthesis, which are
discussed in detail below.
Channels and Devices
[0123] In one aspect of the invention, a device is provided that
includes one or more substrates comprising a first channel
comprising an inlet separated from an outlet; optionally, one or
more secondary channels (or branch channels) in fluid communication
with the first channel, at least one carrier-fluid reservoir in
fluid communication with the first channel, at least two plug-fluid
reservoirs in fluid communication with the first channel, and a
means for applying continuous pressure to a fluid within the
substrate.
[0124] A device according to the invention preferably comprises at
least one substrate.
[0125] A substrate may include one or more expansions or areas
along a channel wherein plugs can be trapped. The substrates of the
present invention may comprise an array of connected channels.
[0126] The device may have one or more outlet ports or inlet ports.
Each of the outlet and inlet ports may also communicate with a well
or reservoir. The inlet and outlet ports may be in fluid
communication with the channels or reservoirs that they are
connecting or may contain one or more valves. Fluid can be
introduced into the channels via the inlet by any means. Typically,
a syringe pump is used, wherein the flow rate of the fluid into the
inlet can be controlled.
[0127] A plug-forming region generally comprises a junction between
a plug-fluid inlet and a channel containing the carrier-fluid such
that plugs form which are substantially similar in size to each
other and which have cross-sections which are substantially similar
in size to the cross-section of the channel in the plug-forming
region. In one embodiment, the substrate may contain a plurality of
plug-forming regions.
[0128] The different plug-forming regions may each be connected to
the same or different channels of the substrate. Preferably, the
sample inlet intersects a first channel such that the pressurized
plug fluid is introduced into the first channel at an angle to a
stream of carrier-fluid passing through the first channel. For
example, in preferred embodiments, the sample inlet and first
channel intercept at a T-shaped junction; i.e., such that the
sample inlet is perpendicular (i.e. at an angle of 90.degree.) to
the first channel. However, the sample inlet may intercept the
first channel at any angle.
[0129] A first channel may in turn communicate with two or more
branch channels at another junction or "branch point", forming, for
example, a T-shape or a Y-shape. Other shapes and channel
geometries may be used as desired. In exemplary embodiments the
angle between intersecting channels is in the range of from about
60.degree. to about 120.degree.. Particular exemplary angles are
45.degree., 60.degree., 90.degree., and 120.degree.. Precise
boundaries for the discrimination region are not required, but are
preferred.
[0130] The first and branch channels of the present invention can,
each independently, be straight or have one or more bends. The
angle of a bend, relative to the substrate, can be greater than
about 10.degree., preferably greater than about 135.degree.,
180.degree., 270.degree., or 360.degree..
[0131] In one embodiment of the invention, a substrate comprises at
least one inlet port in communication with a first channel at or
near a plug-forming region, a detection region within or coincident
with all or a portion of the first channel or plug-forming region,
and a detector associated with the detection region. In certain
embodiments the device may have two or more plug-forming regions.
For example, embodiments are provided in which the analysis unit
has a first inlet port in communication with the first channel at a
first plug-forming region, a second inlet port in communication
with the first channel at a second plug-forming region (preferably
downstream from the first plug-forming region), and so forth.
[0132] In another embodiment, a substrate according to the
invention may comprise a first channel through which a pressurized
stream or flow of a carrier-fluid is passed, and two or more inlet
channels which intersect the first channel at plug-forming regions
and through which a pressurized stream or flow of plug fluids pass.
Preferably, these inlet channels are parallel to each other and
each intercept the first channel at a right angle. In specific
embodiments wherein the plugs introduced through the different plug
forming regions are mixed, the inlet channels are preferably close
together along the first channel. For example, the first channel
may have a diameter of 60 .mu.m that tapers to 30 .mu.m at or near
the plug-forming regions. The inlet channels then also preferably
have a diameter of about 30 .mu.m and, in embodiments where plug
mixing is preferred, are separated by a distance along the first
channel approximately equal to the diameter of the inlet channel
(i.e., about 30 .mu.m).
[0133] In an embodiment according to the invention, the substrate
also has a detection region along a channel. There may be a
plurality of detection regions and detectors, working independently
or together, e.g., to analyze one or more properties of a chemical
such as a reagent.
[0134] A detection region is within, communicating, or coincident
with a portion of a first channel at or downstream of the
plug-forming region and, in sorting embodiments, at or upstream of
the discrimination region or branch point. Precise boundaries for
the detection region are not required, but are preferred.
[0135] A typical substrate according to the invention comprises a
carrier-fluid inlet that is part of and feeds or communicates
directly with a first channel, along with one or more plug fluid
inlets in communication with the first channel at a plug-forming
region situated downstream from the main inlet (each different
plug-fluid inlet preferably communicates with the first channel at
a different plug-forming region).
[0136] Plugs formed from different plug-fluids or solutions may be
released in any order. For example, an aqueous solution containing
a first plug-fluid may be released through a first inlet at a first
plug-forming region. Subsequently, plugs of an aqueous second
plug-fluid may be released through a second inlet at a second
plug-forming region downstream of the first inlet.
Fabrication of Channels, Substrates, and Devices
[0137] The substrates and devices according to the invention are
fabricated, for example by etching a silicon substrate, chip, or
device using conventional photolithography techniques or
micromachining technology, including soft lithography. The
fabrication of microfluidic devices using polydimethylsiloxane has
been previously described. These and other fabrication methods may
be used to provide inexpensive miniaturized devices, and in the
case of soft lithography, can provide robust devices having
beneficial properties such as improved flexibility, stability, and
mechanical strength. Preferably, when optical detection is
employed, the invention also provides minimal light scatter from,
for example, plugs, carrier-fluid, and substrate material. Devices
according to the invention are relatively inexpensive and easy to
set up.
[0138] Machining methods (e.g., micromachining methods) that may be
used to fabricate channels, substrates, and devices according to
the invention are well known in the art and include film deposition
processes, such as spin coating and chemical vapor deposition,
laser fabrication or photolithographic techniques, or etching
methods, which may be performed either by wet chemical or plasma
processes.
[0139] Channels may be molded onto optically transparent silicone
rubber or polydimethylsiloxane (PDMS), preferably PDMS. This can be
done, for example, by casting the channels from a mold by etching
the negative image of these channels into the same type of
crystalline silicon wafer used in semiconductor fabrication. The
same or similar techniques for patterning semiconductor features
can be used to form the pattern of the channels. In one method of
channel fabrication, an uncured PDMS is poured onto the molds
placed in the bottom of, for example, a Petri dish. To accelerate
curing, the molds are preferably baked. After curing the PDMS, it
is removed from on top of the mold and trimmed. Holes may be cut
into the PDMS using, for example, a tool such as a cork borer or a
syringe needle. Before use, the PDMS channels may be placed in a
hot bath of HCl if it is desired to render the surface hydrophilic.
The PDMS channels can then be placed onto a microscope cover slip
(or any other suitable flat surface), which can be used to form the
base/floor or top of the channels.
[0140] A substrate according to the invention is preferably
fabricated from materials such as glass, polymers, silicon
microchip, or silicone elastomers. The dimensions of the substrate
may range, for example, between about 0.3 cm to about 7 cm per side
and about 1 micron to about 1 cm in thickness, but other dimensions
may be used.
[0141] A substrate can be fabricated with a fluid reservoir or well
at the inlet port, which is typically in fluid communication with
an inlet channel. A reservoir preferably facilitates introduction
of fluids into the substrate and into the first channel. An inlet
port may have an opening such as in the floor of the substrate to
permit entry of the sample into the device. The inlet port may also
contain a connector adapted to receive a suitable piece of tubing,
such as Teflon.RTM. tubing, liquid chromatography or HPLC tubing,
through which a fluid may be supplied. Such an arrangement
facilitates introducing the fluid under positive pressure in order
to achieve a desired pressure at the plug-forming region.
[0142] A substrate containing the fabricated flow channels and
other components is preferably covered and sealed, preferably with
a transparent cover, e.g., thin glass or quartz, although other
clear or opaque cover materials may be used. Silicon is a preferred
substrate material due to well-developed technology permitting its
precise and efficient fabrication, but other materials may be used,
including polymers such as polytetrafluoroethylenes. Analytical
devices having channels, valves, and other elements can be designed
and fabricated from various substrate materials. When external
radiation sources or detectors are employed, the detection region
is preferably covered with a clear cover material to allow optical
access to the fluid flow. For example, anodic bonding of a silicon
substrate to a PYREX.RTM. cover slip can be accomplished by washing
both components in an aqueous H.sub.2SO.sub.4/H.sub.2O.sub.2 bath,
rinsing in water, and then, for example, heating to about
350.degree. C. while applying a voltage of 450 V.
[0143] A variety of channels for sample flow and mixing can be
fabricated on the substrate and can be positioned at any location
on the substrate, chip, or device as the detection and
discrimination or sorting points. Channels can also be designed
into the substrate that place the fluid flow at different
times/distances into a field of view of a detector. Channels can
also be designed to merge or split fluid flows at precise
times/distances.
[0144] A group of manifolds (a region consisting of several
channels that lead to or from a common channel) can be included to
facilitate the movement of plugs from different analysis units,
through the plurality of branch channels and to the appropriate
solution outlet. Manifolds are preferably fabricated into the
substrate at different depth levels. Thus, devices according to the
invention may have a plurality of analysis units that can collect
the solution from associated branch channels of each unit into a
manifold, which routes the flow of solution to an outlet. The
outlet can be adapted for receiving, for example, a segment of
tubing or a sample tube, such as a standard 1.5 ml centrifuge tube.
Collection can also be done using micropipettes.
Methods of Forming Plugs
[0145] The various channels, substrates, and devices according to
the invention are primarily used to form and manipulate plugs.
[0146] In a preferred embodiment, plug-fluids do not significantly
mix at or before they are introduced into the first channel. The
plug-fluids may form distinct laminar streams at or before the
inlet. They may be separated by an additional fluid. Alternatively,
they may be introduced into the carrier-fluid via inlets of
differing size. The concentration of plug-fluids in the plugs may
be adjusted by adjusting volumetric flow rates of the plug-fluids.
Further, the diameters of the first channel and the branch
channel(s) may differ.
[0147] FIG. 2A is a schematic diagram contrasting laminar flow
transport and plug transport in a channel. In the lower figure
which depicts the transport of plugs, two aqueous reagents (marked
in red and blue) form laminar streams that are separated by a
"divider" aqueous stream. The three streams enter a channel with
flowing oil, at which point plugs form and plug fluids mix. During
plug transport, rapid mixing of the plug-fluids typically occurs
within the plugs. In contrast, in laminar flow transport, fluid
mixing occurs slowly, and with high dispersion, as shown in the
upper figure. In the upper figure, the time t at a given point
d.sub.1 can be estimated from t.sub.1.apprxeq.d.sub.1/U, where d1
is the distance from d=0 and U is the flow velocity. In the lower
figure, the time t is given by t.sub.1=d.sub.1/U.
[0148] FIG. 2B shows a photograph and a schematic diagram that
depict mixing in water/oil plugs (upper schematic and photograph)
and in laminar streams (lower schematic and photograph) comprising
only aqueous plug-fluids. The oil (carrier-fluid in this case) is
introduced into channel 200 of a substrate. Instead of oil, water
is introduced into the corresponding channel 207 in the case of
mixing using laminar streams. The three aqueous plug-fluids are
introduced by inlet ports 201, 202, 203 into the carrier-fluid (and
by inlet ports 204, 205, 206 in the case of laminar streams). A
preferred scheme is one in which the aqueous plug-fluids initially
coflow preferably along a short or minimal distance before coming
in contact with the carrier-fluid. In a preferred embodiment, the
distance traversed by the coflowing plug-fluids is approximately or
substantially equal to the width of the channel.
[0149] The middle or second aqueous plug-fluid in the top figure
may be plain water, buffer, solvent, or a different plug-fluid. The
middle aqueous plug-fluid would preferably initially separate the
two other aqueous plug-fluids before the aqueous fluids come into
contact with the carrier-fluid. Thus, the intervening aqueous
plug-fluid would prevent, delay, or minimize the reaction or mixing
of the two outer aqueous plug-fluids before they come in contact
with the carrier-fluid. The plugs that form in the plug-forming
region can continue along an unbranched channel, can split and
enter a channel, can merge with plugs from another channel, or can
exit the substrate through an exit port. It can be seen in FIG. 2
that, in the absence of an oil, the aqueous plug-fluids flow in
laminar streams without significant mixing or with only partial
mixing. In contrast, plug-fluids mix substantially or completely in
the plugs.
[0150] FIG. 3 shows photographs and schematic diagrams that depict
a stream of plugs from an aqueous plug-fluid and an oil
(carrier-fluid) in curved channels at flow rates of 0.5 .mu.L/min
(top schematic diagram and photograph) and 1.0 .mu.L/min (bottom
schematic diagram and photograph). This scheme allows enhanced
mixing of reagents in the elongated plugs flowing along a curved
channel with smooth corners or curves. The carrier-fluid is
introduced into an inlet port 300, 307 of a substrate while the
three aqueous plug-fluids are introduced in separate inlet ports
301-306. As in FIG. 2, a preferred scheme would be one in which the
plug-fluids initially coflow preferably along a short or minimal
distance before coming in contact with the carrier-fluid. In a
preferred embodiment, the distance traversed by the coflowing
plug-fluids (e.g., aqueous plug-fluids) is approximately or
substantially equal to the width of the channel. The middle or
second aqueous plug-fluid may comprise plain water, buffer,
solvent, or a plug-fluid, and the middle aqueous plug-fluid
preferably initially separates the two other aqueous plug-fluids
before the aqueous plug-fluids come into contact with the
carrier-fluid which, in this case, is an oil. Thus, the intervening
aqueous plug-fluid would prevent, delay, or minimize the reaction
or mixing of the two outer aqueous plug-fluids before they come in
contact with the oil (or carrier-fluid).
[0151] FIG. 4 shows a photograph and schematic diagram that
illustrate plug formation through the injection of oil and multiple
plug-fluids. Although FIG. 4 shows five separate plug-fluids, one
may also separately introduce less than or more than five
plug-fluids into the substrate. The reagents or solvents comprising
the plug-fluids may be different or some of them may be identical
or similar. As in FIG. 2, the oil is introduced into an inlet port
400 of a substrate while the aqueous plug-fluid is introduced in
separate inlet ports 401-405. The water plugs then flow through
exit 406. A preferred scheme is one in which the aqueous
plug-fluids would initially coflow preferably along a short or
minimal distance before coming in contact with the oil. In a
preferred embodiment, the distance traversed by the coflowing
plug-fluids is approximately or substantially equal to the width of
the channel. One or more of the aqueous plug-fluids may comprise
plain water, buffer, solvent, or a plug-fluid, and at least one
aqueous plug-fluid would preferably initially separate at least two
other aqueous streams before the aqueous plug-fluid comes into
contact with the oil. Thus, the at least one intervening aqueous
plug-fluid would prevent, delay, or minimize the reaction or mixing
of the two outer aqueous streams before the aqueous streams come in
contact with the oil. FIG. 5 shows a microfluidic network, which is
similar to that shown in FIG. 4, in which several reagents can be
introduced into the multiple inlets. In addition, FIG. 5 shows a
channel having a winding portion through which the plugs undergo
mixing of the four reagents A, B, C, and D. As shown in FIG. 5, the
reagents A, B, C, and D are introduced into inlet ports 501, 503,
505, and 507, while aqueous streams are introduced into inlet ports
502, 504, 506. FIG. 5 shows plugs through the various stages of
mixing, wherein mixture 50 corresponds to the initial A+B mixture,
mixture 51 corresponds to the initial C+D mixture, mixture 52
corresponds to the mixed A+B mixture, mixture 53 corresponds to the
mixed C+D mixture, and mixture 54 corresponds to the A+B+C+D
mixture.
[0152] The formation of the plugs preferentially occurs at low
values of the capillary number C.n., which is given by the
equation
C.n.=U.mu./.gamma. Eqn. (1)
where U is the flow velocity, .mu. is the viscosity of the plug
fluid or carrier-fluid, and .gamma. is the surface tension at the
water/surfactant interface.
[0153] The plugs may be formed using solvents of differing or
substantially identical viscosities. Preferably, the conditions and
parameters used in an experiment or reaction are such that the
resulting capillary number lies in the range of about
0.001.ltoreq.C.n..ltoreq.about 10. Preferably, the values of
parameters such as viscosities and velocities are such that plugs
can be formed reliably. Without wishing to be bound by theory, it
is believed that as long as flow is not stopped, the C.n. is
.ltoreq.about 0.2, and as long as the surface tension of the
plug-fluid/carrier-fluid interface is lower than the surface
tension of the solution/wall interface, plug formation will
persist. The C.n. number is zero when flow is stopped.
[0154] In one embodiment, in which perfluorodecaline was used as
the carrier-fluid and the plug-fluid was aqueous, it was found that
this system can be operated at values of C.n. up to .about.0.1 (at
300 mm s.sup.-1). In this system, as the value of the C.n.
increased above .about.0.2, the formation of plugs became
irregular. The viscosity of perfluorodecaline is
5.10.times.10.sup.-3 kg m.sup.-3 s.sup.-1, the surface tension at
the interface between the plugs and the carrier-fluid was
13.times.10.sup.-3 N m.sup.-1.
[0155] The length of the plugs can be controlled such that their
sizes can range from, for example, about 1 to 4 times a
cross-sectional dimension (d, where d is a channel cross-sectional
dimension) of a channel using techniques such as varying the ratio
of the plug-fluids and carrier-fluids or varying the relative
volumetric flow rates of the plug-fluid and carrier-fluid streams.
Short plugs tend to form when the flow rate of the aqueous stream
is lower than that of a carrier-fluid stream. Long plugs tend to
form when the flow rate of the plug-fluid stream is higher than
that of the carrier stream.
[0156] In one approximation, the volume of a plug is taken equal to
about 2.times.d.sup.3, where d is a cross-sectional dimension of a
channel. Thus, the plugs can be formed in channels having
cross-sectional areas of, for example, from 20.times.20 to
200.times.200 .mu.m.sup.2, which correspond to plug volumes of
between about 16 picoliters (pL) to 16 nanoliters (nL). The size of
channels may be increased to about 500 .mu.m (corresponding to a
volume of about 250 nL) or more. The channel size can be reduced
to, for example, about 1 .mu.m (corresponding to a volume of about
1 femtoliter). Larger plugs are particularly useful for certain
applications such as protein crystallizations, while the smaller
plugs are particularly useful in applications such as ultrafast
kinetic measurements.
[0157] In one preferred embodiment, plugs conform to the size and
shape of the channels while maintaining their respective volumes.
Thus, as plugs move from a wider channel to a narrower channel they
preferably become longer and thinner, and vice versa.
[0158] Plug-fluids may comprise a solvent and optionally, a
reactant. Suitable solvents for use in the invention, such as those
used in plug-fluids, include organic solvents, aqueous solvents,
oils, or mixtures of the same or different types of solvents, e.g.
methanol and ethanol, or methanol and water. The solvents according
to the invention include polar and non-polar solvents, including
those of intermediate polarity relative to polar and non-polar
solvents. In a preferred embodiment, the solvent may be an aqueous
buffer solution, such as ultrapure water (e.g., 18 M.OMEGA.
resistivity, obtained, for example, by column chromatography), 10
mM Tris HCl, and 1 mM EDTA (TE) buffer, phosphate buffer saline or
acetate buffer. Other solvents that are compatible with the
reagents may also be used.
[0159] Suitable reactants for use in the invention include
synthetic small molecules, biological molecules (i.e., proteins,
DNA, RNA, carbohydrates, sugars, etc.), metals and metal ions, and
the like.
[0160] The concentration of reagents in a plug can be varied. In
one embodiment according to the invention, the reagent
concentration may be adjusted to be dilute enough that most of the
plugs contain no more than a single molecule or particle, with only
a small statistical chance that a plug will contain two or more
molecules or particles. In other embodiments, the reagent
concentration in the plug-fluid is adjusted to concentrate enough
that the amount of reaction product can be maximized.
[0161] Suitable carrier-fluids include oils, preferably fluorinated
oils. Examples include viscous fluids, such as perfluorodecaline or
perfluoroperhydrophenanthrene; nonviscous fluids such as
perfluorohexane; and mixtures thereof (which are particularly
useful for matching viscosities of the carrier-fluids and
plug-fluids). Commercially available fluorinated compounds such as
Fluorinert.TM. liquids (3M, St. Paul, Minn.) can also be used.
[0162] The carrier-fluid or plug-fluid, or both may contain
additives, such as agents that reduce surface tensions (e.g.,
surfactants). Other agents that are soluble in a carrier-fluid
relative to a plug-fluid can also be used when the presence of a
surfactant in the plug fluid is not desirable. Surfactants may be
used to facilitate the control and optimization of plug size, flow
and uniformity. For example, surfactants can be used to reduce the
shear force needed to extrude or inject plugs into an intersecting
channel. Surfactants may affect plug volume or periodicity, or the
rate or frequency at which plugs break off into an intersecting
channel. In addition, surfactants can be used to control the
wetting of the channel walls by fluids. In one embodiment according
to the invention, at least one of the plug-fluids comprises at
least one surfactant.
[0163] Preferred surfactants that may be used include, but are not
limited to, surfactants such as those that are compatible with the
carrier and plug-fluids. Exemplary surfactants include Tween.TM.,
Span.TM., and fluorinated surfactants (such as Zonyl.TM. (Dupont,
Wilmington Del.)). For example, fluorinated surfactants, such as
those with a hydrophilic head group, are preferred when the
carrier-fluid is a fluorinated fluid and the plug-fluid is an
aqueous solution.
[0164] However, some surfactants may be less preferable in certain
applications. For instance, in those cases where aqueous plugs are
used as microreactors for chemical reactions (including biochemical
reactions) or are used to analyze and/or sort biomaterials, a water
soluble surfactant such as SDS may denature or inactivate the
contents of the plug.
[0165] The carrier-fluid preferably wets the walls of the channels
preferentially over the plugs. If this condition is satisfied, the
plug typically does not come in contact with the walls of the
channels, and instead remains separated from the walls by a thin
layer of the carrier-fluid. Under this condition, the plugs remain
stable and do not leave behind any residue as they are transported
through the channels. The carrier-fluid's preferential wetting of
the channel walls over the plug-fluid is achieved preferably by
setting the surface tension by, for example, a suitable choice of
surfactant. Preferably, the surface tension at a plug fluid/channel
wall interface (e.g., about 38 mN/m surface tension for a
water/PDMS interface) is set higher than the surface tension at a
plug fluid/carrier-fluid interface (e.g., about 13 mN/m for a
water/carrier-fluid interface with a surfactant such as 10% 1H,
1H,2H,2H-perfluorooctanol in perfluorodecaline as the
carrier-fluid). If this condition is not satisfied, plugs tend to
adhere to the channel walls and do not undergo smooth transport
(e.g., in the absence of 1H,1H,2H,2H-perfluorooctanol the surface
tension at the water/perfluorodecaline interface is about 55 mN/m,
which is higher than the surface tension of the water/PDMS
interface (e.g., about 38 mN/m)), and plugs adhere to the walls of
the PDMS channels. Because the walls of the channels (PDMS, not
fluorinated) and the carrier-fluid (fluorinated oil) are
substantially different chemically, when a fluorinated surfactant
is introduced, the surfactant reduces the surface tension at the
oil-water interface preferentially over the wall-water interface.
This allows the formation of plugs that do not stick to the channel
walls.
[0166] The surface tension at an interface may be measured using
what is known as a hanging drop method, although one may also use
other methods. Preferably, the surface tension is sufficiently high
to avoid destruction of the plugs by shear.
[0167] The plug-fluids and carrier-fluids may be introduced through
one or more inlets. Specifically, fluids may be introduced into the
substrate through pneumatically driven syringe reservoirs that
contain either the plug-fluid or carrier-fluid. Plugs may be
produced in the carrier-fluid stream by modifying the relative
pressures such that the plug-fluids contact the carrier-fluid in
the plug-forming regions then shear off into discrete plugs.
[0168] In the invention, plugs are formed by introducing the
plug-fluid, at the plug-forming region, into the flow of
carrier-fluid passing through the first channel. The force and
direction of flow can be controlled by any desired method for
controlling flow, for example, by a pressure differential, or by
valve action. This permits the movement of the plugs into one or
more desired branch channels or outlet ports.
[0169] In preferred embodiments according to the invention, one or
more plugs are detected, analyzed, characterized, or sorted
dynamically in a flow stream of microscopic dimensions based on the
detection or measurement of a physical or chemical characteristic,
marker, property, or tag.
[0170] The flow stream in the first channel is typically, but not
necessarily continuous and may be stopped and started, reversed or
changed in speed. Prior to sorting, a non-plug-fluid can be
introduced into a sample inlet port (such as an inlet well or
channel) and directed through the plug-forming region, e.g., by
capillary action, to hydrate and prepare the device for use.
Likewise, buffer or oil can also be introduced into a main inlet
port that communicates directly with the first channel to purge the
substrate (e.g., of "dead" air) and prepare it for use. If desired,
the pressure can be adjusted or equalized, for example, by adding
buffer or oil to an outlet port.
[0171] The pressure at the plug-forming region can also be
regulated by adjusting the pressure on the main and sample inlets,
for example with pressurized syringes feeding into those inlets. By
controlling the difference between the oil and water flow rates at
the plug-forming region, the size and periodicity of the plugs
generated may be regulated. Alternatively, a valve may be placed at
or coincident to either the plug-forming region or the sample inlet
connected thereto to control the flow of solution into the
plug-forming region, thereby controlling the size and periodicity
of the plugs. Periodicity and plug volume may also depend on
channel diameter and/or the viscosity of the fluids.
Mixing in Plugs
[0172] FIG. 7(a)-(b) show microphotographs (10 .mu.s exposure)
illustrating rapid mixing inside plugs (a) and negligible mixing in
a laminar flow (b) moving through winding channels at the same
total flow velocity. Aqueous streams were introduced into inlets
700-705 in FIGS. 7(a)-(b). In FIGS. 7(c) and 7(e), Fluo-4 was
introduced into inlets 706, 709, buffer was introduced into inlets
707, 710, and CaCl.sub.2 was introduced into inlets 708, 711. FIG.
7(c) shows a false-color microphotograph (2 s exposure, individual
plugs are invisible) showing time-averaged fluorescence arising
from rapid mixing inside plugs of solutions of Fluo-4 (54 .mu.M)
and CaCl.sub.2 (70 .mu.M) in aqueous sodium morpholine
propanesulfonate buffer (20 .mu.M, pH 7.2); this buffer was also
used as the middle aqueous stream. FIG. 7(d) shows a plot of the
relative normalized intensity (I) of fluorescence obtained from
images such as shown in (c) as a function of distance (left)
traveled by the plugs and of time required to travel that distance
(right) at a given flow rate. The total intensity across the width
of the channel was measured. Total PFD/water volumetric flow rates
(in .mu.L min.sup.-1) were 0.6:0.3, 1.0:0.6, 12.3:3.7, 10:6, and
20:6. FIG. 7(e) shows a false-color microphotograph (2 s exposure)
of the weak fluorescence arising from negligible mixing in a
laminar flow of the solutions used in (c). All channels were 45
.mu.m deep; inlet channels were 50 .mu.m and winding channels 28
.mu.m wide; Re.about.5.3 (water), .about.2.0 (PFD).
[0173] FIG. 8 shows photographs and schematics that illustrate fast
mixing at flow rates of about 0.5 .mu.L/min (top schematic diagram
and photograph) and about 1.0 .mu.L/min (lower schematic diagram
and photograph) using 90.degree.-step channels while FIG. 9
illustrates fast mixing at flow rates of about 1.0 .mu.L/min (top
schematic diagram and photograph) and about 0.5 .mu.L/min (lower
schematic diagram and photograph) using 135.degree.-step channels.
Aqueous streams are introduced into inlets 800-805 in FIG. 8
(inlets 900-905 in FIG. 9), while a carrier fluid is introduced
into channels 806, 807 (channels 906, 907 in FIG. 9). The plugs
that form then flow through exits 808, 809 (FIG. 8) and exits 908,
909 (FIG. 9). As can be seen in FIG. 8 and FIG. 9, the plugs are
transported along multi-step channels, instead of channels with
smooth curves (as opposed to channels with sharp corners). An
advantage of these multi-step configurations of channels is that
they may provide further enhanced mixing of the substances within
the plugs.
[0174] Several approaches may be used to accelerate or improve
mixing. These approaches may then be used to design channel
geometries that allow control of mixing. Flow can be controlled by
perturbing the flow inside a moving plug so that it differs from
the symmetric flow inside a plug that moves through a straight
channel. For example, flow perturbation can be accomplished by
varying the geometry of a channel (e.g., by using winding
channels), varying the composition of the plug fluid (e.g., varying
the viscosities), varying the composition of the carrier-fluid
(e.g., using several laminar streams of carrier-fluids that are
different in viscosity or surface tension to form plugs; in this
case, mixing is typically affected, and in some cases enhanced),
and varying the patterns on the channel walls (e.g., hydrophilic
and hydrophobic, or differentially charged, patches would interact
with moving plugs and induce time-periodic flow inside them, which
should enhance mixing).
[0175] Various channel designs can be implemented to enhance mixing
in plugs. FIG. 1A shows a schematic of a basic channel design,
while FIG. 1B shows a series of periodic variations of the basic
channel design. FIG. 1C shows a series of aperiodic combinations
resulting from a sequence of alternating elements taken from a
basic design element shown in FIG. 1A and an element from the
periodic variation series shown in FIGS. 1B(1)-(4). When the
effects of these periodic variations are visualized, aperiodic
combinations of these periodic variations are preferably used to
break the symmetries arising from periodic flows (see FIG. 1C).
Here, the relevant parameters are channel width, period, radius of
curvature, and sequence of turns based on the direction of the
turns. The parameters of the basic design are defined such that c
is the channel width, l is the period, and r is the radius of
curvature. For the basic design, the sequence can be defined as
(left, right, left, right), where left and right is relative to a
centerline along the path taken by a plug in the channel.
[0176] FIGS. 1B(1)-4) show schematic diagrams of a series of
periodic variations of the basic design. At least one variable
parameter is preferably defined based on the parameters defined in
FIG. 1a). In FIG. 1B(1), the channel width is c/2; in FIG. 1B(2),
the period is 2 l; and in FIG. 1B(3), and the radius of curvature
is 2r. In FIG. 1B(4), the radius of curvature is r/2 and the
sequence is (left, left, right, right).
[0177] FIGS. 1C(1)-(4) show a schematic diagram of a series of
aperiodic combinations formed by combining the basic design element
shown in FIG. 1A with an element from the series of periodic
variations in FIG. 1B(1)-(4). In FIG. 1C(1), the alternating
pattern of a period of the basic design shown in FIG. 1A (here
denoted as "a") and a period of the channel in FIG. 1B(1) (here
denoted as "b1") is given by a+b1+a+ . . . . In FIG. 1C(2), the
aperiodic combination is given by a+b2+a. In the channel shown in
FIG. 1C(3) (here denoted as "c3"), the aperiodic combination is
given by a+c3+a. In the channel shown in FIG. 1C(4) (here denoted
as "c4"), a (right, left) sequence is introduced with a kink in the
pattern. A repeating (left, right) sequence would normally be
observed. By adding this kink, the sequence becomes (left, right,
left, right)+(right, left)+(left, right, left, right).
[0178] Another approach for accelerating mixing relies on
rationally-designed chaotic flows on a microfluidic chip using what
is known as the baker's transformation. Reorientation of the fluid
is critical for achieving rapid mixing using the baker's
transformation. The baker's transformation leads to an exponential
decrease of the striation thickness (the distance over which mixing
would have to occur by diffusion) of the two components via a
sequence of stretching and folding operations. Typically, every
stretch-fold pair reduces the striation thickness by a factor of 2,
although this factor may have a different value. The striation
thickness (ST) can be represented, in an ideal case, by Eqn. (2)
below. Thus, in the ideal case, in a sequence of n
stretch-fold-reorient operations, the striation thickness undergoes
an exponential decrease given by
ST(t.sub.n)=ST(t.sub.0).times.2.sup.-n Eqn. (2)
where ST(t.sub.n) represents the striation thickness at time
t.sub.n, ST(t.sub.0) represents the initial striation thickness at
time t.sub.0, and n is the number of stretch-fold-reorient
operations.
[0179] In accordance with the invention, the baker's transformation
is preferably implemented by creating channels composed of a
sequence of straight regions and sharp turns. FIG. 11 shows a
schematic diagram of a channel geometry designed to implement and
visualize the baker's transformation of plugs flowing through
microfluidic channels. Other designs could also be used. The angles
at the channel bends and the lengths of the straight portions are
chosen so as to obtain optimal mixing corresponding to the flow
patterns shown. Different lengths of straight paths and different
turns may be used depending on the particular application or
reaction involved.
[0180] A plug traveling through every pair of straight part 112 and
sharp-turn part 111 of the channel, which is equivalent to one
period of a baker's transformation, will experience a series of
reorientation, stretching and folding. In a straight part of the
channel, a plug will experience the usual recirculating flow. At a
sharp turn, a plug normally rolls and reorients due to the much
higher pressure gradient across the sharp internal corner and also
due to larger travel path along the outside wall. This method of
mixing based on the baker's transformation is very efficient and is
thus one of the preferred types of mixing. In particular, this type
of mixing leads to a rapid reduction of the time required for
reagent mixing via diffusion.
[0181] It is believed that plug formation can be maintained at
about the same flow rate in channels of different sizes because the
limit of a flow rate is typically set by the capillary number,
C.n., which is independent of the channel size. At a fixed flow
rate, the mixing time t.sub.mix may decrease as the size of the
channel (d) is reduced. First, it is assumed that it takes the same
number n of stretch-fold-reorient cycles to mix reagents in both
large and small channels. This assumption (e.g., for n.about.5) is
in approximate agreement with previously measured mixing in d=55
and d=20 micrometer (.mu.m) channels. Each cycle requires a plug to
travel over a distance of approximately 2 lengths of the plug
(approximately 3d). Therefore, mixing time is expected to be
approximately equal to the time it takes to travel 15d, and will
decrease linearly with the size of the channel, t.sub.mix.about.d.
A method that provides mixing in about 1 ms in 25-.mu.m channels
preferably provides mixing in about 40 .mu.s in 1-.mu.m channels.
Achieving microsecond mixing times generally requires the use of
small channels. High pressures are normally required to drive a
flow through small channels.
[0182] Without wishing to be bound by theory, theoretical modeling
indicates that the number of cycles it takes for mixing to occur in
a channel with diameter d is given approximately by
n.times.2.sup.2n.apprxeq.dU/D Eqn. (3)
where n is the number of cycles, U is the flow velocity, D is the
diffusion constant, one cycle is assumed to be equal to 6d, and
mixing occurs when convection and diffusion time scales are
matched. The mixing time is primarily determined by the number of
cycles. This result indicates that mixing will be accelerated more
than just in direct proportion to the channel diameter. For
example, when d decreases by a factor of 10, mixing time decreases
by a factor of d.times.Log(d)=10.times.Log(10). With properly
designed channels, mixing times in 1-.mu.m channels can be limited
to about 20 .mu.s. Even at low flow rates or long channels (such as
those involving protein crystallization), however, significant
mixing can still occur. In addition, without being bound by theory,
it is expected that increasing the flow rate U by a factor of 10
will decrease the mixing time by a factor of Log(U)/U=(Log
(10))/10.
[0183] To visualize mixing in a channel according to the invention,
a colored marker can be used in a single plug-fluid. The initial
distribution of the marker in the plug has been observed to depend
strongly on the details of plug formation. As the stationary
aqueous plug was extruded into the flowing carrier-fluid, shearing
interactions between the flow of the carrier-fluid and the
plug-fluid induced an eddy that redistributed the solution of the
marker to different regions of the plug. The formation of this eddy
is referred to here as "twirling" (see FIG. 27b)). Twirling is not
a high Reynolds number (R.sub.e) phenomenon (see FIG. 30) since it
was observed at substantially all values of R.sub.e and at
substantially all velocities. However, the flow pattern of this
eddy appears to be slightly affected by the velocity.
[0184] Various characteristics and behavior of twirling were
observed. Twirling redistributed the marker by transferring it from
one side of the plug to the other, e.g., from the right to the left
side of the plug. The most efficient mixing was observed when there
was minimal fluctuations in intensity, i.e., when the marker was
evenly distributed across the plug. While twirling was present
during the formation of plugs of all lengths that were
investigated, its significance to the mixing process appears to
depend on the length of the plug. For example, the extent of
twirling was observed to be significantly greater for short plugs
than for long plugs. Twirling was also observed to affect only a
small fraction of the long plugs and had a small effect on the
distribution of the marker in the plugs. Moreover, twirling
occurred only at the tip of the forming plug before the tip made
contact with the right wall of the microchannel. Also, the amount
of twirling in a plug was observed to be related to the amount of
the carrier-fluid that flowed past the tip. The results of
experiments involving twirling and its effect on mixing show that
twirling is one of the most important factors, if not the most
important factor, in determining the ideal conditions for mixing
occurring within plugs moving through straight channels. By
inducing twirling, one may stimulate mixing; by preventing
twirling, one may suppress complete mixing. Suppressing mixing may
be important in some of the reaction schemes, for example those
shown in FIG. 5 and FIG. 6. In these reaction schemes, selective
mixing of reagents A with reagent B, and also reagent C with
reagent D, can occur without mixing of all four reagents. Mixing of
all four reagents occurs later as plugs move through, for example,
the winding part of the channel. This approach allows several
reactions to occur separated in time. In addition, suppressing
mixing may be important when interfaces between plug fluids have to
be created, for example interfaces required for some methods of
protein crystallization (FIG. 20).
[0185] The eddy at the tip of a developing plug may complicate
visualization and analysis of mixing. This eddy is normally
significant in short plugs, but only has a minor effect on long
plugs. For applications involving visualization of mixing, the
substrate is designed to include a narrow channel in the
plug-forming region is designed such that narrow, elongated plugs
form. Immediately downstream from the plug-forming region, the
channel dimension is preferably expanded. In the expanded region of
the channel(s), plugs will expand and become short and rounded
under the force of surface tension; this preserves the distribution
of the marker inside the plugs. This approach affords a relatively
straightforward way of visualizing the mixing inside plugs of
various sizes. Video microscopy may be used to observe the
distribution of colored markers inside the drops. A confocal
microscope may also be used to visualize the average
three-dimensional distribution of a fluorescent marker.
Visualization can be complemented or confirmed using a
Ca.sup.2+/Fluo-4.sup.-4 reaction. At millimolar concentrations,
this reaction is expected to occur with a half-life of about 1
.mu.s. Thus, it can be used to measure mixing that occurs on time
scales of about 10 .mu.s and longer.
[0186] The following discussion describes at least one method for
three-dimensional visualization of flows in plugs. Visualization of
chaotic transport in three-dimensions is a challenging task
especially on a small scale. Predictions based on two-dimensional
systems may be used to gain insight about plugs moving through a
three-dimensional microfluidic channel. Experiments and simulations
involving a two-dimensional system can aid in the design of
channels that ensure chaotic flow in two-dimensional liquid plugs.
Confocal microscopy has been used to quantify steady, continuous
three-dimensional flows in channels. However, due to instrumental
limitations of an optical apparatus such as a confocal microscope,
it is possible that the flow cannot be visualized with sufficiently
high-resolution to observe, for example, self-similar fractal
structures characteristic of chaotic flow. Nonetheless, the overall
dynamics of the flow may still be captured and the absence of
non-chaotic islands confirmed. Preferably, the channels (periodic
or aperiodic) used in the visualization process are fabricated
using soft lithography in PDMS. A PDMS replica is preferably sealed
using a thin glass cover slip to observe the flow using confocal
microscopy.
[0187] In one experiment according to the invention, a series of
line scans are used to obtain images of a three-dimensional
distribution of fluorescent markers within the plugs. FIG. 10a) is
a schematic diagram depicting a three-dimensional confocal
visualization of chaotic flows in plugs. Plugs are preferably
formed from three laminar streams. The middle stream 11 preferably
contains fluorescent markers. Preferably, the middle stream 11 is
injected into the channel system at a low volumetric flow rate. The
volumetric flow rates of the two side streams 10, 12 are preferably
adjusted to position the marker stream in a desired section of the
channel. Preferably, a confocal microscope such as a Carl Zeiss LSM
510 is used. The LSM 510 is capable of line scans at about 0.38
ms/512 pixel line or approximately 0.2 ms/100 pixel line.
Fluorescent microspheres, preferably about 0.2 .mu.m, and
fluorescently labeled high-molecular weight polymers are preferably
used to visualize the flow with minimal interference from
diffusion. A channel such as one with 100 .mu.m wide and 100 .mu.m
deep channel may be used. The line scan technique may be applied to
various sequences such as one that has about 200-.mu.m long plugs
separated by about 800-.mu.m long oil stream.
[0188] A beam is preferably fixed in the x and z-directions and
scanned repeatedly back and forth along the y-direction. The
movement of the plug in the x-direction preferably provides
resolution along the x-direction. Line scan with 100 pixels across
a 100 .mu.m-wide channel will provide a resolution of about 1
.mu.m/pixel in the y-direction. Approximately 200 line scans per
plug are preferably used to give a resolution of about 1
.mu.m/pixel in the x-direction. For a 200 .mu.m plug moving at
about 2000 .mu.m/s, about 200 line scans are preferably obtained
over a period of about (200 .mu.m)/(2000 .mu.m/s)=0.1 s, or about
0.5 ms per line.
[0189] The sequence shown in FIG. 10b) is preferably used for
visualization of a three dimensional chaotic flow. Each line scan
preferably takes about 0.2 ms with about 0.3 ms lag between the
scans to allow the plug to move by about 500 .mu.m. Some optical
distortions may result during the approximately 0.2 ms scan as the
plug is translated along the x-direction by about 0.2 .mu.m.
However, these distortions are believed to be comparable to the
resolution of the method. For a given position along the
x-direction, a series of line scans are preferably obtained for
about 10 seconds for each point along the z-direction to obtain an
x-y cross-sections of ten plugs. Scans along the z-direction are
preferably taken in 1 .mu.m increments to obtain a full
three-dimensional image of the distribution of the fluorescent
marker in the plug. This procedure is preferably repeated at
different positions along the x-direction to provide information
such as changes in the three-dimensional distribution of the
fluorescent marker inside the plug as the plug moves along the
channels.
[0190] In case of periodic perturbations, the fluorescent
cross-sections of the plug in the y-z plane recovered from the
above procedure represent Poincare sections corresponding to the
evolution of the initial thin sheet of dye. The twirling of the
aqueous phase upon formation of the small plugs could distribute
the dye excessively throughout the plug and could make
visualization less conclusive. This twirling is prevented
preferably by designing a small neck in the plug-forming region,
and then beginning the first turn in a downward direction. This
approach has been successfully applied to flow visualization, and
may be useful for conducting reactions.
Merging Plugs
[0191] The invention also provides a method of merging of plugs
within a substrate (see upper portion of FIG. 12). Plugs are formed
as described above. Plugs containing different reagents can be
formed by separately introducing different plug-fluids into a
channel. The plugs containing different reagents may be
substantially similar in viscosity or may differ. The plugs
containing different reagents may be substantially similar in size
or they may differ in size. Provided that the relative velocities
of the plugs containing different reagents differ, the plugs will
merge in the channels. The location of merging can be controlled in
a variety of ways, for example by varying the location of
plug-fluid inlet ports, by varying the location of channel
junctions (if one of the plug forming fluids is introduced into a
secondary channel), varying the size of the plugs, adjusting the
speed at which different sets of plugs are transported varying the
viscosity or surface tension of plugs having substantially the same
size, etc.
[0192] As shown in FIG. 12 (top photograph), plugs may be merged by
directing or allowing the plugs 120, 121 to pass through a T-shaped
channel or a T-shaped region of a channel. The resulting merged
plugs 122 flow in separate channels or channel branches which may
be perpendicular, as shown in FIG. 12, or nonperpendicular (FIG.
33). The merged plugs 122 may undergo further merging or undergo
splitting, or they may be directed to other channels, channel
branches, area, or region of the substrate where they may undergo
one or more reactions or "treatments" such as one or more types of
characterizations, measurements, detection, sorting, or
analysis.
[0193] In one embodiment, large and small plugs flow along separate
channels or channel branches towards a common channel where they
merge. In a case where a large and a small plug do not converge at
the same point at the same time, they eventually form a merged plug
as the larger plug, which moves faster than the smaller plug,
catches up with the small plug and merges with it. In the case
where the larger and smaller plugs meet head on at the same point
or region, they immediately combine to form a merged plug. The
merged plugs may undergo splitting, described below, or further
merging in other channels or channel regions, or they may be
directed to other channels, channel branches, area, or region of
the substrate where they may undergo one or more types of
characterizations, measurements, detection, sorting, or
analysis.
[0194] In another embodiment, plugs can be merged by controlling
the arrival time of the plugs flowing in opposite directions
towards a common point, area, or region of the channel so that each
pair of plugs arrive at the common point, area, or region of the
channel at around the same time to form a single plug.
[0195] In another embodiment, an arched, semi-circular, or circular
channel provides a means for increasing the efficiency of plug
merging. Thus, for example, a greater frequency of merging would
occur within a more compact area or region of the substrate. Using
this scheme, plugs flowing along separate channels towards a common
channel may merge within a shorter distance or a shorter period of
time because the arched, semi-circular, or circular channel or
channel branch converts or assists in converting initially
out-of-phase plug pairs to in-phase plug pairs. Specifically, the
arched, semi-circular, or circular channel or channel branch would
allow a lagging plug to catch up and merge with a plug ahead of it,
thereby increasing the number of merged plugs in a given period or
a given area or region of a substrate.
Splitting and/or Sorting Plugs
[0196] The present invention also provides a method for splitting
of plugs within a substrate. Plugs can be split by passing a first
portion of a plug into a second channel through an opening, wherein
the second channel is downstream of where the plug is formed.
Alternatively, plugs may be split at a "Y" intersection in a
channel. In both embodiment, the initial plug splits into a first
portion and a second portion and thereafter each portion passes
into separate channel (or outlet). Either initially formed plugs
can be split or, alternatively, merged plugs can be split. FIG. 6
shows a schematic diagram illustrating part of a microfluidic
network that uses multiple inlets (inlets 601, 603, 605, 607 for
reagents A, B, C, and D; inlets 602, 604, 606 for aqueous streams)
and that allows for both splitting and merging of plugs. This
schematic diagram shows two reactions that are conducted
simultaneously. A third reaction (between the first two reaction
mixtures) is conducted using precise time delay. Plugs can be split
before or after a reaction has occurred. In addition, FIG. 6 shows
plugs at various stages of mixing from the initial mixture 60 (A+B)
and initial mixture 61 (C+D) through the mixed solutions 62 (A+B),
63 (C+D), and the 4-component mixture 64 (A+B+C+D).
[0197] As shown in FIG. 12 (lower photograph), plugs may be split
by directing or allowing the plugs 123, 124 to pass through a
T-shaped channel or a T-shaped region of a channel. In a preferred
embodiment, the area or junction at which the plugs undergo
splitting may be narrower or somewhat constricted relative to the
diameter of the plugs a certain distance away from the junction.
The resulting split plugs 125 flow in separate channels or channel
branches which may be perpendicular, as shown in FIG. 12, or
nonperpendicular (FIG. 33). The split 125 plugs may undergo merging
or further splitting, or they may be directed to other channels,
channel branches, area, or region of the substrate where they may
undergo one or more reactions or "treatments" such as one or more
types of characterizations, measurements, detection, sorting, or
analysis.
[0198] In another embodiment, aqueous plugs can be split or sorted
from an oil carrier fluid by using divergent hydrophilic and
hydrophobic channels. The channels are rendered hydrophilic or
hydrophobic by pretreating a channel or region of a channel such
that a channel or channel surface becomes predominantly hydrophilic
or hydrophobic. As discussed in more detail below, substrates with
hydrophilic channel surfaces may be fabricated using methods such
as rapid prototyping in polydimethylsiloxane. The channel surface
can be rendered hydrophobic either by silanization or heat
treatment. For example,
(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (United
Chemical Technologies, Inc.) vapor may be applied to the inlets of
the substrate with dry nitrogen as a carrier gas to silanize the
channel surface.
[0199] Once plugs have been split into separate channels, further
reactions can be performed by merging the split plugs with other
plugs containing further reactants.
[0200] Manipulation of plugs and reagents/products contained
therein can also be accomplished in a fluid flow using methods or
techniques such as dielectrophoresis. Dielectrophoresis is believed
to produce movement of dielectric objects, which have no net
charge, but have regions that are positively or negatively charged
in relation to each other. Alternating, nonhomogeneous electric
fields in the presence of plugs and/or particles, cause the plugs
and/or particles to become electrically polarized and thus to
experience dielectrophoretic forces. Depending on the dielectric
polarizability of the particles and the suspending medium,
dielectric particles will move either toward the regions of high
field strength or low field strength. Using conventional
semiconductor technologies, electrodes can be fabricated onto a
substrate to control the force fields in a micro fabricated device.
Dielectrophoresis is particularly suitable for moving objects that
are electrical conductors. The use of AC current is preferred, to
prevent permanent alignment of ions. Megahertz frequencies are
suitable to provide a net alignment, attractive force, and motion
over relatively long distances.
[0201] Radiation pressure can also be used in the invention to
deflect and move plugs and reagents/products contained therein with
focused beams of light such as lasers. Flow can also be obtained
and controlled by providing a thermal or pressure differential or
gradient between one or more channels of a substrate or in a method
according to the invention.
[0202] Preferably, both the fluid comprising the plugs and the
carrier fluid have a relatively low Reynolds Number, for example
10.sup.-2. The Reynolds Number represents an inverse relationship
between the density and velocity of a fluid and its viscosity in a
channel of given cross-sectional dimension. More viscous, less
dense, slower moving fluids will have a lower Reynolds Number, and
are easier to divert, stop, start, or reverse without turbulence.
Because of the small sizes and slow velocities, fabricated fluid
systems are often in a low Reynolds number regime
(R.sub.e<<1). In this regime, inertial effects, which cause
turbulence and secondary flows, are negligible and viscous effects
dominate the dynamics. These conditions are advantageous for
analysis, and are provided by devices according to the invention.
Accordingly the devices according to the invention are preferably
operated at a Reynolds number of less than 100, typically less than
50, preferably less than 10, more preferably less than 5, most
preferably less than 1.
Detection and Measurement
[0203] The systems of the present invention are well suited for
performing optical measurements using an apparatus such as a
standard microscope. For example, PDMS is transparent in the
visible region. When it is used to construct a substrate, a glass
or quartz cover slip can be used to cover or seal a PDMS network,
thereby constructing a set of channels that can be characterized
using visible, UV, or infrared light. Preferably, fluorescent
measurements are performed, instead of absorption measurements,
since the former has a higher sensitivity than the latter. When the
plugs are being monitored by optical measurements, the refractive
index of the carrier-fluid and the plug-fluids are preferably
substantially similar, but they can be different in certain
cases.
[0204] In a plug-based system according to the invention, the
relative concentrations (or changes in concentrations) can be
typically measured in a straightforward fashion. In some instances,
the use of plugs to perform quantitative optical measurements of,
for example, absolute concentrations is complicated by the presence
of non-horizontal oil/water interfaces surrounding the plugs. These
curved interfaces act as lenses, and may lead to losses of emitted
light or optical distortions. Such distortions may adversely affect
or prevent visual observation of growing protein crystals, for
example. Exact modeling of these losses is usually difficult
because of the complicated shape that this interface may adopt at
the front and back of a plug moving in a non-trivial pressure
gradient.
[0205] This problem can be overcome or minimized in accordance with
the invention by using a technique such as refractive index
matching. The losses and distortions depend on the difference
between the refractive index (.eta..sub.D) of the aqueous phase and
the refractive index of the immiscible carrier-fluid. Preferably,
the carrier-fluid used in an analysis have refractive indices that
are substantially similar to those of water and aqueous buffers
(TABLE 1), e.g., fluorinated oils having refractive indices near
that of water close to the sodium D line at 589 nm.
[0206] Preferably, for applications involving detection or
measurement, the carrier-fluids used are those having refractive
indices that match those of commonly used aqueous solutions at the
wavelengths used for observation. To calibrate a system for
quantitative fluorescence measurements, the plugs preferably
contain known concentrations of fluorescein. Preferably, the
fluorescence originating from the plugs are measured and then
compared with the fluorescence arising from the same solution of
fluorescein in the channel in the absence of oil. It is believed
that when the refractive indexes are matched, the intensity (I) of
fluorescence arising from the plugs will be substantially similar
or equal to the intensity of the fluorescence from the aqueous
solutions after making adjustments for the fraction of the aqueous
stream:
I.sub.plug=I.sub.solution*V.sub.water/(V.sub.water+V.sub.oil) Eqn.
(3)
where V is the volumetric flow rate of the fluid streams. It is
expected that smaller plugs with a higher proportion of curved
interfaces will show larger deviations from ideal plug behavior,
i.e., those smaller plugs will tend to cause greater optical
distortion. If necessary, measurements are performed partly to
determine the errors associated with refractive index mismatch.
Information from these measurements is useful when unknown fluids
are analyzed, or when a compromise between matching the refractive
index and matching the viscosities of the two fluids is
required.
TABLE-US-00001 TABLE 1 Physical properties of some fluids used in
certain embodiments of the microfluidic devices. Refractive index,
Viscosity, Fluid .eta..sub.D .mu. [mPa-s] water 1.3330 1.00 aqueous
PBS buffer, 1% 1.3343 1.02 aqueous PBS buffer, 10% 1.3460 1.25
perfluorohexane 1.251 0.66 perfluoro(methylcyclohexane) 1.30 1.56
perfluoro(1,3- 1.2895 1.92 dimethylcyclohexane) perfluorodecaline
1.314 5.10 perfluoroperhydrofluorene 1.3289 9.58
perfluoroperhydrophenanthrene 1.3348 28.4 perfluorotoluene 1.3680
N/A hexafluorobenzene 1.3770 N/A
[0207] The detector can be any device or method for evaluating a
physical characteristic of a fluid as it passes through the
detection region. Examples of suitable detectors include CCD
detectors. A preferred detector is an optical detector, such as a
microscope, which may be coupled with a computer and/or other image
processing or enhancement devices to process images or information
produced by the microscope using known techniques. For example,
molecules can be analyzed and/or sorted by size or molecular
weight. Reactions can be monitored by measuring the concentration
of a product produced or the concentration of a reactant remaining
at a given time. Enzymes can be analyzed and/or sorted by the
extent to which they catalyze a chemical reaction of an enzyme's
substrate (conversely, an enzyme's substrate can be analyzed (e.g.,
sorted) based on the level of chemical reactivity catalyzed by an
enzyme). Biological particles or molecules such as cells and
virions can be sorted according to whether they contain or produce
a particular protein, by using an optical detector to examine each
cell or virion for an optical indication of the presence or amount
of that protein. A chemical itself may be detectable, for example
by a characteristic fluorescence, or it may be labeled or
associated with a tag that produces a detectable signal when, for
example, a desired protein is present, or is present in at least a
threshold amount.
[0208] Practically any characteristic of a chemical can be
identified or measured using the techniques according to the
invention, provided that the characteristic or characteristics of
interest for analysis can be sufficiently identified and detected
or measured to distinguish chemicals having the desired
characteristic(s) from those which do not. For example, particulate
size, hydrophobicity of the reagent versus carrier-fluids, etc. can
be used as a basis for analyzing (e.g., by sorting) plug-fluids,
reaction products or plugs.
[0209] In a preferred embodiment, the plugs are analyzed based on
the intensity of a signal from an optically detectable group,
moiety, or compound (referred to here as "tag") associated with
them as they pass through a detection window or detection region in
the device. Plugs having an amount or level of the tag at a
selected threshold or within a selected range can be directed into
a predetermined outlet or branch channel of the substrate. The tag
signal may be collected by a microscope and measured by a detector
such as a photomultiplier tube (PMT). A computer is preferably used
to digitize the PMT signal and to control the flow through methods
such as those based on valve action. Alternatively, the signal can
be recorded or quantified as a measure of the tag and/or its
corresponding characteristic or marker, e.g., for the purpose of
evaluation and without necessarily proceeding to, for example, sort
the plugs.
[0210] In one embodiment according to the invention, a detector
such as a photodiode is larger in diameter than the width of the
channel, forming a detection region that is longer (along the
length of channel) than it is wide. The volume of such a detection
region is approximately equal to the cross sectional area of the
channel above the diode multiplied by the diameter of the
diode.
[0211] To detect a chemical or tag, or to determine whether a
chemical or tag has a desired characteristic, the detection region
may include an apparatus (e.g., a light source such as a laser,
laser diode, high intensity lamp such as mercury lamp) for
stimulating a chemical or tag for that characteristic to, for
example, emit measurable light energy. In embodiments where a lamp
is used, the channels are preferably shielded from light in all
regions except the detection region. In embodiments where a laser
is used, the laser can be set to scan across a set of detection
regions. In addition, laser diodes may be fabricated into the same
substrate that contains the analysis units. Alternatively, laser
diodes may be incorporated into a second substrate (i.e., a laser
diode chip) that is placed adjacent to the analysis or sorter
substrate such that the laser light from the diodes shines on the
detection region(s).
[0212] In preferred embodiments, an integrated semiconductor laser
and/or an integrated photodiode detector are included on the
silicon wafer in the device according to the invention. This design
provides the advantages of compactness and a shorter optical path
for exciting and/or emitted radiation, thus minimizing, for
example, optical distortion.
[0213] As each plug passes into the detection region, it may be
examined for a characteristic or property, e.g., a corresponding
signal produced by the plug, or the chemicals contained in the
plugs, may be detected and measured to determine whether or not a
given characteristic or property is present. The signal may
correspond to a characteristic qualitatively or quantitatively.
Typically, the amount of signal corresponds to the degree to which
a characteristic is present. For example, the strength of the
signal may indicate the size of a molecule, the amount of
products(s) formed in a reaction, the amount of reactant(s)
remaining, the potency or amount of an enzyme expressed by a cell,
a positive or negative reaction such as binding or hybridization of
one molecule to another, or a chemical reaction of a substrate
catalyzed by an enzyme. In response to the signal, data can be
collected and/or a flow control can be activated, for example, to
direct a plug from one channel to another. Thus, for example,
chemicals present in a plug at a detection region may be sorted
into an appropriate branch channel according to a signal produced
by the corresponding examination at a detection region. Optical
detection of molecular characteristics or the tag associated with a
characteristic or property that is chosen for sorting, for example,
may be used. However, other detection techniques, for instance
electrochemistry, or nuclear magnetic resonance, may also be
employed.
[0214] In one embodiment according to the invention, a portion of a
channel corresponds to an analysis unit or detection region and
includes a detector such as a photodiode preferably located in the
floor or base of the channel. The detection region preferably
encompasses a receive field of the photodiode in the channel, which
receive field has a circular shape. The volume of the detection
region is preferably the same as, or substantially similar, to the
volume of a cylinder with a diameter equal to the receive field of
the photodiode and a height equal to the depth of the channel above
the photodiode.
[0215] The signals from the photodiodes may be transmitted to a
processor via one or more lines representing any form of electrical
communication (including e.g. wires, conductive lines etched in the
substrate, etc.). The processor preferably acts on the signals, for
example by processing them into values for comparison with a
predetermined set of values for analyzing the chemicals. In one
embodiment, a value corresponds to an amount (e.g., intensity) of
optically detectable signal emitted from a chemical which is
indicative of a particular type or characteristic of a chemical
giving rise to the signal. The processor preferably uses this
information (i.e., the values) to control active elements in a
discrimination region, for example to determine how to sort the
chemicals (e.g., valve action).
[0216] When more than one detection region is used, detectors such
as photodiodes in a laser diode substrate are preferably spaced
apart relative to the spacing of the detection regions in the
analysis unit. That is, for more accurate detection, the detectors
are placed apart at the same spacing as the spacing of the
detection region.
[0217] A processor can be integrated into the same substrate that
contains at least one analysis unit, or it can be separate, e.g.,
an independent microchip connected to the analysis unit containing
substrate via electronic leads that connect to the detection
region(s) and/or to the discrimination region(s), such as by a
photodiode. The processor can be a computer or microprocessor, and
is typically connected to a data storage unit, such as computer
memory, hard disk, or the like, and/or a data output unit, such as
a display monitor, printer and/or plotter.
[0218] The types and numbers of chemicals based on the detection
of, for example, a tag associated with or bound to the chemical
passing through the detection region, can be calculated or
determined, and the data obtained can be stored in the data storage
unit. This information can then be further processed or routed to a
data outlet unit for presentation, e.g. histograms representing,
for example, levels of a protein, saccharide, or some other
characteristic of a cell surface in the sample. The data can also
be presented in real time as the sample flows through a
channel.
[0219] If desired, a substrate may contain a plurality of analysis
units, i.e., more than one detection region, and a plurality of
branch channels that are in fluid communication with and that
branch out from the discrimination regions. It will be appreciated
that the position and fate of the reagents in the discrimination
region can be monitored by additional detection regions installed,
for example, immediately upstream of the discrimination region
and/or within the branch channels immediately downstream of the
branch point. The information obtained by the additional detection
regions can be used by a processor to continuously revise estimates
of the velocity of the reagents in the channels and to confirm that
molecules, particles, and substances having a selected
characteristic enter the desired branch channel.
[0220] In one embodiment, plugs are detected by running a
continuous flow through a channel, taking a spatially resolved
image with a CCD camera, and converting the relevant distance
traversed by the plugs into time.
[0221] In another embodiment, plugs are detected following their
exit through a channel point leading to a mass spectrometer (MS),
e.g., an electrospray MS. In this embodiment, time-resolved
information (e.g., mass spectrum) can be obtained when the flow
rate and the distance traversed by the plugs are known. This
embodiment is preferable when one wants to avoid using a label.
Varying the Concentration of Reagents Inside Plugs
[0222] The various devices and methods according to the invention
allow the control and manipulation of plug composition and
properties. For example, they allow the variation of reagent
concentration inside plugs. In one aspect according to the
invention, the concentrations of the reagents in the plugs are
varied by changing the relative flow rates of the plug-fluids. This
is possible in conventional systems, but is complicated by problems
of slow mixing and dispersion. Methods according to the invention
are convenient for simultaneously testing a large number of
experimental conditions ("screening") because the concentrations
can be changed within a single setup. Thus, for example, syringes
do not have to be disconnected or reconnected, and the inlets of a
system according to the invention do not have to be refilled when
using the above technique for varying the reagent concentrations in
plugs.
[0223] The concentration of aqueous solutions inside plugs can be
varied by changing the flow rates of the plug-fluid streams (see
FIG. 25, discussed in detail in Example 11). In FIG. 25, water is
introduced into inlets 251-258 at various flow rates while
perfluorodecaline flows through channels 259-261. In aqueous
laminar flows, the ratio of flow rates of laminar streams in a
microfluidic channel may be varied from about 1000:1 and 1:1000,
preferably 100:1 to 1:100, more preferably 1:20 to 20:1.
[0224] The actual relative concentrations may be quantified using a
solution of known concentration of fluorescein. In this example,
the intensity of a fluorescein stream can be used as a reference
point to check for fluctuations of the intensity of the excitation
lamp.
[0225] To illustrate an advantage offered by the invention over
other techniques, consider the following example. The method(s)
described in this example may be modified or incorporated for use
in various types of applications, measurements, or experiments. Two
or more reagents, such as reagents A, B, C, are to be screened for
the effects of different concentrations of reagents on some
process, and the conditions under which an inhibitor can terminate
the reaction of the enzyme with a substrate at various enzyme and
substrate concentrations is of interest. If A is an enzyme, B a
substrate, and C an inhibitor, a substrate with 5 inlets such as
A/water/B/water/C inlets can be used, and the flow rates at which
A, B and C are pumped into the substrate can be varied. Preferably,
the size of the plug is kept constant by keeping the total flow
rate of all plug-fluids constant. Because different amounts of A,
B, C are introduced, the concentrations of A, B, C in the plugs
will vary. The concentrations of the starting solutions need not be
changed and one can rapidly screen all combinations of
concentrations, as long as an enzymatic reaction or other reactions
being screened can be detected or monitored. Because the solutions
are flowing and the transport is linear, one can determine not only
the presence or absence of an interaction or reaction, but also
measure the rate at which a reaction occurs. Thus, both qualitative
and quantitative data can be obtained. In accordance with the
invention, the substrate typically need not be cleaned between runs
since most, if not all, reagents are contained inside the plugs and
leave little or no residue.
[0226] To extend the range over which concentrations can be varied,
one may use a combination of, say, reagents A, B, C, D, E and
prepare a micromolar solution of A, a mM solution of B, and a M
solution of C, and so on. This technique may be easier than
controlling the flow rate over a factor of, say, more than
10.sup.6. Using other known methods is likely to be more difficult
in this particular example because changing the ratio of reagents
inside the plug requires changing the size of the plugs, which
makes merging complicated.
[0227] In another example, one may monitor RNA folding in a
solution in the presence of different concentrations of Mg.sup.2+
and H.sup.+. Previously, this was done using a stopped-flow
technique, which is time consuming and requires a relatively large
amount of RNA. Using a method according to the invention, an entire
phase space can be covered in a relatively short period of time
(e.g., approximately 15 minutes) using only .mu.L/minute runs
instead of the usual ml/shot runs.
[0228] These particular examples highlight the usefulness according
to the invention in, for example, the study of protein/protein
interaction mediation by small molecules, protein/RNA/DNA
interaction mediation by small molecules, or binding events
involving a protein and several small molecules. Other interactions
involving several components at different concentrations may also
be studied using the method according to the invention.
Generating Gradients in a Series of Plugs
[0229] In one aspect according to the invention, dispersion in a
pressure-driven flow is used to generate a gradient in a continuous
stream of plug-fluid. By forming plugs, the gradient is "fixed",
i.e., the plugs stop the dispersion responsible for the formation
of the gradient. Although the stream does not have to be aqueous,
an aqueous stream is used as a non-limiting example below.
[0230] FIG. 44 illustrates how an initial gradient may be created
by injecting a discrete aqueous sample of a reagent B into a
flowing stream of water. In FIG. 44a), the water+B mixture flowed
through channel 441. Channels 443 and 445 contain substantially
non-flowing water+B mixture. Water streams were introduced into
inlets 440, 442, 444, 446-448 while oil streams flowed through
channels 449-452. FIG. 44d) shows a multiple-inlet system through
which reagents A, B, and C are introduced through inlets 453, 454,
and 455. A pressure-driven flow is allowed to disperse the reagent
along the channel, thus creating a gradient of B along the channel.
The gradient can be controlled by suitable adjustments or control
of the channel dimensions, flow rates, injection volume, or
frequency of sample or reagent addition in the case of multiple
injections. This gradient is then "fixed" by the formation of
plugs. Several of these channels are preferably combined into a
single plug-forming region or section. In addition, complex
gradients with several components may be created by controlling the
streams. This technique may be used for various types of analysis
and synthesis. For example, this technique can be used to generate
plugs for protein or lysozome crystallization. FIG. 42 shows an
experiment involving the formation of gradients by varying the flow
rates (the experimental details are described in Example 17). FIG.
43 illustrates the use of gradients to form lysozyme crystals (the
experimental details are described in Example 18).
Formation and Isolation of Unstable Intermediates
[0231] The devices and methods according to the present invention
may also be used for synthesizing and isolating unstable
intermediates. The unstable intermediates that are formed using a
device according to the invention are preferably made to undergo
further reaction and/or analysis or directed to other parts of the
device where they may undergo further reaction and/or analysis. In
one aspect, at least two different plug-fluids, which together
react to form an unstable intermediate, are used. As the unstable
intermediates form along the flow path of the substrate,
information regarding, for example, the reaction kinetics can be
obtained. Such unstable intermediates can be further reacted with
another reagent by merging plugs containing the unstable
intermediate with another plug-fluid. Examples of unstable
intermediates include, but are not limited to, free radicals,
organic ions, living ionic polymer chains, living organometallic
polymer chains, living free radical polymer chains, partially
folded proteins or other macromolecules, strained molecules,
crystallization nuclei, seeds for composite nanoparticles, etc.
[0232] One application of devices according to the invention that
involves the formation of unstable intermediates is
high-throughput, biomolecular structural characterization. It can
be used in both a time-resolved mode and a non-time resolved mode.
Unstable (and/or reactive) intermediates (for example hydroxyl
radicals (OH)) can be generated in one microfluidic stream (for
example using a known reaction of metal ions with peroxides). These
reactive species can be injected into another stream containing
biomolecules, to induce reaction with the biomolecules. The sites
on the biomolecule where the reaction takes place correlate with
how accessible the sites are. This can be used to identify the
sites exposed to the solvent or buried in the interior of the
biomolecule, or identify sites protected by another biomolecule
bound to the first one. This method could be applied to
understanding structure in a range of biological problems. Examples
include but are not limited to protein folding, protein-protein
interaction (protein footprinting), protein-RNA interaction,
protein-DNA interactions, and formation of protein-protein
complexes in the presence of a ligand or ligands (such as a small
molecule or another biomolecule). Interfacing such a system to a
mass-spectrometer may provide a powerful method of analysis.
[0233] Experiments involving complex chemical systems can also be
performed in accordance with the invention. For example, several
unstable intermediates can be prepared in separate plugs, such as
partially folded forms of proteins or RNA. The reactivity of the
unstable intermediates can then be investigated when, for example,
the plugs merge.
Dynamic Control of Surface Chemistry
[0234] Control of surface chemistry is particularly important in
microfluidic devices because the surface-to-volume ratio increases
as the dimensions of the systems are reduced. In particular,
surfaces that are generally inert to the adsorption of proteins and
cells are invaluable in microfluidics. Polyethylene glycols (PEG)
and oligoethylene glycols (OEG) are known to reduce non-specific
adsorption of proteins on surfaces. Self-assembled monolayers of
OEG-terminated alkane thiols on gold have been used as model
substrates to demonstrate and carefully characterize resistance to
protein adsorption. Surface chemistry to which the solutions are
exposed can be controlled by creating self-assembled monolayers on
surfaces of silicone or grafting PEG-containing polymers on PDMS
and other materials used for fabrication of microfluidic devices.
However, such surfaces may be difficult to mass-produce, and they
may become unstable after fabrication, e.g., during storage or
use.
[0235] In one aspect according to the invention, the reagents
inside aqueous plugs are exposed to the carrier-fluid/plug-fluid
interface, rather than to the device/plug-fluid interface. Using
perfluorocarbons as carrier-fluids in surface studies are
attractive because they are in some cases more biocompatible than
hydrocarbons or silicones. This is exemplified by the use of
emulsified perfluorocarbons as blood substitutes in humans during
surgeries. Controlling and modifying surface chemistry to which the
reagents are exposed can be achieved simply by introducing
appropriate surfactants into the fluorinated PFD phase.
[0236] In addition, the use of surfactants can be advantageous in
problems involving unwanted adsorption of substances or particles,
for example, on the channel walls. Under certain circumstances or
conditions, a reaction may occur in one or more channels or regions
of the substrate that give rise to particulates that then adhere to
the walls of the channels. When they collect in sufficient number,
the adhering particulates may thus lead or contribute to channel
clogging or constriction. Using methods according to the invention,
such as using one or more suitable surfactants, would prevent or
minimize adhesion or adsorption of unwanted substances or particles
to the channel walls thereby eliminating or minimizing, for
example, channel clogging or constriction.
[0237] Encapsulated particulates may be more effectively prevented
from interfering with desired reactions in one or more channels of
the substrate since the particulates would be prevented from
directly coming into contact with reagents outside the plugs
containing the particulates.
[0238] Fluorosurfactants terminated with OEG-groups have been shown
to demonstrate biocompatibility in blood substitutes and other
biomedical applications. Preferably, oil-soluble fluorosurfactants
terminated with oligoethylene groups are used to create interfaces
in the microfluidic devices in certain applications. Surfactants
with well-defined composition may be synthesized. This is
preferably followed by the characterization of the formation of
aqueous plugs in the presence of those surfactants. Their inertness
towards nonspecific protein adsorption will also be characterized.
FIG. 24 shows examples of fluorinated surfactants that form
monolayers that are: resistant to protein adsorption; positively
charged; and negatively charged. For OEG-terminated surfactants,
high values of n (.gtoreq.16) are preferred for making these
surfactants oil-soluble and preventing them from entering the
aqueous phase. In FIG. 24, compounds that have between about 3 to 6
EG units attached to a thiol are sufficient to prevent the
adsorption of proteins to a monolayer of thiols on gold, and are
thus preferred for inertness. In addition, surfactants that have
been shown to be biocompatible in fluorocarbon blood substitutes
may also be used as additives to fluorinated carrier fluids.
Applications: Kinetic Measurements and Assays
[0239] The devices and methods of the invention can be also used
for performing experiments typically done in, for example, a
microtiter plate where a few reagents are mixed at many
concentrations and then monitored and/or analyzed. This can be
done, for example, by forming plugs with variable composition,
stopping the flow if needed, and then monitoring the plugs. The
assays may be positionally encoded, that is, the composition of the
plug may be deduced from the position of the plug in the channel.
The devices and methods of the invention may be used to perform
high-throughput screening and assays useful, for example, in
diagnostics and drug discovery. In particular, the devices and
methods of the invention can be used to perform relatively fast
kinetic measurements.
[0240] The ability to perform fast measurements has revolutionized
the field of biological dynamics. Examples include studies of
protein C folding and cytochrome C folding. These measurements are
performed using fast kinetics instruments that rely on turbulence
to mix solutions rapidly. To achieve turbulence, the channels and
the flow rates normally have to be large, which require large
sample volumes. Commercially available instruments for performing
rapid kinetics studies can access times on the order of 1 ms. The
improved on-chip version of a capillary glass-ball mixer gives a
dead time of about 45 .mu.s with a flow rate of more than about
0.35 mL/sec. The miniaturization of these existing methods is
generally limited by the requirement of high flow rate to generate
turbulence. Miniaturization afforded by devices and methods
according to the invention is advantageous because it allows, for
example, quantitative characterization, from genetic manipulation
and tissue isolation, of a much wider range of biomolecules
including those available only in minute quantities, e.g.,
microgram quantities. In addition, these new techniques and
instruments afford a wide range of accessible time scales for
measurements.
[0241] Time control is important in many chemical and biochemical
processes. Typically, stopped-flow type instruments are used to
measure reaction kinetics. These types of instruments typically
rely on turbulent flow to mix the reagents and transport them while
minimizing dispersion. Because turbulent flow occurs in tubes with
relatively large diameters and at high flow rates, stopped-flow
instruments tend to use large volumes of reagents (e.g., on the
order of ml/s). A microfluidic analog of a stopped-flow instrument
that consumes small volumes of reagents, e.g., on the order of
.mu.L/min, would be useful in various applications such as
diagnostics. Thus far, microfluidic devices have not been able to
compete with stopped-flow instruments because EOF is usually too
slow (although it has less dispersion), and pressure-driven flows
tend to suffer from dispersion. In addition, mixing is usually very
slow in both systems.
[0242] Stopped-flow instruments typically have sub-millisecond
mixing, and could be useful for experiments where such fast mixing
is required. The devices and methods of the invention allow
sub-millisecond measurements as well. In particular, the present
invention can be advantageous for reactions that occur on a
sub-second but slower than about 1 or about 10 millisecond (ms)
time scale or where the primary concern is the solute volume
required to perform a measurement.
[0243] Further, if a plug is generated with two reactive
components, it can serve as a microreactor as the plug is
transported down a channel. A plug's property, such as its optical
property, can then be measured or monitored as a function of
distance from a given point or region of a channel or substrate.
When the plugs are transported at a constant flow rate, a reaction
time can be directly determined from a given distance. To probe the
composition of the plug as it exits a channel, the contents of the
plugs may be injected into a mass spectrometer (e.g., an
electrospray mass spectrometer) from an end of the channel. The
time corresponding to the end of the channel may be varied by
changing the flow rate. Multiple outlets may be designed along the
channels to probe, for example, the plug contents using a mass
spectrometer at multiple distance and time points.
[0244] An advantage of the devices and methods of the invention is
that when plugs are formed continuously, intrinsically slow methods
of observation can be used. For example, plugs flowing at a flow
rate of about 10 cm/s through a distance of about 1 mm from a point
of origin would be about 10 ms old. In this case, the invention is
particularly advantageous because it allows the use of a relatively
slow detection method to repeatedly perform a measurement of, for
example, 10 ms-old plugs for virtually unlimited time. In contrast,
to observe a reaction in a stopped-flow experiment at a time, say,
between about 9 and 11 ms, one only has about 2 ms to take data.
Moreover, the present invention allows one to obtain information
involving complex reactions at several times, simultaneously,
simply by observing the channels at different distances from the
point of origin.
[0245] The reaction time can be monitored at various points along a
channel--each point will correspond to a different reaction or
mixing time. Given a constant fluid flow rate u, one may determine
a reaction time corresponding to the various times t.sub.1,
t.sub.2, t.sub.3, . . . t.sub.n along the channel. Thus, if the
distance between each pair of points n and n-1, which correspond to
time t.sub.n and t.sub.n-1, are the same for a given value of n,
then the reaction time corresponding to point n along the channel
may be calculated from t.sub.n=nl/u. Thus, one can conveniently and
repeatedly monitor a reaction at any given time t.sub.n. In
principle, the substrate of the present invention allows one to
cover a greater time period for monitoring a reaction by simply
extending the length of the channel that is to be monitored at a
given flow rate or by decreasing the flow rate over a given channel
distance (see, for example, FIG. 22). In FIG. 22, the following can
be introduced into the following inlets: enzyme into inlets 2201,
2205, 2210, 2215; buffer into inlets 2202, 2206, 2211, 2216;
substrate into inlets 2203, 2207, 2212, 2217; buffer into inlets
2204, 2208, 2213, 2218; inhibitor into inlets 2228, 2209, 2214,
2219. In FIG. 22, a carrier fluid flows through the channel
portions 2220, 2221, 2222, 2223 from left to right. The channel
portions enclosed by the dotted square 2224, 2225, 2226, 2227
represent fields of view for the purpose of monitoring a reaction
at various points along the channel.
[0246] The same principle applies to an alternate embodiment of the
present invention, where the distance corresponding to a point n
from a common point of origin along the channel differs from that
corresponding to another channel by a power or multiples of 2. This
can be seen more clearly from the following discussion. Given a
constant fluid flow rate u, one may determine a reaction time
corresponding to the various times t.sub.1, t.sub.2, t.sub.3, . . .
t.sub.n along the channel. Thus, if the distance between each pair
of points n and n-1, which correspond to time t.sub.n and
t.sub.n-1, are the same for a given value of n, then the reaction
time corresponding to point n along the channel may be calculated
from t.sub.n=nl/u. In a relatively more complex channel geometry
such as the one shown in FIG. 22(c), the corresponding equation is
given by t.sub.n=2.sup.(n-1)l/u, which shows that the reaction
times at various points n varies as a power or multiples of 2.
[0247] In one aspect, channels according to the invention are used
that place into a field of view different regions that correspond
to different time points of a reaction. The channels according to
the invention allow various measurements such as those of a
complete reaction profile, a series of linearly separated time
points (such as those required for the determination of an initial
reaction velocity in enzymology), and a series of exponentially
separated time points (e.g., first-order kinetic measurements or
other exponential analysis). Time scales in an image frame can be
varied from microseconds to seconds by, for example, changing the
total flow rate and channel length.
[0248] FIG. 22A-D show various examples of geometries of
microfluidic channels according to the invention for obtaining
kinetic information from single optical images. The illustrated
channel systems are suitable for studies such as measurements of
enzyme kinetics in the presence of inhibitors. The device shown in
FIG. 22D has multiple outlets that can be closed or opened. In the
device shown in FIG. 22D, preferably only one outlet is open at a
time. At the fastest flow rates, the top outlet is preferably open,
providing reduced pressure for flow through a short fluid path l.
As flow rates are reduced, other outlets are preferably opened to
provide a longer path and a larger dynamic range for measurements
at the same total pressure.
[0249] In FIG. 22, n is the number of segments for a given channel
length l traveled by the reaction mixture in time t.sub.n (see p.
73, second full paragraph for a related discussion of reaction
times and channel lengths). These systems allow the control of the
ratio of reagents by varying the flow rates. The systems also allow
a quick quantification of enzyme inhibition.
[0250] For example, ribonuclease A can be used with known
inhibitors such as nucleoside complexes of vanadium and oxovanadium
ions and other small molecules such as 5'-diphosphoadenosine
3'-phosphate and 5'-diphosphoadenosine 2'-phosphate. The kinetics
may be characterized by obtaining data and making Lineweaver-Burk,
Eadie-Hofstee, or Hanes-Wolfe plots in an experiment. The
experiment can be accomplished using only a few microliters of the
protein and inhibitor solutions. This capability is particularly
useful for characterizing new proteins and inhibitors that are
available in only minute quantities, e.g., microgram
quantities.
[0251] Kinetic measurements of reactions producing a fluorescent
signal can be performed according to the invention by analyzing a
single image obtained using, for example, an optical microscope.
Long exposures (i.e., about 2 seconds) have been used to measure
fast (i.e., about 2 milliseconds) kinetics. This was possible
because in a continuous flow system, time is simply equal to the
distance divided by the flow rate. In the continuous flow regime in
accordance with the invention, the accessible time scales can be as
slow as about 400 seconds, which can be extended to days or weeks
if the flow is substantially slowed down or stopped. Typically, the
time scale depends on the length of the channel (e.g., up to about
1 meter on a 3-inch diameter chip) at a low flow rate of about 1
mm/s, which is generally limited by the stability of the syringe
pumps, but may be improved using pressure pumping. The fastest time
scale is typically limited by the mixing time, but it may be
reduced to about 20 .mu.s in the present invention. Mixing time is
generally limited by two main factors: (1) the mixing distance
(e.g., approximately 10-15 times the width of the channel); and (2)
the flow rates (e.g., approximately 400 mm/s, depending on the
capillary number and the pressure drop required to drive the flow).
Mixing distance is normally almost independent of the flow rate. By
using suitable designs of microfluidic channels, or networks of
microfluidic channels, a wide range of kinetic experiments can be
performed.
[0252] Reducing the channel size generally reduces the mixing time
but it also increases the pressure required to drive a flow. The
equation below describes the pressure drop, .DELTA.P (in units of
Pa), for a single-phase flow in a rectangular capillary:
.DELTA.P=28.42U.mu.l/ab Eqn. (9)
where U (m/s) is the velocity of the flow, .mu.
(kilogram/meter-second, kg m.sup.-1s.sup.-1) is the viscosity of
the fluid, l (m) is the length of the capillary, a (m) is the
height of the capillary, and b (m) is the width of the capillary.
There is generally a physical limitation on how much pressure a
microfluidic device can withstand, e.g., about 3 atm for PDMS and
about 5 atm for glass and Si. This limitation becomes crucial for
very small channels and restricts the total length of the channel
and thus the dynamic range (the total distance through which this
flow rate can be maintained at a maximum pressure divided by the
mixing distance) of the measurement.
[0253] FIG. 23 depicts a microfluidic network according to the
invention with channel heights of 15 and 2 .mu.m. The channel
design shown in FIG. 23 illustrates how a dynamic range of about
100 can be achieved by changing the cross-section of the channels.
Under these conditions, mixing time in the winding channel is
estimated to be about 25 .mu.s and observation time in the
serpentine channels are estimated to be about 3 ms.
[0254] As FIG. 23 shows, rapid mixing occurs in the 2 .mu.m.times.1
.mu.m (height.times.width) channels and measurements are taken in
the 2 .mu.m.times.3 .mu.m) channels. The table in FIG. 23 shows the
distribution of the pressure drop, flow velocity, and flow time as
a function of the channel cross-section dimensions. A transition
from a 1-.mu.m wide to 3-.mu.m wide channels should occur smoothly,
with plugs maintaining their stability and decreasing their
velocity when they move from a 20-.mu.m wide into a 50-.mu.m wide
channel. Changing the width of the channel can be easily done and
easily incorporated into a mask design. The height of the channel
can be changed by, for example, using photoresist layers having two
different heights that are sequentially spun on, for example, a
silicon wafer. A two-step exposure method may then be used to
obtain a microfluidic network having the desired cross-section
dimensions.
[0255] In another example of the application of the devices and
methods of the present invention, the folding of RNase P catalytic
domain (P RNA C-domain) of Bacillus subtilis ribozyme can be
investigated using channels according to the invention. RNA folding
is an important problem that remains largely unsolved due to
limitations in existing technology. Understanding the rate-limiting
step in tertiary RNA folding is important in the design,
modification, and elucidation of the evolutionary relationship of
functional RNA structures.
[0256] The folding of P RNA C-domain is known to involve three
populated species: unfolded (U), intermediate (I), and native (N,
folded) states. Within the first millisecond, the native secondary
structure and some of the tertiary structure would have already
folded (the RNA is compacted to about 90% of the native dimension)
but this time regime cannot be resolved using conventional
techniques such as stopped-flow. Using channels and substrates
according to the invention, the time-dependence of the P RNA
folding kinetics upon the addition of Mg.sup.2+ can be studied.
[0257] Various types of assays (e.g., protein assays) known in the
art, including absorbance assays, Lowry assays, Hartree-Lowry
assays, Biuret assays, Bradford assays, BCA assays, etc., can be
used, or suitably adapted for use, in conjunction with the devices
and methods of the invention. Proteins in solution absorb
ultraviolet light with absorbance maxima at about 280 and 200 nm.
Amino acids with aromatic rings are the primary reason for the
absorbance peak at 280 nm. Peptide bonds are primarily responsible
for the peak at 200 nm. Absorbance assays offer several advantages.
Absorbance assays are fast and convenient since no additional
reagents or incubations are required. No protein standard need be
prepared. The assay does not consume the protein and the
relationship of absorbance to protein concentration is linear.
Further, the assay can be performed using only a UV
spectrophotometer.
[0258] The Lowry assay is an often-cited general use protein assay.
It was the method of choice for accurate protein determination for
cell fractions, chromatography fractions, enzyme preparations, and
so on. The bicinchoninic acid (BCA) assay is based on the same
principle, but it can be done in one step. However, the modified
Lowry is done entirely at room temperature. The Hartree version of
the Lowry assay, a more recent modification that uses fewer
reagents, improves the sensitivity with some proteins, is less
likely to be incompatible with some salt solutions, provides a more
linear response, and is less likely to become saturated.
[0259] In the Hartree-Lowry assay, the divalent copper ion forms a
complex with peptide bonds under alkaline conditions in which it is
reduced to a monovalent ion. Monovalent copper ion and the radical
groups of tyrosine, tryptophan, and cysteine react with Folin
reagent to produce an unstable product that becomes reduced to
molybdenum/tungsten blue. In addition to standard liquid handling
supplies, the assay only requires a spectrophotometer with infrared
lamp and filter. Glass or inexpensive polystyrene cuvettes may be
used.
[0260] The Biuret assay is similar in principle to that of the
Lowry, however it involves a single incubation of 20 minutes. In
the Biuret assay, under alkaline conditions, substances containing
two or more peptide bonds form a purple complex with copper salts
in the reagent. The Biuret assay offer advantages in that there are
very few interfering agents (ammonium salts being one such agent),
and there were fewer reported deviations than with the Lowry or
ultraviolet absorption methods. However, the Biuret consumes much
more material. The Biuret is a good general protein assay for
batches of material for which yield is not a problem. In addition
to standard liquid handling supplies, a visible light
spectrophotometer is needed, with maximum transmission in the
region of 450 nm. Glass or inexpensive polystyrene cuvettes may be
used.
[0261] The Bradford assay is very fast and uses about the same
amount of protein as the Lowry assay. It is fairly accurate and
samples that are out of range can be retested within minutes. The
Bradford is recommended for general use, especially for determining
protein content of cell fractions and assessing protein
concentrations for gel electrophoresis. Assay materials including
color reagent, protein standard, and instruction booklet are
available from Bio-Rad Corporation. The assay is based on the
observation that the absorbance maximum for an acidic solution of
Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when
binding to protein occurs. Both hydrophobic and ionic interactions
stabilize the anionic form of the dye, causing a visible color
change. The assay is useful since the extinction coefficient of a
dye-albumin complex solution is constant over a 10-fold
concentration range. In addition to standard liquid handling
supplies, a visible light spectrophotometer is needed, with maximum
transmission in the region of 595 nm, on the border of the visible
spectrum (no special lamp or filter usually needed). Glass or
polystyrene cuvettes may be used, but the color reagent stains
both. Disposable cuvettes are recommended.
[0262] The bicinchoninic acid (BCA) assay is available in kit form
from Pierce (Rockford, Ill.). This procedure is quite applicable to
microtiter plate methods. The BCA is used for the same reasons the
Lowry is used. The BCA assay is advantageous in that it requires a
single step, and the color reagent is stable under alkaline
conditions. BCA reduces divalent copper ion to the monovalent ion
under alkaline conditions, as is accomplished by the Folin reagent
in the Lowry assay. The advantage of BCA is that the reagent is
fairly stable under alkaline condition, and can be included in the
copper solution to allow a one step procedure. A
molybdenum/tungsten blue product is produced as with the Lowry. In
addition to standard liquid handling supplies, a visible light
spectrophotometer is needed with transmission set to 562 nm. Glass
or inexpensive polystyrene cuvettes may be used.
[0263] The range of concentrations that can be measured using the
above assays range from about 20 micrograms to 3 mg for absorbance
at 280, between about 1-100 micrograms for absorbance at 205 nm,
between about 2-100 micrograms for the Modified Lowry assay,
between about 1-10 mg for the Biuret assay, between about 1-20
micrograms for the Bradford assay, and between about 0.2-50
micrograms for BCA assay. Many assays based on fluorescence or
changes in fluorescence have been developed and could be performed
using methods and devices of the invention.
[0264] A detailed description of various physical and chemical
assays is provided in Remington: The Science and Practice of
Pharmacy, A. R. Gennaro (ed.), Mack Publishing Company, chap. 29,
"Analysis of Medicinals," pp. 437-490 (1995) and in references
cited therein while chapter 30 of the same reference provides a
detailed description of various biological assays. The assays
described include titrimetric assays based on acid-base reactions,
precipitation reactions, redox reactions, and complexation
reactions, spectrometric methods, electrochemical methods,
chromatographic methods, and other methods such as gasometric
assays, assays involving volumetric measurements and measurements
of optical rotation, specific gravity, and radioactivity. Other
assays described include assays of enzyme-containing substances,
proximate assays, alkaloidal drug assays, and biological tests such
as pyrogen test, bacterial endotoxin test, depressor substances
test, and biological reactivity tests (in-vivo and in-vitro).
[0265] In addition, Remington: The Science and Practice of
Pharmacy, A. R. Gennaro (ed.), Mack Publishing Company, chap. 31,
"Clinical Analysis," pp. 501-533 (1995) and references cited
therein provide a detailed description of various methods of
characterizations and quantitation of blood and other body fluids.
In particular, the reference includes a detailed description of
various tests and assays involving various body fluid components
such as erythrocytes, hemoglobin, thrombocyte, reticulocytes, blood
glucose, nonprotein nitrogen compounds, enzymes, electrolytes,
blood-volume and erythropoeitic mechanisms, and blood
coagulation.
Nonlinear and Stochastic Sensing
[0266] Stochastic behavior has been observed in many important
chemical reactions, e.g., autocatalytic reactions such as inorganic
chemical reactions, combustion and explosions, and in
polymerization of sickle-cell hemoglobin that leads to sickle-cell
anemia. Crystallization may also be considered an autocatalytic
process. Several theoretical treatments of these reactions have
been developed. These reactions tend to be highly sensitive to
mixing.
[0267] Consider the extensively studied stochastic autocatalytic
chemical reaction between NaClO.sub.2 and Na.sub.2S.sub.2O.sub.3
(chlorite-thiosulfate reaction). The mechanism of this reaction can
be described by reactions (1) and (2),
4S.sub.2O.sub.3.sup.2-+ClO.sub.2.sup.-+4H+.fwdarw.2S.sub.4O.sub.6.sup.2--
+2H.sub.2O+Cl.sup.-rate (.nu.).alpha.[H.sup.+] (1)
S.sub.2O.sub.3.sup.2-+2ClO.sub.2.sup.-+H.sub.2O.fwdarw.2SO.sub.4.sup.2-+-
2H.sup.++2Cl.sup.-rate (.nu.).alpha.[H.sup.+].sup.2[Cl] (2)
where [H.sup.+] stands for the concentration of H.sup.+. At a
slightly basic pH=7.5, the slow reaction (1) dominates and
maintains a basic pH of the reaction mixture (since the rate of
this reaction .nu. is directly proportional [H.sup.+], this
reaction consumes H.sup.+ and is auto-inhibitory). Reaction (2)
dominates at acidic pH (since the rate of this reaction varies in
proportion to [H.sup.+].sup.2[Cl.sup.-], this reaction produces
both H.sup.+ and Cl.sup.- and is superautocatalytic). FIG. 21 shows
the reaction diagram for two reactions corresponding to the curves
211, 212. The rates of the two reactions (referred to here as
reaction 211 and reaction 212) are equal at an unstable critical
point at a certain pH. The lifetime of the reaction mixtures of
NaClO.sub.2 and NaS.sub.2O.sub.3 at this critical point crucially
depends on stirring. In the absence of stirring, stochastic
fluctuations of [H.sup.+] in solution generate a localized increase
in [H.sup.+]. This increase in [H.sup.+] marginally increases the
rate of reaction 212, but it has a much stronger accelerating
effect on reaction 211 because of the higher-order dependence on
[H.sup.+] of this reaction. Therefore, in the region where local
fluctuations increase local [H.sup.+], reaction 211 becomes
dominant, and more H.sup.+ is produced (which rapidly diffuses out
of the region of the initial fluctuation). The initiated chemical
wave then triggers the rapid reaction of the entire solution.
Unstirred mixtures of NaClO.sub.2 and NaS.sub.2O.sub.3 are stable
only for a few seconds, and these fluctuations arise even in the
presence of stirring.
[0268] FIG. 21 depicts a reaction diagram illustrating an unstable
point in the chlorite-thiosulfate reaction. At [H.sup.+] values
below the critical point, the slow reaction (1) dominates. At
[H.sup.+] values above the critical point, the autocatalytic
reaction (2) dominates. The reaction mixture at the [H.sup.+] value
equal to the critical point is metastable in the absence of
fluctuations. Under perfect mixing, the effects of small
fluctuations average out and the system remains in a metastable
state. Under imperfect mixing, fluctuations that reduce [H.sup.+]
grow more slowly than those that increase [H.sup.+] due to the
autocatalytic nature of reaction (2), and the reaction mixture thus
rapidly becomes acidic.
[0269] It is known that chaotic flows should have a strong effect
on diffusive transport within the fluid ("anomalous diffusion"). It
is also known that chaotic dynamics can lead to non-Gaussian
transport properties ("strange kinetics"). In one aspect according
to the invention, these highly unstable mixtures are stabilized in
the presence of chaotic mixing using channels according to the
invention because this mixing can effectively suppress
fluctuations. This invention can be used to understand the effects
of mixing on the stochastic behavior of such systems, including for
example, the chlorite thiosulfate system.
[0270] In a laminar flow, the flow profile in the middle of the
channel is flat and there is virtually no convective mixing.
Fluctuations involving [H.sup.+] that arise in the middle of the
channel can grow and cause complete decomposition of the reaction
mixture. Slow mixing reduces the probability of fluctuations in
plugs moving through straight channels. When fluctuations that
occur in the centers of vortices are not efficiently mixed away,
one or more spontaneous reactions involving some of the plugs can
take place. In the present invention, chaotic mixing in plugs
moving through winding channels efficiently mix out fluctuations,
and thus substantially fewer or no spontaneous reactions are
expected to occur.
[0271] In a simple laminar flow, there is normally very little or
no velocity gradient and substantially no mixing at the center of
the channel. Thus, fluctuations that arise in the
chlorite-thiosulfate reaction mixture prepared at the critical
[H.sup.+] are able to grow and lead to rapid decomposition of the
reaction mixture. Propagation of chemical fronts in autocatalytic
reactions occurring in laminar flows has been described with
numerical simulations, and back-propagation has been predicted
(that is, a reaction front traveling upstream of the direction of
the laminar flow). Using the method of the present invention, this
back-propagation involving the reaction between NaClO.sub.2 and Na
S.sub.2O.sub.3 under laminar flow conditions was observed.
[0272] In accordance with the invention, chaotic flow within plugs
that flow through winding channels suppresses fluctuations and
gives rise to stable reaction mixtures. There exists, of course, a
finite probability that fluctuations can arise even in a
chaotically stirred plug. In one aspect according to the invention,
the details of the evolution of these reactions are monitored using
a high-speed digital camera. The plugs are preferably separated by
the oil and are not in communication with each other, so the
reaction of one plug will not affect the behavior of the
neighboring plug. Statistics covering the behavior of thousands of
plugs can be obtained quickly under substantially identical
experimental conditions.
[0273] Whether a fluctuation would be able to trigger an
autocatalytic reaction depends on factors such as the magnitude of
a fluctuation and its lifetime. The lifetime of a fluctuation is
typically limited by the mixing time in the system. In an unstirred
solution, mixing is by diffusion and quite slow, and fluctuations
may persist and lead to autocatalytic reactions. In a stirred
solution, the lifetime of a fluctuation is relatively short, and
only large fluctuations have sufficient time to cause an
autocatalytic reaction.
[0274] Mixing time and the lifetime of fluctuations typically
depend on the size of the plugs. As plug size decreases, mixing is
accelerated and fluctuations are suppressed. However, very small
plugs (e.g., about 1 .mu.m.sup.3 or 10.sup.-15 L) in a solution
containing about 10.sup.-8 mole/liter concentration of H.sup.+
(pH=8) will contain only a few H.sup.+ ions per plug (about
10.sup.-23 moles or about 6H.sup.+ ions). When such small plugs are
formed, the number of H.sup.+ ions in them will have a Poisson
distribution.
[0275] An important experimental challenge is to establish that the
stochastic behavior in these systems is due mainly to internal
fluctuations of concentrations. Other factors that may act as
sources of noise and instability are: (1) temporal fluctuations in
the flow rates of the incoming reagent streams, which can lead to
the formation of plugs with varying amounts of reagents; (2)
temperature fluctuations in solutions in a microfluidic device,
which may arise due to, for example, illumination by a microscope;
and (3) fluctuations due to impurities in carrier-fluids leading to
variations in the surface properties of different plugs.
[0276] Microfluidic systems according to the invention may be used
to probe various chemical and biochemical processes, such as those
that show stochastic behavior in bulk due to their nonlinear
kinetics. They can also be used in investigating processes that
occur in systems with very small volumes (e.g., about 1
.mu.m.sup.3, which corresponds to the volume of a bacterial cell).
In systems with very small volumes, even simple reactions are
expected to exhibit stochastic behavior due to the small number of
molecules localized in these volumes.
[0277] Autocatalytic reactions present an exciting opportunity for
highly sensitive detection of minute amounts of autocatalysts.
Several systems are known to operate on this principle,
silver-halide photography being the most widely used. In
silver-halide photography, the energy of photons of light is used
to decompose an emulsion of silver halide AgX into nanometer-sized
particles of metallic silver. A film that is embedded with the
silver particles is then chemically amplified by the addition of a
metastable mixture of a soluble silver(I) salt and a reducing agent
(hydroquinone). Metallic silver particles catalyze reduction of
silver(I) by hydroquinone, leading to the growth of the initial
silver particles. Another example of an autocatalytic reaction is
the polymerase-chain reaction (PCR), which is a very effective
amplification method that has been widely used in the biological
sciences.
[0278] However, a dilemma occurs when designing systems with very
high sensitivity and amplification. To achieve a very highly
sensitive amplification, the system typically has to be made very
unstable. On the other hand, an unstable system is very sensitive
to noise and has a very short lifetime. Also, in unstable systems,
it is difficult to distinguish between spontaneous decomposition
and a reaction caused by the analyte. In one aspect, microfluidic
devices according to the invention, which allow chaotic mixing and
compartmentalization, are used to overcome this problem.
[0279] To demonstrate the potential of microfluidic systems
according to the present invention, a microfluidic system according
to the invention is used to handle unstable mixtures. In one
application, a microfluidic system according to the invention is
preferably used to control a stochastic reaction between
NaClO.sub.2 and NaS.sub.2O.sub.3. In particular, this reaction is
preferably used for a highly sensitive amplification process.
[0280] If a plug containing an unstable reaction mixture of
NaClO.sub.2 and NaS.sub.2O.sub.3 is merged with a small plug
containing an amount of H.sup.+ sufficient to bring the local
concentration of H.sup.+ above critical, a rapid autocatalytic
reactions is generally triggered. This autocatalytic reaction
typically leads to the production of large amounts of H.sup.+.
Thus, a weak chemical signal, e.g., a small amount of H.sup.+, is
rapidly amplified by an unstable reaction mixture. Thus, for
example, this approach can be used to investigate biological
reactions such as those that involve enzymes, in which small
amounts of H.sup.+ are produced.
[0281] The above autocatalytic system possesses several features
that contribute to its novelty and usefulness. In one aspect, an
unstable amplifying reaction mixture is prepared in-situ and is
used within milliseconds before it has a chance to decompose.
Preferably, the system is compartmentalized so a reaction that
occurs in one compartment does not affect a reaction in another
compartment. This compartmentalization allows thousands of
independent experiments to be conducted in seconds using only
minute quantities of samples. Importantly, chaotic mixing in the
system reduces fluctuations and stabilizes the reaction
mixture.
[0282] The applications of controlled autocatalytic amplification
in accordance with the invention are not limited to the detection
of protons or Co.sup.2+ ions. For example, the
(Co(III)-5-Br-PAPS)/peroxomonosulfate oxidation reaction can also
be used indirectly, for example, for a detection of small amounts
of peroxidase, which can be used as a labeling enzyme bound to an
antibody. The (Co(III)-5-Br-PAPS)/peroxomonosulfate oxidation
reaction, which has been characterized analytically, involves the
autocatalytic decomposition of violet
bis[2-(5-bromo-pyridylazo)-5-(N-propyl-N-sulfopropyl-amino-phenolato]
cobaltate, (Co(III)-5-Br-PAPS), upon oxidation with potassium
peroxomonosulfate to produce colorless Co.sup.2+ ions, which serve
as the autocatalyst (the order of autocatalysis has not been
established for this reaction). (Endo et al., "Kinetic
determination of trace cobalt(II) by visual autocatalytic
indication," Talanta, 1998, vol. 47, pp. 349-353; Endo et al.,
"Autocatalytic decomposition of cobalt complexes as an indicator
system for the determination of trace amounts of cobalt and
effectors," Analyst, 1996, vol. 121, pp. 391-394.)
Co(III)-[5-Br-PAPS]reduced+HSO.sub.5.sup.-.fwdarw.Co.sup.2++[5-Br-PAPS]o-
xidized+HSO.sup.4-
[0283] Addition of small amounts of Co.sup.2+ to the violet mixture
of the (Co(III)-5-Br-PAPS and peroxomonosulfate produces an abrupt
loss of color to give a colorless solution. The time delay before
this decomposition depends on the amount of the Co.sup.2+ added to
the solution. This reaction has been used to detect concentrations
of Co.sup.2+ as low as 1.times.10.sup.-10 mole/L. The reaction
shows good selectivity in the presence of other ions (V(V),
Cr(III), Cr(VI), Mn(II), Fe(II), Ni(II), Cu(II) and Zn(II)).
[0284] The devices and methods according to the invention may be
applied to other autocatalytic reactions, some of which have been
described in inorganic, organic and biological chemistry. Reactions
of transition metal ions such as Cr(III) (B82) Mn.sup.2+ or
colloidal MnO.sub.2, and reactions of halides and oxohalides are
often autocatalytic. Autocatalysis involving lanthanides
(Eu.sup.2+) and actinides (U.sup.4+) has also been reported. All of
these elements are potential targets for detection and monitoring
in chemical waste, drinking water, or biological fluids. Intriguing
possibilities arise from using asymmetric autocatalytic reactions
to detect minute amounts of optically active, chiral impurities,
such as biomolecules.
[0285] It is also possible to design new autocatalytic reactions.
Autocatalysis is abundant in biology, and many enzymes are
autocatalytic (e.g., caspases involved in programmed cell death,
kinases involved in regulation and amplification, and other enzymes
participating in metabolism, signal transduction, and blood
coagulation. Emulsions of perfluorocarbons such as
perfluorodecaline (PFD) are used as blood substitutes in humans
during surgeries and should be compatible with a variety of
biological molecules. Since the feasibility of quantitative
measurements of enzyme kinetics has been demonstrated using plugs
formed according to the invention, plugs formed according to the
invention may also be applied to the detection of biological
autocatalysts.
[0286] The devices and methods according to the present invention
are not limited to the detection of the autocatalyst itself. For
example, the labeling of an analyte using an autocatalyst is also
within the scope of the present invention. Biomolecules are often
labeled with metallic nanoparticles. Such metallic nanoparticles
are highly effective autocatalysts for the reduction of metal ions
to metals. Preferably, the systems and methods of the present
invention are used in the visual detection of a single molecule of
DNA, RNA, or protein labeled with nanoparticles via an
autocatalytic pathway. In preliminary experiments in accordance
with the invention, clean particle formation and transport within
plugs were observed.
[0287] In addition, the generation of metal (e.g., copper, silver,
gold, nickel) deposits and nanoparticles upon chemical reduction
also proceed by an autocatalytic mechanism. These reactions are
commonly used for electroless deposition of metals and should be
useful for the detection of minute amounts of metallic particles.
The presence of metallic particles in water can be indicative of
the presence of operating mechanical devices. In one aspect
according to the invention, devices and methods according to the
invention are used to detect the presence of minute or trace
quantities of metallic particles.
[0288] The devices in accordance with the present invention are
simple in design, consume minute amounts of material, and robust.
They do not require high voltage sources and can be operated, for
example, using gravity or a pocket-sized source of compressed air.
In one aspect, the systems according to the invention are used in
portable and hand-held devices.
[0289] Autocatalytic reactions show a threshold response, that is,
there is a very abrupt temporal change from unreacted mixture to
reacted mixture. In the case where time is equal to distance, this
abrupt transition over a short distance can be observed using the
devices and methods of the invention. The time (and distance) is
very sensitive to the initial concentration of the catalyst, and
thus it should be easy to determine the concentration of the
autocatalyst in the sample by noting how far the reaction system
traveled before it reacted.
[0290] One example of an autocatalytic process is blood
coagulation. It is very sensitive to flow and mixing, therefore
experimenting with it in the absence of flow gives unreliable
results or results that have little relevance to the real function
of the coagulation cascade. A typical microfluidic system may be
difficult to use with blood because once coagulation occurs, it
blocks the channel and stops the flow in the microfluidic device.
In addition, coagulated blood serves as an autocatalyst; even small
amounts of coagulated blood in the channels can make measurements
unreliable.
[0291] These problems can be overcome using the devices of the
present invention. Using plugs, autocatalytic reactions can be
easily controlled, and the formation of solid clots would not be a
problem because any solids formed will be transported inside the
plugs out of the channel without blocking the channel and without
leaving autocatalytic residue. In addition, flow inside plugs can
be easily controlled and adjusted to resemble flow under
physiological conditions.
[0292] To address the sensitivity of blood coagulation to surfaces
(the cascade is normally initiated on the surface), microscopic
beads containing immobilized tissue factor (the cascade initiator)
on the surface may be added to one of the streams and transported
inside the plugs. Also, surfactants may be used to control surface
chemistry.
[0293] Thus, the devices and methods of the invention may be used,
for example, to test how well the coagulation cascade functions
(e.g., for hemophilia or the tendency to form thrombus) under
realistic flow conditions. This test would be particularly valuable
in diagnostics. Blood may be injected in one stream, and a known
concentration of a molecule known to induce coagulation (e.g.,
factor VIIa) can be added through another stream prior to plug
formation. At a given flow rate, normal blood would coagulate at a
certain distance (which corresponds to a given time), which can be
observed optically by light scattering or microscopy. Blood of
hemophiliac patients would coagulate at a later time. This type of
testing would be useful before surgical operations. In particular,
this type of testing is important for successful child delivery,
especially when hemophilia is suspected. Fetal testing may be
performed since only minute amounts of blood are required by
systems according to the invention. The blood may be injected
directly from the patient or collected in the presence of
anticoagulating agent (for example EDTA), and then reconstituted in
the plug by adding Ca.sup.2+. In some cases, the addition of
Ca.sup.2+ may be sufficient to initiate the coagulation
cascade.
[0294] The devices and methods of the invention may also be used to
evaluate the efficacy of anticoagulating agents under realistic
flow conditions. Plugs can be formed from normal blood (which may
be used directly or reconstituted by adding Ca.sup.2+ or other
agents), an agent known to induce coagulation, and an agent (or
several agents that need to be compared) being tested as an
anticoagulation agent. The concentrations of these agents can be
varied by varying the flow rates. The distance at which coagulation
occurs is noted, and the efficacy of various agents to prevent
coagulation is compared. The effects of flow conditions and
presence of various compounds in the system on the efficacy of
anticoagulation agents can be investigated quickly. The same
techniques may also be used to evaluate agents that cause, rather
prevent, coagulation. These tests could be invaluable in evaluating
drug candidates.
Synthesis
[0295] In accordance with the present invention, a method of
conducting a reaction within a substrate is provided. The reaction
is initiated by introducing two or more plug-fluids containing
reactants into the substrate of the present invention.
[0296] In one aspect, the plug-fluids include a reagent and solvent
such that mixing of the plug-fluids results in the formation of a
reaction product. In another embodiment, one of the plug-fluids may
be reagent free and simply contain fluid. In this embodiment,
mixing of the plug-fluids will allow the concentration of the
reagent in the plug to be manipulated.
[0297] The reaction can be initiated by forming plugs from each
plug-fluid and subsequently merging these different plugs.
[0298] When plugs are merged to form merged plugs, the first and
second set of plugs may be substantially similar or different in
size. Further, the first and second set of plugs may have different
relative velocities. In one embodiment, large arrays of
microfluidic reactors are operated in parallel to provide
substantial throughput.
[0299] The devices and methods of the invention can be used for
synthesizing nanoparticles. Nanoparticles that are monodisperse are
important as sensors and electronic components but are difficult to
synthesize (Trindade et al., Chem. Mat. 2001, vol. 13, pp.
3843-3858). In one aspect, monodisperse nanoparticles of
semiconductors and noble metals are synthesized under time control
using channels according to the invention (Park et al, J. Phys.
Chem. B, 2001, vol. 105, pp. 11630-11635). Fast nucleation is
preferably induced by rapid mixing, thereby allowing these
nanoparticles to grow for a controlled period of time. Then their
growth is preferably quickly terminated by passivating the surfaces
of the particles with, for example, a thiol. Nanoparticles of
different sizes are preferably obtained by varying the flow rate
and therefore the growth time. In addition, devices according to
the invention can be used to monitor the synthesis of
nanoparticles, and thus obtain nanoparticles with the desired
properties. For example, the nanoparticle formation may be
monitored by measuring the changes in the color of luminescence or
absorption of the nanoparticles. In addition, the growth of
nanoparticles may be stopped by introducing a stream of quenching
reagent at a certain position along the main channel.
[0300] Rapid millisecond mixing generated in channels according to
the invention can help ensure the formation of smaller and much
more monodisperse nanoparticles than nanoparticles synthesized by
conventional mixing of solutions. FIG. 13 shows the UV-VIS spectra
of CdS nanoparticles formed by rapid mixing in plugs (lighter shade
spectrum with sharp absorption peak) and by conventional mixing of
solutions (darker shade spectrum). The sharp absorption peak
obtained for synthesis conducted in plugs indicates that the
nanoparticles formed are highly monodisperse. In addition, the
blue-shift (shift towards shorter wavelengths) of the absorption
peak indicates that the particles formed are small.
[0301] FIG. 14A-B illustrates the synthesis of CdS nanoparticles
performed in PDMS microfluidic channels in single-phase aqueous
laminar flow (FIG. 14A) and in aqueous plugs that were surrounded
by water-immiscible perfluorodecaline (FIG. 14B). In FIGS. 14A-B,
Cd.sup.2+ was introduced into inlets 1400, 1403, aqueous stream was
introduced into inlets 1401, 1404, and S.sup.2- was introduced into
inlets 1402, 1405. In FIG. 14A, an aqueous stream flowed through
channel 1406 while in FIG. 14B, oil flowed through channel 1407.
FIG. 14A shows portions of the channels 1408 and 1410 at time t=6
minutes and portions of the channels 1409, 1411 at time t=30
minutes. It can be seen in FIG. 14A that when laminar flow is used
in the synthesis, large amounts of CdS precipitate form on the
channel walls. When plugs were used for the synthesis, all CdS
formed inside the plugs, and no surface contamination was observed.
FIG. 15 illustrates a technique for the synthesis of CdS
nanoparticles, which is discussed in detail in Example 13
below.
[0302] The following methods according to the invention can be used
in synthesis involving nanoparticles:
[0303] (a) using self-assembled monolayers to nucleate
nanoparticles with crystal structures not accessible under
homogeneous nucleation conditions (e.g., controlling polymorphism
by controlling the surface at which nucleation takes place).
[0304] (b) using merging of plugs to create core-shell
nanoparticles with a range of core and shell sizes. In a stream of
plugs of a first channel, small core nanoparticles such as CdSe
particles can be synthesized in a matter of few milliseconds. The
CdSe particles can then be used as seeds for mixing with solutions
such as those containing Zn.sup.+2 and S.sup.-2. The CdSe
particles, acting as seeds for the formation of ZnS, thus allow the
formation of CdSe(core)/ZnS(shell) nanoparticles. Core-shell
particles with more than two layers may be obtained by simply
repeating the merging process more than once.
[0305] (c) using merging of plugs to create composite
nanoparticles. For example, small nanoparticles of CdSe and ZnS can
be formed using streams of plugs from two separate channels.
Merging of these streams leads to aggregation of these particles to
form larger nanoparticles containing CdSe/ZnS composite. The
composite nanoparticles that contain only a few of the original
nanoparticles can be made non-centrosymmetric, which may have
interesting photophysical properties.
[0306] (d) using the devices and methods according to the invention
to synthesize medically important nanoparticles, such as
encapsulated drugs and composite drugs.
[0307] (e) combinatorial synthesis of core-shell particles and
other complex systems. For example, the luminescence of CdSe/ZnS
particles may be monitored and the conditions adjusted to produce
particles with various core and shell sizes, various doping
impurities in the core and shell, and various ligand composition on
the surface of the particles. These can be conducted in real time
using a device according to the invention. The entire process can
also be automated.
[0308] The devices and methods according to the present invention
may also be used for synthesizing polymers. Since the invention
allows precise control of the timing of a polymerization reaction,
one or more properties of a polymer such as molecular weight,
polydispersity and blockiness can be readily controlled or
adjusted. In addition, use of the substrate of the present
invention allows the user to precisely form block copolymers by
merging plugs within a device, since the path length of the channel
will correspond to a specific duration of the polymerization
reaction. Similarly, a living polymer chain can be terminated with
a specific end group to yield polymers with a discrete subset of
molecular weights.
[0309] In addition, combinatorial libraries of drug candidates may
be synthesized using similar approaches. The library may be encoded
using the position of plugs in a channel. Plugs of variable
composition may be created by varying flow rates. Combination of
synthesis of the library may be combined with screening and assays
performed on the same microfluidic chip according to the present
invention. In some embodiments, merging, splitting and sorting of
plugs may be used during synthesis, assays, etc.
[0310] All of the above synthesis methods of the present invention
can be used to form macroscopic quantities of one or more reaction
products by running multiple reactions in parallel.
Particle Separation/Sorting Using Plugs
[0311] The flow within the moving plugs can be used for separation
of polymers and particles. Plugs can be used for separation by
first using flow within a moving plug to establish a distribution
of the polymers or particles inside the plug (for example, an
excess of the polymer inside the front, back, right or left side of
the plug) and then using splitting to separate and isolate the part
of the plug containing higher concentration of the polymers or
particles. When two polymers or particles are present inside the
plug and establish different distributions, slitting can be used to
separate the polymers or particles. This approach may be useful,
for example, in achieving on a microfluidic chip any of, but not
limited to, the following: separation, purification, concentration,
membrane-less dialysis, and filtration.
Crystallization
[0312] The devices and methods of the invention allow fast,
inexpensive miniaturization of existing crystallization methods and
other methods that can be adapted into, for example, novel protein
screening and crystallization techniques. The crystallization
methods according to the invention may be applied to various drugs,
materials, small molecules, macromolecules, colloidal and
nanoparticles, or any of their combinations. Many relevant protein
structures remain undetermined due to their resistance to
crystallization. Also, many interesting proteins are only available
in microgram quantities. Thus, a screening process must permit the
use of small amounts protein for analysis. Current crystallization
screening technologies generally determine the ideal conditions for
protein crystallization on a milligram scale. Devices and methods
according to the invention improve current bench-top methodology
available to single users, and enables higher throughput automated
systems with improved speed, sample economy, and entirely new
methods of controlling crystallization.
[0313] A microfluidic system according to the invention can be
applied to the crystallization of small molecules or macromolecules
and their complexes.
[0314] For example, systems and methods in accordance with the
present invention may include but are not limited to: (1)
biological macromolecules (cytosolic proteins, extracellular
proteins, membrane proteins, DNA, RNA, and complex combinations
thereof); (2) pre- and post-translationally modified biological
molecules (including but not limited to, phosphorylated,
sulfolated, glycosylated, ubiquitinated, etc. proteins, as well as
halogenated, abasic, alkylated, etc. nucleic acids); (3)
deliberately derivatized macromolecules, such as heavy-atom labeled
DNAs, RNAs, and proteins (and complexes thereof),
selenomethionine-labeled proteins and nucleic acids (and complexes
thereof), halogenated DNAs, RNAs, and proteins (and complexes
thereof); (4) whole viruses or large cellular particles (such as
the ribosome, replisome, spliceosome, tubulin filaments, actin
filaments, chromosomes, etc.); (5) small-molecule compounds such as
drugs, lead compounds, ligands, salts, and organic or
metallo-organic compounds; (6) small-molecule/biological
macromolecule complexes (e.g., drug/protein complexes,
enzyme/substrate complexes, enzyme/product complexes,
enzyme/regulator complexes, enzyme/inhibitor complexes, and
combinations thereof); (7) colloidal particles; and (8)
nanoparticles.
[0315] Preferably, a general crystallization technique according to
the present invention involves two primary screening steps: a crude
screen of crystallization parameters using relatively small
channels with a large number of small plugs, and a fine screen
using larger channels and larger plugs to obtain
diffraction-quality crystals. For example, ten crude screens
performed using channels with a (50 .mu.m).sup.2 cross-sectional
dimension and with more or less one thousand 150-picoliter (pL)
plugs corresponding to 10 mg/mL final concentration of a protein
(10,000 trials total) will typically require about 1.5 .mu.L of
solution, produce crystals up to about (10 .mu.m).sup.3 in size,
and will consume approximately 15 .mu.g of protein. Up to 300 or
more of such plugs can be formed in about 1 second in these
microfluidic networks. A fine screen around optimal conditions in
(500 .mu.m).sup.2 channels is expected to use more or less 50
plugs. Another .about.5 .mu.L of solution and another 50 .mu.g of
the protein are expected to be consumed. This can produce crystals
up to (100 .mu.m).sup.3 in size. Approximately 30 plugs can be
formed about every second or so. The throughput of the system will
generally be determined by the rate of plug formation, and may be
limited by how rapidly the flow rates can be varied. Pressure
control methods that operate at frequencies of 100 Hz are available
and may be applied to PDMS microfluidic networks (Unger et al.,
"Monolithic fabricated valves and pumps by multilayer soft
lithography," Science 2000, vol. 288, pp. 113-116).
[0316] Crystal properties such as appearance, size, optical
quality, and diffractive properties may be characterized and
measured under different conditions. For example, a Raxis IIc X-ray
detector mounted on a Rigaku RU 200 rotating anode X-ray generator,
which is equipped with double focusing mirrors and an MSC
cryosystem, may be used for at least some of the characterizations
and measurements. A synchrotron beam may be useful for
characterization of small crystals. Also, these devices and methods
may be used to build microfluidic systems according to the
invention that are compatible with structural studies using x-ray
beams.
[0317] A significant problem involving current crystallization
approaches is determining the conditions for forming crystals with
optimal diffractive properties. Normally crystals have to be grown,
isolated, mounted, and their diffractive properties determined
using an x-ray generator or a synchrotron. Microfluidic systems
with thin, non-scattering walls would be desirable for determining
the diffractive properties of crystals inside a microfluidic
system. Preferably, crystallization is carried out inside this
system using methods according to the invention, which are
described herein. The crystals are exposed to x-ray beams either to
determine their structure or diffractive properties (the screening
mode). For example, a PDMS membrane defining two side walls of the
channels could be sandwiched between two very thin glass plates
(defining the top and bottom walls of the channels) that do not
significantly scatter X-rays. Thus, the devices of the invention
offer a further advantage in that structural characterization could
be conducted while the sample is inside the microfluidic device.
Thus, the sample can be characterized without the need to take out
the sample, e.g., crystal, from the device.
[0318] The present system enables higher throughput automated
systems with improved speed, sample economy, and entirely new
methods of controlling crystallization. Microfluidic versions of
microbatch, vapor phase diffusion and FID techniques may be carried
out using the present invention, as described below, or using a
combination of these techniques or other techniques. In addition,
the nucleation and growth phases may be carried out in discrete
steps through merging plugs, as described herein.
[0319] Screening for protein crystallization can involve varying a
number of parameters. During crystallization screening, a large
number of chemical compounds may be employed. These compounds
include salts, small and large molecular weight organic compounds,
buffers, ligands, small-molecule agents, detergents, peptides,
crosslinking agents, and derivatizing agents. Together, these
chemicals can be used to vary the ionic strength, pH, solute
concentration, and target concentration in the plug, and can even
be used to modify the target. The desired concentration of these
chemicals to achieve crystallization is variable, and can range
from nanomolar to molar concentrations.
[0320] A typical crystallization mix may contain a set of fixed,
but empirically-determined, types and concentrations of
precipitation agent, buffers, salts, and other chemical additives
(e.g., metal ions, salts, small molecular chemical additives,
cryoprotectants, etc.). Water is a key solvent in many
crystallization trials of biological targets, as many of these
molecules may require hydration to stay active and folded.
Precipitation agents act to push targets from a soluble to
insoluble state, and may work by volume exclusion, changing the
dielectric constant of the solvent, charge shielding, and molecular
crowding. Precipitation agents compatible with the PDMS material of
certain embodiments according to the invention include, but are not
limited to, nonvolatile salts, high molecular weight polymers,
polar solvents, aqueous solutions, high molecular weight alcohols,
divalent metals.
[0321] Precipitation agents, which include large and small
molecular weight organics, as well as certain salts, may be used
from under 1% to upwards of 40% concentration, or from <0.5M to
greater than 4M concentration. Water itself can act in a
precipitating manner for samples that require a certain level of
ionic strength to stay soluble. Many precipitation agents may also
be mixed with one another to increase the chemical diversity of the
crystallization screen. Devices according to the invention are
readily compatible with a broad range of such compounds.
[0322] A nonexclusive list of salts that may be used as
precipitation agents is as follows: tartrates (Li, Na, K, Na/K,
NH.sub.4); phosphates (Li, Na, K, Na/K, NH.sub.4); acetates (Li,
Na, K, Na/K, Mg, Ca, Zn, NH.sub.4); formates (Li, Na, K, Na/K, Mg,
NH.sub.4); citrates (Li, Na, K, Na/K, NH.sub.4); chlorides (Li, Na,
K, Na/K, Mg, Ca, Zn, Mn, Cs, Rb, NH.sub.4); sulfates (Li, Na, K,
Na/K, NH.sub.4); maleates (Li, Na, K, Na/K, NH.sub.4); glutamates
(Li, Na, K, Na/K, NH.sub.4.
[0323] A nonexclusive list of organic materials that may be used as
precipitation agents is as follows: PEG 400; PEG 1000; PEG 1500;
PEG 2K; PEG 3350; PEG 4K; PEG 6K; PEG 8K; PEG 10K; PEG 20K; PEG-MME
550; PEG-MME 750; PEG-MME 2K; PEGMME 5K; PEG-DME 2K; dioxane;
methanol; ethanol; 2-butanol; n-butanol; t-butanol; jeffamine
m-600; isopropanol; 2-methyl-2,4-pentanediol; 1,6 hexanediol.
[0324] Solution pH can be varied by the inclusion of buffering
agents; typical pH ranges for biological materials lie anywhere
between values of 3 and 10.5 and the concentration of buffer
generally lies between 0.01 and 0.25 M. The microfluidics devices
described in this document are readily compatible with a broad
range of pH values, particularly those suited to biological
targets.
[0325] A nonexclusive list of possible buffers that may be used
according to the invention is as follows: Na-acetate; HEPES;
Na-cacodylate; Na-citrate; Na-succinate; Na--K-phosphate; TRIS;
TRIS-maleate; imidazole-maleate; bistrispropane; CAPSO, CHAPS, MES,
and imidazole.
[0326] Additives are small molecules that affect the solubility
and/or activity behavior of the target. Such compounds can speed up
crystallization screening or produce alternate crystal forms or
polymorphs of the target. Additives can take nearly any conceivable
form of chemical, but are typically mono and polyvalent salts
(inorganic or organic), enzyme ligands (substrates, products,
allosteric effectors), chemical crosslinking agents, detergents
and/or lipids, heavy metals, organometallic compounds, trace
amounts of precipitating agents, and small molecular weight
organics.
[0327] The following is a nonexclusive list of additives that may
be used in accordance with the invention: 2-butanol; DMSO;
hexanediol; ethanol; methanol; isopropanol; sodium fluoride;
potassium fluoride; ammonium fluoride; lithium chloride anhydrous;
magnesium chloride hexahydrate; sodium chloride; calcium chloride
dihydrate; potassium chloride; ammonium chloride; sodium iodide;
potassium iodide; ammonium iodide; sodium thiocyanate; potassium
thiocyanate; lithium nitrate; magnesium nitrate hexahydrate; sodium
nitrate; potassium nitrate; ammonium nitrate; magnesium formate;
sodium formate; potassium formate; ammonium formate; lithium
acetate dihydrate; magnesium acetate tetrahydrate; zinc acetate
dihydrate; sodium acetate trihydrate; calcium acetate hydrate;
potassium acetate; ammonium acetate; lithium sulfate monohydrate;
magnesium sulfate heptahydrate; sodium sulfate decahydrate;
potassium sulfate; ammonium sulfate; di-sodium tartrate dihydrate;
potassium sodium tartrate tetrahydrate; di-ammonium tartrate;
sodium dihydrogen phosphate monohydrate; di-sodium hydrogen
phosphate dihydrate; potassium dihydrogen phosphate; di-potassium
hydrogen phosphate; ammonium dihydrogen phosphate; di-ammonium
hydrogen phosphate; tri-lithium citrate tetrahydrate; tri-sodium
citrate dihydrate; tri-potassium citrate monohydrate; diammonium
hydrogen citrate; barium chloride; cadmium chloride dihydrate;
cobaltous chloride dihydrate; cupric chloride dihydrate; strontium
chloride hexahydrate; yttrium chloride hexahydrate; ethylene
glycol; Glycerol anhydrous; 1,6 hexanediol; MPD; polyethylene
glycol 400; trimethylamine HCl; guanidine HCl; urea;
1,2,3-heptanetriol; benzamidine HCl; dioxane; ethanol;
iso-propanol; methanol; sodium iodide; L-cysteine; EDTA sodium
salt; NAD; ATP disodium salt; D(+)-glucose monohydrate;
D(+)-sucrose; xylitol; spermidine; spermine tetra-HCl;
6-aminocaproic acid; 1,5-diaminopentane diHCl; 1,6-diaminohexane;
1,8-diaminooctane; glycine; glycyl-glycyl-glycine; hexaminecobalt
trichloride; taurine; betaine monohydrate; polyvinylpyrrolidone K5;
non-detergent sulfo-betaine 195; non-detergent sulfo-betaine 201;
phenol; DMSO; dextran sulfate sodium salt; Jeffamine M-600; 2,5
Hexanediol; (+/-)-1,3 butanediol; polypropylene glycol P400; 1,4
butanediol; tert-butanol; 1,3 propanediol; acetonitrile; gamma
butyrolactone; propanol; ethyl acetate; acetone; dichloromethane;
n-butanol; 2,2,2 trifluoroethanol; DTT; TCEP; nonaethylene glycol
monododecyl ether, nonaethylene glycol monolauryl ether;
polyoxyethylene (9) ether; octaethylene glycol monododecyl ether,
octaethylene glycol monolauryl ether; polyoxyethylene (8) lauryl
ether; Dodecyl-.beta.-D-maltopyranoside; Lauric acid sucrose ester;
Cyclohexyl-pentyl-.beta.-D-maltoside; Nonaethylene glycol
octylphenol ether; Cetyltrimethylammonium bromide;
N,N-bis(3-D-gluconamidopropyl)-deoxycholamine;
Decyl-.beta.-D-maltopyranoside; Lauryldimethylamine oxide;
Cyclohexyl-pentyl-.beta.-D-maltoside; n-Dodecylsulfobetaine,
3-(Dodecyldimethylanimonio)propane-1-sulfonate;
Nonyl-.beta.-D-glucopyranoside; Octyl-.beta.-D-thioglucopyranoside,
OSG; N,N-Dimethyldecylamine-.beta.-oxide; Methyl
0-(N-heptylcarbamoyl)-.alpha.-D-glucopyranoside; Sucrose
monocaproylate;
n-Octanoyl-.beta.-D-fructofuranosyl-.alpha.-D-glucopyranoside;
Heptyl-.beta.-D-thioglucopyranoside;
Octyl-.beta.-D-glucopyranoside, OG;
Cyclohexyl-propyl-.beta.-D-maltoside;
Cyclohexylbutanoyl-N-hydroxyethylglucamide; n-decylsulfobetaine,
3-(Decyldimethylammonio)propane-Isulfonate;
Octanoyl-N-methylglucamide, OMEGA; Hexyl-.beta.-D-glucopyranoside;
Brij 35; Brij 58; Triton X-114; Triton X-305; Triton X-405; Tween
20; Tween 80; polyoxyethylene(6)decyl ether;
polyoxyethylene(9)decyl ether; polyoxyethylene(10)dodecyl ether;
polyoxyethylene(8)tridecyl ether; Decanoyl-N-hydroxyethylglucamide;
Pentaethylene glycol monooctyl ether;
3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate;
3-[(3-Cholamidopropyl)-dimethylammonio]hydroxy-1-propane sulfonate;
Cyclohexylpentanoyl-N-hydroxyethylglucamide;
Nonanoyl-N-hydroxyethyglucamide;
Cyclohexylpropanol-N-hydroxyethylglucamide;
Octanoyl-N-hydroxyethylglucamide;
Cyclohexylethanoyl-N-hydroxyethylglucamide; Benzyldimethyldodecyl
ammonium bromide; n-Hexadecyl-.beta.-D-maltopyranoside;
n-Tetradecyl-.beta.-D-maltopyranoside;
n-Tridecyl-.beta.-D-maltopyranoside;
Dodecylpoly(ethyleneglycoether); n-Tetradecyl-N,N-dimethyl
ammonio-1-propanesulfonate; n-Undecyl-.beta.-D-maltopyranoside;
n-Decyl D-thiomaltopyranoside; n-dodecylphosphocholine;
.alpha.-D-glucopyranoside, .beta.-D-fructofuranosyl monodecanoate,
sucrose mono-caprate; 1-s-Nonyl-.beta.-D-thioglucopyranoside;
n-Nonyl-.beta.-D-thiomaltoyranoside;
N-Dodecyl-N,N-(dimethlammonio)butyrate;
n-Nonyl-.beta.-D-maltopyranoside; Cyclohexyl-butyl D-maltoside;
n-Octyl-.beta.-D-thiomaltopyranoside; n-Decylphosphocholine;
n-Nonylphosphocholine; Nonanoyl-N-methylglucamide;
1-s-Heptyl-.beta.-D-thioglucopyranoside; n-Octylphosphocholine;
Cyclohexyl-ethyl D-maltoside; n-Octyl-N,N-dimethyl
ammonio-1-propanesulfonate;
Cyclohexyl-methyl-.beta.-D-maltoside.
[0328] Cryosolvents are agents that stabilize a target crystal to
flash-cooling in a cryogen such as liquid nitrogen, liquid propane,
liquid ethane, or gaseous nitrogen or helium (all at approximately
100-120.degree. K) such that crystal becomes embedded in a vitreous
glass rather than ice. Any number of salts or small molecular
weight organic compounds can be used as a cryoprotectant, and
typical ones include but are not limited to: MPD, PEG-400 (as well
as both PEG derivatives and higher molecular-weight PEG compounds),
glycerol, sugars (xylitol, sorbitol, erythritol, sucrose, glucose,
etc.), ethylene glycol, alcohols (both short- and long chain, both
volatile and nonvolatile), LiOAc, LiCl, LiCHO.sub.2, LiNO.sub.3,
Li.sub.2SO.sub.4, Mg(OAc).sub.2, NaCl, NaCHO.sub.2, NaNO.sub.3,
etc. Again, materials from which microfluidics devices in
accordance with the present invention are fabricated may be
compatible with a range of such compounds.
[0329] Many of these chemicals can be obtained in predefined
screening kits from a variety of vendors, including but not limited
to Hampton Research of Laguna Niguel, Calif., Emerald Biostructures
of Bainbridge Island, Wash., and Jena BioScience of Jena, Germany,
that allow the researcher to perform both sparse matrix and grid
screening experiments. Sparse matrix screens attempt to randomly
sample as much of precipitant, buffer, and additive chemical space
as possible with as few conditions as possible. Grid screens
typically consist of systematic variations of two or three
parameters against one another (e.g., precipitant concentration vs.
pH). Both types of screens have been employed with success in
crystallization trials, and the majority of chemicals and chemical
combinations used in these screens are compatible with the chip
design and matrices in accordance with embodiments of the present
invention. Moreover, current and future designs of microfluidic
devices may enable flexible combinatorial screening of an array of
different chemicals against a particular target or set of targets,
a process that is difficult with either robotic or hand screening.
This latter aspect is particularly important for optimizing initial
successes generated by first-pass screens.
[0330] In addition to chemical variability, a host of other
parameters can be varied during crystallization screening. Such
parameters include but are not limited to: (1) volume of
crystallization trial; (2) ratio of target solution to
crystallization solution; (3) target concentration; (4)
cocrystallization of the target with a secondary small or
macromolecule; (5) hydration; (6) incubation time; (7) temperature;
(8) pressure; (9) contact surfaces; (10) modifications to target
molecules; and (11) gravity.
[0331] Although the discussion below refers to proteins, the
particular devices or methods described can also be used or
suitably adapted for the crystallization of other types of samples
such as those mentioned above (e.g., small molecules, other
macromolecules, nanoparticles, colloidal particles, etc.). In one
aspect of the present invention, protein crystallization is
conducted using miniaturized microbatch conditions. The process
consists of two steps. First, plugs are preferably formed wherein
the concentrations of the protein, precipitant, and additive are
adjusted by varying the relative flow rates of these solutions.
This step corresponds to a screening step. Once the optimal
concentrations have been found, the flow rates can then be kept
constant at the optimal conditions. In this step, plugs are
preferably transported through the channel as they form. Second,
the flow is preferably stopped once the desired number of plugs are
formed. The plugs are then preferably allowed to incubate. In some
embodiments according to the invention the flow may be continued,
rather than stopped. In those embodiments, the flow is maintained
sufficiently slow and the channels are made sufficiently long that
plugs spend sufficient time in the channels for crystallization to
occur (from tens of minutes to weeks, but may be faster or
slower).
[0332] In one aspect, upon formation of the plugs, they are trapped
using expansions in the channels. The expansions act as dead volume
elements while the plugs are being formed in the presence of flow.
Thus, the expansions do not interfere with the flow of the plugs
through the channel. Once the flow is stopped, surface tension
drives plugs into the expansions where surface tension is
minimized. The expansions may be, but are not limited to, oval,
round, square, rectangular, or star-shaped. In particular, a
star-shaped expansion may prevent adherence of the plug or of a
crystal to the walls of the expansion. The ratio of the size of the
expansion opening to the width of the channel may be varied based
on empirical results for a particular set of conditions. FIG. 16 is
a schematic illustration of a microfluidic device according to the
invention that illustrates the trapping of plugs. In experiments,
plugs were sustained in perfluorodecaline inside a channel for one
day, and did not appear to change during that time (a refractive
index mismatch between the fluorinated and aqueous phase was
introduced to aid in visualization of plugs).
[0333] The method described above allows a high degree of control
over protein and precipitant concentrations. It also allows a high
degree of control over a range of time scales through the control
of plug size and composition. FIG. 17 shows a schematic of a
microfluidic method for forming plugs with variable compositions
for protein crystallization. Continuously varied flow rates of the
incoming streams are preferably used to form plugs with various
concentrations of the protein, precipitation agents, and additives.
In FIG. 17, for example, the following can be introduced into the
various inlets: buffers into inlets 171, 172; PEG into inlet 173;
salt into inlet 174; solvent into inlet 175; and protein into inlet
176. These various solutions can enter a channel 177 through which
a carrier fluid such as perfluorodecaline flows. For example, a
1-meter long channel with a 200.times.80 .mu.m.sup.2 cross section
can be used to form approximately two hundred 6 nL (nanoliter)
plugs. If each plug contains enough protein to form a
40-.mu.m.sup.3 crystal, 200 trials will consume only about 1.2
.mu.L of approximately 10 mg/mL protein solution (12 .mu.g of
protein). About one minute may be sufficient to form plugs in these
trials.
[0334] In another aspect according to the invention, after plugs
are formed as described above for the microbatch system, slow
evaporation through a very thin PDMS membrane (or another membrane
with slight water permeability) is preferably used for added
control over the crystallization process. A slow decrease in the
volume of the plug during evaporation is expected to produce a
trajectory of the solution through the crystallization phase space
similar to that in a vapor diffusion experiment. Hence, this
method, in addition to microbatch methods, can be used to
miniaturize and optimize vapor diffusion methods.
[0335] In the vapor diffusion method, a drop containing protein,
stabilizing buffers, precipitants, and/or crystallization agents is
allowed to equilibrate in a closed system with a much larger
reservoir. The reservoir usually contains the same chemicals minus
the protein but at an over all higher concentration so that water
preferentially evaporates from the drop. If conditions are right,
this will produce a gradual increase in protein concentration such
that a few crystals may form.
[0336] Vapor diffusion can be performed in several ways. The one
most often used is called Hanging Drop Technique. The drop is
placed on a glass coverslip, which is then inverted and used to
seal a small reservoir in a Linbro Plate. After a period of several
hours to weeks, microscopic crystals may form and continue to grow.
The other set up is known as Sitting Drop. In this method a drop
(usually >10 uL) is placed in a depression in either a Micro
Bridge in a Linbro Plate or a glass plate and again placed in a
closed system to equilibrate with a much larger reservoir. One
usually uses the sitting drop technique if the drop has very low
surface tension, making it hard to turn upside down or if the drops
need to be larger than 20 uL. Also, in some cases, crystals will
grow better using one technique or the other.
[0337] In another embodiment, the plugs are preferably formed and
transported such that excessive mixing of the protein with the
precipitation agent is minimized or prevented. For example, gentle
mixing using spiral channels may be used to achieve this and also
to create interfaces between the protein and the precipitation
agent. Alternatively, combining two streams of plugs in a
T-junction without merging may be used to create plugs that diffuse
and combine without significant mixing to establish a free
interface after the flow is stopped. Diffusion of the proteins and
precipitates through the interface induces crystallization. This is
an analogue of the Free-Interface Diffusion method. It may be
performed under either the microbatch or vapor diffusion conditions
as described above.
[0338] Preferably, the spacing between plugs can be increased or
the oil composition changed to reduce plug-plug diffusion. For
example, a spacing of about 2.5 mm in paraffin oil can be used,
which has been shown to be an effective barrier to aqueous
diffusion in crystallization trials.
[0339] Visually identifying small crystals inside plugs with curved
surfaces can be a challenge when performing microbatch experiments.
In an aspect according to the invention, a method based on matching
the refractive indices of carrier-fluid with that of the plug fluid
to enhance visualization is used. Microscopic detection is
preferably performed by using shallow channels and by matching the
refractive indices of carrier-fluid mixtures to those of the
aqueous solutions.
[0340] In addition, at least three other novel methods of
controlling protein crystallization are described below: (1) using
surface chemistry to effect nucleation of protein crystals; (2)
using different mixing methods to effect crystallization; and (3)
performing protein crystals seeding by separating nucleation and
growth phases in space.
[0341] Control of nucleation is one of the difficult steps in
protein crystallization. Heterogeneous nucleation is statistically
a more favorable process than its solution-phase counterpart. Ideal
surfaces for heterogeneous nucleation have complementary
electrostatic maps with respect to their macromolecular
counterparts. Critical nuclei are more stable on such surfaces than
in solution. Further, the degree of supersaturation required for
heterogeneous nucleation is much less than that required for the
formation of solution-phase nuclei. Surfaces such as silicon,
crystalline minerals, epoxide surfaces, polystyrene beads, and hair
are known to influence the efficiency of protein crystallization.
Few studies have been done, but promising results have been shown
for protein crystallization at the methyl, imidazole, hydroxyl, and
carboxylic acid termini of self-assembled monolayers on gold. Using
self-assembled monolayers, proteins were crystallized over a
broader range of crystallization conditions and at faster rates
than when using the traditional silanized glass.
[0342] FIG. 18 is a schematic illustration of a method for
controlling heterogeneous nucleation by varying the surface
chemistry at the interface of an aqueous plug-fluid and a
carrier-fluid. In FIG. 18, plugs are formed in the presence of
several solutions of surfactants that possess different functional
groups (left side of the diagram). The right side of FIG. 18 shows
the aqueous phase region in which a precipitant, solvent, and
protein may be introduced into inlets 180, 181, and 182,
respectively. The composition of the surfactant monolayer is
preferably controlled by varying the flow rates. In another
application of the method illustrated in FIG. 18, the surface
chemistry can be varied continuously. The manipulation and control
of the surface chemistry can be used for screening, assays,
crystallizations, and other applications where surface chemistry is
important.
[0343] In one aspect of the invention, heterogeneous nucleation of
proteins is controlled by forming aqueous plugs in a carrier-fluid,
preferably containing fluoro-soluble surfactants if the
carrier-fluid is a fluorocarbon. Varying the relative flow rates of
the surfactant solutions may generate a wide variety of
liquid-liquid interface conditions that can lead to the formation
of mixed monolayers or mixed phase-separated monolayers.
Preferably, several surfactants are used to control the
heterogeneous nucleation of protein crystals. Ethylene-glycol
monolayers are preferably used to reduce heterogeneous nucleation,
and monolayers with electrostatic properties complementary to those
of the protein are preferably used to enhance heterogeneous
nucleation. These methods for controlling heterogeneous nucleation
are designed to induce or enhance the formation of crystals that
are normally difficult to obtain. These methods may also be used to
induce or enhance the formation of different crystal polymorphs
that are relatively more stable or better ordered.
[0344] As mentioned above, control of nucleation is highly desired
in an advanced crystallization screen. One method that can be used
to achieve control of nucleation involves the transfer of
nucleating crystals from one concentration to another via dilution.
This method, which has been applied in macroscopic systems
primarily to vapor diffusion, was intended to allow decoupling of
the nucleation and growth phases. This method is difficult to
perform using traditional methods of crystallization because
nucleation occurs long before the appearance of microcrystals.
[0345] FIG. 19 illustrates a method of separating nucleation and
growth using a microfluidic network according to the present
invention using proteins as a non-limiting example. The left side
of FIG. 19 shows plugs that are formed preferably using high
concentrations of protein and precipitant. In FIG. 19, the
following can be introduced into the various inlets shown: buffer
into inlets 191, 196; PEG into inlets 192, 197; precipitant into
inlets 193, 198; solvent into inlets 194, 199; and protein into
inlets 195, 200. Oil flows through the channels 201, 202 from left
to right. The portions 203, 204, and 205 of the channel correspond
to regions where fast nucleation occurs (203), no nucleation occurs
(204), and where crystal growth occurs (205). The concentrations
used are those that correspond to the nucleating region in the
phase diagram. Nucleation occurs as the plugs move through the
channel to the junction over a certain period. Preferably, these
plugs are then merged with plugs containing a protein solution at a
point corresponding to a metastable (growth, rather than
nucleation) region (right side of FIG. 19). This step ends
nucleation and promotes crystal growth. When the combined channel
has been filled with merged plugs, the flow is preferably stopped
and the nuclei allowed to grow to produce crystals.
[0346] Nucleation time can be varied by varying the flow rate along
the nucleation channel. The nucleus is preferably used as a seed
crystal for a larger plug with solution concentrations that
correspond to a metastable region. Existing data indicate the
formation of nuclei within less than about 5 minutes.
[0347] Fluid mixing is believed to exert an important effect in
crystal nucleation and growth. Methods according to the invention
are provided that allow a precise and reproducible degree of
control over mixing. FIG. 20 illustrates two of these methods. A
method of mixing preferably places the solution into a nucleation
zone of the phase diagram without causing precipitation.
Preferably, gentle mixing (FIG. 20, left side) is used to achieve
this by preventing, reducing, or minimizing contact between
concentrated solutions of the protein and precipitant.
Alternatively, rapid mixing (FIG. 20, right side) is used to
achieve this by allowing passage through the precipitation zone
sufficiently quickly to cause nucleation but not precipitation. The
two methods used as examples involve the use of spiraling channels
for gentle mixing and serpentine channels for rapid mixing.
[0348] The two methods in accordance with the invention depicted in
FIG. 20 can be used to determine the effect of mixing on protein
crystallization. In addition, the various methods for controlling
mixing described previously (e.g., slow mixing in straight
channels, chaotic mixing in non-straight channels, or mixing in
which twirling may or may not occur) can be applied to
crystallization, among other things.
[0349] After obtaining the crystals using any of the above
described techniques, the crystals may be removed from the
microfluidic device for structure determination. In other systems,
the fragile and gelatinous nature of protein crystals makes crystal
collection difficult. For example, removing protein crystals from
solid surfaces can damage them to the point of uselessness. The
present invention offers a solution to this problem by nucleating
and growing crystals in liquid environments. In an aspect according
to the invention, a thin wetting layer of a carrier-fluid covered
with a surfactant is used to enable or facilitate the separation of
a growing crystal from a solid surface. When the crystals form,
they may be separated from the PDMS layer by using a thin layer of
a carrier-fluid.
[0350] In one aspect, a microfluidic device of the present system
can include further include capillary tubing suitable for
collecting plugs ("the capillary device"; FIG. 46). The tubing is
preferably composed of a material that prevents uncontrolled
evaporation of solutions (such as water) through its wall. Further,
use of the capillary tubing can enable direct screening of crystals
by x-ray diffraction analysis or other spectrophotometric
detection/analysis means employing e.g., optical or infrared
detection. Plugs in the capillary tubing have been found to be
stable and did not show signs of evaporation over several months,
even in the absence of humidity control. Therefore, the capillary
device can be incubated for a much longer time than all-PDMS
microfluidic chips. Water diffusion can be controlled by varying
the starting salt concentration differences as well the distance
between plugs. Production of crystals directly inside the capillary
tubes can facilitate on-chip diffraction without having to move the
crystal around.
[0351] Upon formation of plugs in the PDMS portion and their
transfer into capillary tubing, the flow rates are stopped, the
capillary tubing is disconnected from the PDMS portion and the ends
are sealed by capillary wax. The capillary tubing may be incubated
under suitable crystallization conditions (e.g, temperature etc.)
until crystals form inside the plugs. Formation of crystals can be
monitored using optical detection and/or x-ray diffraction methods.
Crystals grown at the fluid-fluid interface can be easily removed
from the capillary by gentle flow, or by breaking the capillary and
wicking the liquid out. Upon formation of suitable crystals, the
capillaries are frozen and structures are directly determined from
inside the capillary using e.g., synchrotron radiation. Because
this method obviates the problem of handling and mounting crystals
and because it can facilitate the determination of structure
directly from within the capillary, it may be especially suitable
for high-throughput, fully automated crystallization.
[0352] The plugs in the capillary tubing can be stable in both
hydrophilic (e.g., treated with by chromic acid) or hydrophobic
(e.g., silanized) capillaries for over a month, even if the
capillary is placed vertically for over three days.
[0353] The use of x-ray capillary tubing for protein
crystallization can also be applied to a controlled vapor diffusion
process which lends itself to direct monitoring and structural
determination of protein crystals in the capillary tubing (FIG.
49). In this modified vapor-diffusion process an array of plugs is
generated in the channel portion of a capillary device (as
described above) where the protein and precipitant plugs alternate
with plugs containing a high concentration of precipitant. Syringe
pumps attached to the capillary device cause the plugs to flow into
suitable x-ray capillary tubing. At the conclusion of the
experiment, the flow is stopped, the capillary is disconnected from
the PDMS portion and the ends are sealed with capillary wax. The
x-ray capillary is incubated under optimal conditions until
crystals form inside the plugs.
[0354] The use of carrier fluid (oil) permeable to water causes the
water from the plugs to diffuse through from the oil from the plugs
that are low in osmolarity into plugs that are higher in
osmolarity, thereby increasing the concentration of the protein and
precipitants in the plugs for crystallization. The rate of water
transfer from the plugs and the amount of water transferred between
the two types of plugs may be controlled by using oils having
different water permeabilities, by changing the size or distance
between plugs or by altering the precipitant concentrations between
the different types of plugs (i.e., changing the difference in
osmolarity between the different plug types). All of these
parameters can be conveniently altered by changing the relative
flow rates of the aqueous and carrier-fluid (oil) solutions.
Poly-3,3,3-trifluoropropylmethylsiloxane (FMS-121) can be a
suitable carrier-oil fluid for this procedure.
[0355] One scheme for generating alternating plugs by vapor
diffusion involves attaching four different syringes to a PDMS
device, each syringe associated with a syringe pump for introducing
each of aqueous solutions A, B into respective aqueous inlet
channels and for introducing each of carrier oil fluids C, D into
respective oil inlet channels. The aqueous solutions can be the
same or different. Multiple, distinct aqueous solutions can also be
co-introduced together in one or both of the two aqueous channels.
In principle, the same oil or different oils may be used in the two
oil inlets. In either case, one oil inlet channel is parallel to
the main channel; the other oil inlet channel is vertical to the
main channel and is positioned between the two aqueous inlet
channels to separate the two aqueous streams into alternating
plugs.
[0356] Importantly, the flow rates of solutions A and B may be
changed in a correlated fashion. Thus, when the flow rate of
solution A.sub.1 is increased and solution A.sub.2 is decreased,
the flow rate of solutions B.sub.1 is also increased and solution
B.sub.2 is also decreased. This can allow one to maintain a
constant difference in osmolarity between the plugs of stream A and
stream B to ensure that transfer from all plugs A to all plugs B
occurs at a constant rate. Moreover, if the flow rates of the
corresponding A and B streams are changed in a correlated fashion,
the composition of plugs B will reflect the composition of plugs A
thereby allowing one to incorporate markers into the B stream plugs
to serve as a code for the plugs in the A stream. Thus, if the two
types of plugs are made in a correlated way, one type of droplet
may be used for crystallization, while the other type of droplet is
used for indexing provided it contains a label conferring a read
out with respect to crystallization. In other words,
absorption/fluorescent dyes or x-ray scattering/absorbing materials
can be incorporated in markers in the B streams to facilitate
optical density quantification or x-ray diffraction analysis to
provide a read out of relative protein and precipitant
concentrations in the A streams. This approach can provide a
powerful means for optimizing crystallization conditions for
subsequent scale-up experiments.
[0357] The use of markers may be performed using an oil that is
impermeable to water (as in a microbatch procedure) to prevent
transfer of water or any other material between the A plugs and B
plugs. Alternatively, the B plugs may additionally incorporate a
high concentration of dehydration agents (salt, other precipitants)
in conjunction with a water-permeable oil as described above. In
this way, the B plugs can serve both as markers for the A plugs and
as sinks for excess water. Oils that are selectively permeable to
materials other than water may also be used to induce transfer of
other materials between the plugs and through the oil.
[0358] Alternating plugs may be generated using a range of channel
geometries. The plugs may also alternate in patterns other than
A:B:A:B. For example, other patterns (such as A:A:A:B:A:A:A:B, etc)
may be obtained where transfer of water from A plugs adjacent to B
plugs is faster than transfer of water from the middle A plug. This
can create conditions favorable for creating multiple, different
sets of crystallization conditions. The alternating droplet systems
may be extended to more than two types of plugs alternating in the
same channel or capillary (for example, A plugs with the
crystallization solutions, B plugs with the dehydrating agents, and
C plugs with markers or with a cryoprotectant).
[0359] The above described capillary systems are not limited to
protein crystallization--other types of crystallizations and
experiments may be performed. For example, the vapor
diffusion/alternating droplet approach can be extended to e.g., a
process for concentrating materials (such as protein). Such a
process would be effected through diffusion of water plugs that are
relatively low in osmolarity into plugs having a higher osmolarity.
It should be noted, however, that solution materials in the
different plug types do not have to be aqueous in nature, but can
be in the form of solvents also. Alternatively, the A and B plugs
do not have to be in solution at all, but can instead be in the
form of emulsions or suspensions.
[0360] It will be clear to one skilled in the art that while the
above techniques are described in detail for the crystallization of
proteins, techniques similar to the ones described above may also
be used for the crystallization of other substances, including
other biomolecules or synthetic chemicals. In addition, the devices
and methods according to the invention may be used to perform
co-crystallization. For example, a crystal comprising more than one
chemical may be obtained, for example, through the use of at least
one stream of protein, a stream of precipitant, and optionally, a
stream comprising a third chemical such as an inhibitor, another
protein, DNA, etc. One may then vary the conditions to determine
those that are optimal for forming a co-crystal.
Particle Separation/Sorting Using Plugs
[0361] The flow within the moving plugs can be used for separation
of polymers and particles. Plugs can be used for separation by
first using flow within a moving plug to establish a distribution
of the polymers or particles inside the plug (for example, an
excess of the polymer inside the front, back, right or left side of
the plug) and then using splitting to separate and isolate the part
of the plug containing higher concentration of the polymers or
particles. When two polymers or particles are present inside the
plug and establish different distributions, splitting can be used
to separate the polymers or particles.
[0362] The invention is further described below, by way of the
following examples. It will be appreciated by persons of ordinary
skill in the art that this example is one of many embodiments and
is merely illustrative. In particular, the device and method
described in this example (including the channel architectures,
valves, switching and flow control devices and methods) may be
readily adapted, e.g., used in conjunction with one or more devices
or methods, so that plugs may be analyzed, characterized,
monitored, and/or sorted as desired by a user.
EXAMPLE
Example 1
Fabrication of Microfluidic Devices and a General Experimental
Procedure
[0363] Microfluidic devices with hydrophilic channel surfaces were
fabricated using rapid prototyping in polydimethylsiloxane. The
channel surfaces were rendered hydrophobic either by silanization
or heat treatment. To silanize the surfaces of channels,
(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (United
Chemical Technologies, Inc.) vapor was applied to the inlets of a
device with dry nitrogen as a carrier gas at around 40-60 mm Hg
above about 1 atm pressure. Vacuum was simultaneously applied to
the outlet of the device at about 650 mm Hg below atmospheric
pressure. The silane vapor was applied for a period of between
about 1-3 hours. To treat the channels using heat, a device was
placed in an oven at approximately 120.degree. C. for about three
hours. Alternatively, a device can be heated in a Panasonic "The
Genius" 1300 Watt microwave oven at power set to "10" for about ten
minutes.
[0364] Oils and aqueous solutions were pumped through devices using
a kdScientific syringe pump (Model 200) or Harvard Apparatus PhD
2000 pump. Hamilton Company GASTIGHT syringes were used (10-250
.mu.l) and Hamilton Company 30 gauge Teflon.RTM. needles were used
to attach the syringes to the devices. Oils and aqueous solutions
were pumped through devices at volumetric flow rates ranging from
about 0.10 .mu.L/min to about 10.0 .mu.L/min.
[0365] Aqueous solutions were colored using Crayola Original
Formula Markers or Ferroin Indicator (0.025 M, Fisher Scientific).
Oils that were used included perfluorodecaline (mixture of cis and
trans, 95%, Acros Organics), perfluoroperhydrophenanthrene (tech.,
Alfa-Aesar), or 1H,1H,2H,2H-perfluorooctanol (98%, Alfa-Aesar). The
experiments were typically performed using 10:1 mixtures of
perfluorodecaline and 1H,1H,2H,2H-perfluorooctanol.
[0366] The experiments were monitored using a Lica MZFLIII
stereoscope with Fostec (Schott-Fostec, LLC) Modulamps. Photographs
of the experiments were taken with a Spot Insight Color Camera,
Model #3.2.0 (Diagnostic Instruments, Inc.). Spot Application
version 3.4.0.0 was used to take the photographs with the
camera.
Example 2
Varying the Concentration of Aqueous Solutions in Plugs
[0367] The left side of each of FIGS. 25A-C shows a schematic
diagram of the microfluidic network and the experimental
conditions. The right side of each of FIGS. 25A-C shows
microphotographs illustrating the formation of plugs using
different concentrations of the aqueous streams. Aqueous solutions
of food dyes (red/dark and green/light) and water constituted the
three streams. The volumetric flow rates of the three solutions
(given in .mu.L/min) are indicated. The dark stream is more viscous
than the light stream. Therefore, the dark (more viscous) stream
moves (measured in mm/s) more slowly and occupies a larger fraction
of the channel at a given volumetric flow rate.
[0368] FIG. 45a) shows a schematic of the microfluidic network used
to demonstrate that on-chip dilutions can be accomplished by
varying the flow rates of the reagents. In FIG. 45a), the reagents
are introduced through inlets 451, 453 while the dilution buffer is
introduced through inlet 452. An oil stream flows through channel
454. The blue rectangle outlines the field of view for images shown
in FIG. 45c)-d). FIG. 45b) shows a graph quantifying this dilution
method by measuring fluorescence of a solution of fluorescein
diluted in plugs in the microchannel. Data are shown for 80
experiments in which fluorescein was flowed through one of the
three inlets, where C.sub.measured and C.sub.theoretical [.mu.M]
are measured and expected fluorescein concentration. FIG. 45(c)
shows photographs illustrating this dilution method with streams of
food dyes 455, 456, 457 having flow rates of 45 nL/s, 10 nL/s, and
10 nL/s, respectively. FIG. 45(d) shows photographs illustrating
this dilution method with streams of food dyes 458, 459, 460 having
flow rates of 10 nL/s, 45 nL/s, and 10 nL/s, respectively. Carrier
fluid was flowed at 60 nL/s.
Example 3
[0369] Networks of microchannels with rectangular cross-sections
were fabricated using rapid prototyping in PDMS. The PDMS used was
Dow Corning Sylgard Brand 184 Silicone Elastomer, and devices were
sealed using a Plasma Prep II (SPI Supplies). The surfaces of the
devices were rendered hydrophobic by baking the devices at
120.degree. C. for 2-4 hours.
[0370] In FIG. 26, the red aqueous streams were McCormick.RTM. red
food coloring (water, propylene glycol, FD&C Red 40 and 3,
propylparaben), the green aqueous streams were McCormick.RTM. green
food coloring (water, propylene glycol, FD&C yellow 5, FD&C
blue 1, propylparaben) diluted 1:1 with water, and the colorless
streams were water. PFD used was a 10:1 mixture of
perfluorodecaline (mixture of cis and trans, 95%, Acros
Organics):1H,1H,2H,2H-perfluorooctanol (Acros Organics). The red
aqueous streams were introduced in inlet 260, 265 while the green
aqueous streams were introduced in inlets 262, 263 in FIG. 26b).
The colorless aqueous stream was introduced in inlets 261, 264. The
dark shadings of the streams and plug are due mainly from the red
dye while the lighter shadings are due mainly from the green
dye.
[0371] Aqueous solutions were pumped using 100 .mu.L Hamilton
Gastight syringes (1700 series, TLL) or 50 .mu.L SGE gastight
syringes. PFD was pumped using 1 mL Hamilton Gastight syringes
(1700 series, TLL). The syringes were attached to microfluidic
devices by means of Hamilton Teflon needles (30 gauge, 1 hub).
Syringe pumps from Harvard Apparatus (PHD 2000 Infusion pumps;
specially-ordered bronze bushings were attached to the driving
mechanism to stabilize pumping) were used to infuse the aqueous
solutions and PFD.
[0372] Microphotographs were taken with a Leica MZ12.5
stereomicroscope and a SPOT Insight Color digital camera (Model
#3.2.0, Diagnostic Instruments, Inc.). SPOT Advanced software
(version 3.4.0 for Windows, Diagnostic Instruments, Inc.) was used
to collect the images. Lighting was provided from a Machine Vision
Strobe X-Strobe X1200 (20 Hz, 12 .mu.F, 600V, Perkin Elmer
Optoelectronics). To obtain an image, the shutter of the camera was
opened for 1 second and the strobe light was flashed once with the
duration of the flash being about 10 .mu.s.
[0373] Images were analyzed using NIH Image software, Image J.
Image J was used to measure periods and lengths of plugs from
microphotographs such as shown in FIG. 27b). Periods corresponded
to the distance from the center of one plug to the center of an
adjacent plug, and the length of a plug was the distance from the
extreme front to the extreme back of the plug (see FIG. 28 for the
definitions of front and back). Measurements were initially made in
pixels, but could be converted to absolute measurements by
comparing them to a measurement in pixels of the 50 .mu.m width of
the channel.
[0374] To make measurements of the optical intensity of
Fe(SCN).sub.x.sup.(3-x)+ complexes in plugs, microphotographs were
converted from RGB to CMYK color mode in Adobe Photoshop 6.0. Using
the same program, the yellow color channels of the microphotographs
were then isolated and converted to grayscale images, and the
intensities of the grayscale images were inverted. The yellow color
channel was chosen to reduce the intensity of bright reflections at
the extremities of the plugs and at the interface between the plugs
and the channel. Following the work done in Photoshop, regions of
plugs containing high concentrations of Fe(SCN).sub.x.sup.(3-x)+
complexes appeared white while regions of low concentration
appeared black. Using Image J, the intensity was measured across a
thin, rectangular region of the plug, located halfway between the
front and back of the plug (white dashed lines in FIG. 27a1)). The
camera used to take the microphotographs of the system was not
capable of making linear measurements of optical density.
Therefore, the measurements of intensity were not quantitative.
Several of the plots of intensity versus relative position across
the channel (FIG. 27c) were shifted vertically by less than 50
units of intensity to adjust for non-uniform illuminations of
different parts of the images. These adjustments were justified
because it was the shape of the distribution that was of interest,
rather than the absolute concentration.
[0375] FIG. 29a)-b) shows plots of the sizes of periods and sizes
of plugs as a function of total flow velocity (FIG. 29a)) and water
fraction (wf) (FIG. 29b)). Values of capillary number (C.n.) were
0.0014, 0.0036, 0.0072 and 0.011, while values of the Reynolds
number (R.sub.e) were 1.24, 3.10, 6.21, and 9.31, each of the C.n.
and R.sub.e value corresponding to a set of data points with water
fractions (wf) 0.20, 0.52, 0.52, and 0.20 (the data points from top
to bottom in FIG. 29A)). In turn, each of these sets of data points
corresponds to a particular flow velocity as shown in FIG. 29a).
Plugs in FIG. 29b) travel at about 50 millimeter/second (mm/s). All
measurements of length and size are relative to the width of the
channels (50 .mu.m).
[0376] FIG. 30 shows microphotographs illustrating weak dependence
of periods, length of plugs, and flow patterns inside plugs on
total flow velocity. The left side of FIG. 30 shows a diagram of
the microfluidic network. Here, the same solutions were used as in
the experiment corresponding to FIG. 27. The
Fe(SCN).sub.x.sup.(3-x)+ solution was introduced into inlet 301
while the colorless aqueous streams were introduced into inlets
302, 303. The same carrier fluid as used in the FIG. 27 experiment
was flowed into channel 304. The right side of FIG. 30 shows
microphotographs of plugs formed at the same water fraction (0.20),
but at different total flow velocities (20, 50, 100, 150 mm/s from
top to bottom). Capillary numbers were 0.0014, 0.0036, 0.0072, and
0.011, respectively, from top to bottom. Corresponding Reynolds
numbers were 1.24, 3.10, 6.21, and 9.31.
[0377] FIG. 31A-C are plots showing the distribution of periods and
lengths of plugs where the water fractions were 0.20, 0.40, and
0.73, respectively. The total flow velocity was about 50 mm/s,
C.n.=0.0036, R.sub.e=3.10 in all cases.
[0378] FIG. 27 shows the effects of initial conditions on mixing by
recirculating flow inside plugs moving through straight
microchannels. FIG. 27a1) shows that recirculating flow (shown by
black arrows) efficiently mixed solutions of reagents that were
initially localized in the front and back halves of the plug.
Notations of front, back, left, and right are the same as that in
FIG. 28. FIG. 27a2) shows that recirculating flow (shown by black
arrows) did not efficiently mix solutions of reagents that were
initially localized in the left and right halves of the plugs. The
left side of FIG. 27b) shows a schematic diagram of the
microfluidic network. The two colorless aqueous streams were
introduced into inlets 271, 272 while a carrier fluid in the form
of perfluorodecaline flowed through channel 273. These solutions
did not perturb the flow patterns inside plugs.
[0379] The right side of FIG. 27b) shows microphotographs of plugs
of various lengths near the plug-forming region of the microfluidic
network for water fractions of from 0.14 up to 1.00. FIG. 27c1)
shows a graph of the relative optical intensity of
Fe(SCN).sub.x.sup.(3-x)+ complexes in plugs of varying lengths. The
intensities were measured from left (x=1.0) to right (x=0.0) across
the width of a plug (shown by white dashed lines in FIG. 27a1)-a2))
after the plug had traveled 4.4 times its length through the
straight microchannel. The gray shaded areas indicate the walls of
the microchannel. FIG. 27c2) is the same as FIG. 27c1) except that
each plug had traversed a distance of 1.3 mm. The d/l of each water
fraction (wf) were 15.2 (wf 0.14), 13.3 (wf 0.20), 11.7 (wf 0.30),
9.7 (wf 0.40), 6.8 (wf 0.60), 4.6 (wf 0.73), and 2.7 (wf 0.84),
where d is the distance traveled by the plug and l is the length of
the plug.
Example 4
Merging of Plugs
[0380] Experiments were conducted to investigate the merging of
plugs using different channel junctions (T- or Y-shaped),
cross-sections, and flow rates (see FIG. 33a-d). The figures on the
left side of FIGS. 33a-d show top views of microfluidic networks
that comprise channels having either uniform or nonuniform
dimension (e.g., the same or different channel diameters). The
corresponding figures on the right are microphotographs that
include a magnified view of two plug streams (from the two separate
channels portions of which form the branches of the Y-shaped
junction) that merges into a common channel.
[0381] In FIG. 33a, the oil-to-water volumetric ratio was 4:1 in
each pair of oil and water inlets. The oil streams were introduced
into inlets 330, 332, while the aqueous streams were introduced
into inlets 331, 333. The flow rates of the combined oil/water
stream past the junction where the oil and water meet was 8.6 mm/s.
The channels, which were rectangular, had dimensions of 50
(width).times.50 (height) .mu.m.sup.2. As shown in FIG. 33a, plugs
that flow in uniform-sized channels typically merged only when they
simultaneously arrived at the T-junction. Thus, plug merging in
these channels occur infrequently. In addition, lagging plugs were
typically not able to catch up with leading plugs along the common
channel.
[0382] FIG. 33b illustrates plug merging occurring between plugs
arriving at different times at the Y-shaped junction (magnified
view shown). The oil streams were introduced into inlets 334, 336,
while the aqueous streams were introduced into inlets 335, 337. In
FIG. 33b, the flow rates for the combined oil/water fluid past the
junction where the oil and water meet were 6.9 mm/s for channel 346
(the 50.times.50 .mu.m.sup.2 channel) and 8.6 mm/s for channel 347
(the 25.times.50 .mu.m.sup.2 channel). The oil-to-water volumetric
ratio was 4:1 in each pair of oil and water inlets. The two
channels (the branch channels) merged into a common channel 348
that had a 100.times.50 .mu.m.sup.2 cross-section. As shown in the
figure, the larger plugs from the bigger channel are able to merge
with the smaller plugs from the narrower channel even when they do
not arrive at the junction at the same time. This is because
lagging larger plugs are able to catch up with the leading smaller
plugs once the plugs are in the common channel.
[0383] FIG. 33c depicts in-phase merging (i.e., plug merging upon
simultaneous arrival of at least two plugs at a junction) of plugs
of different sizes generated using different oil/water ratios at
the two pairs of inlets. The oil streams were introduced into
inlets 338, 340, while the aqueous streams were introduced into
inlets 339, 341. The flow rate corresponding to the fluid stream
through channel 349 resulting from a 1:1 oil-to-water volumetric
ratio was 4.0 mm/s, while that through channel 350 corresponding to
the 4:1 oil-to-water volumetric ratio was 6.9 mm/s. Each branch
channel of the Y-shaped portion of the network (magnified view
shown) had a dimension of 50.times.50 .mu.m while the common
channel 351 (the channel to which the branch channels merge) was
125.times.50 .mu.m.sup.2.
[0384] FIG. 33d illustrates defects (i.e., plugs that fail to
undergo merging when they would normally merge under typical or
ideal conditions) produced by fluctuations in the relative velocity
of the two incoming streams of plugs. The oil streams were
introduced into inlets 342, 344, while the aqueous streams were
introduced into inlets 343, 345. In this experiment, the flow rate
corresponding to the fluid stream through channel 352 resulting
from a 1:1 oil-to-water volumetric ratio was 4.0 mm/s, while that
through channel 353 corresponding to the 4:1 oil-to-water
volumetric ratio was 6.9 mm/s. Each branch channel that formed one
of the two branches of the Y-shaped intersection (magnified view
shown) was 50.times.50 .mu.m.sup.2 while the common channel 354
(the channel to which the two branch channels merge) is
125.times.50 .mu.m.sup.2.
Example 5
Splitting Plugs Using a Constricted Junction
[0385] The splitting of plugs was investigated using a channel
network with a constricted junction. In this case, the plugs split
and flowed past the junction into two separate branch channels (in
this case, branch channels are the channels to which a junction
branches out) that are at a 180.degree.-angle to each other (see
FIGS. 34a-c each of which show a channel network viewed from the
top). In these experiments, the outlet pressures, P.sub.1 and
P.sub.2, past the constricted junction were varied such that either
P.sub.1.apprxeq.P.sub.2 (FIG. 34b) or P.sub.1<P.sub.2 (FIG.
34c). Here, the relative pressures were varied by adjusting the
relative heights of the channels that were under pressures P.sub.1
and P.sub.2. Since longer plugs tend to split more reliably, this
branching point (or junction) was made narrower than the channel to
elongate the plugs. FIG. 34a shows a schematic diagram of the
channel network used in the experiment. The oil and water were
introduced into inlets 3400 and 3401, respectively. The
oil-to-water ratio was 4:1 while the flow rate past the junction
where the oil and water meet was 4.3 mm/s.
[0386] FIG. 34b is a microphotograph showing the splitting of plugs
into plugs of approximately one-half the size of the initial plugs.
The channels 3404, which were rectangular, had a cross-section that
measured 50.times.50 .mu.m.sup.2. The constricted section of the
channel 3402 right next to the branching point measured 25.times.50
.mu.m.sup.2. The outlet pressures, P.sub.1 and P.sub.2, were about
the same in both branch channels. Here, the plugs split into plugs
of approximately the same sizes.
[0387] FIG. 34c is a microphotograph showing the asymmetric
splitting of plugs (i.e., the splitting of plugs into plugs of
different sizes or lengths) which occurred when P.sub.1<P.sub.2.
The microphotograph shows that larger plugs (somewhat rectangular
in shape) flowed along the channel with the lower pressure P.sub.1,
while smaller plugs (spherical in shape) flowed along the channel
with the higher pressure P.sub.2. As in FIG. 34b, each of the
channel 3405 cross-section measured 50.times.50 .mu.m.sup.2. The
constricted section of the channel 3403 at the junction measured
25.times.50 .mu.m.sup.2.
Example 6
Splitting Plugs without Using a Constricted Junction
[0388] The splitting of plugs was investigated using a channel
network without a constriction such as the one shown in FIGS.
35b-c. The channel network used was similar to that shown in FIG.
34(a) except that here the plugs split and flowed past the junction
in two separate channels at a 90.degree.-angle to each other (the
plug flow being represented by arrows). The oil and aqueous streams
(4:1 oil:aqueous stream ratio) were introduced into inlets 3500 and
3501, respectively. An oil-only stream flowed through channel 3502.
All channels had a cross-section of 50.times.50 .mu.m.sup.2. The
flow rate used was 4.3 mm/s. FIGS. 35a-c, which represent top views
of a channel network, show that plugs behave differently compared
to the plugs in Example 3 when they flow past a junction in the
absence of a channel constriction, such as a constriction shown in
FIGS. 35b-c. As FIG. 35c shows, when P.sub.1<P.sub.2, the plugs
remained intact after passing through the junction. Further, the
plugs traveled along the channel that had the lower pressure
(P.sub.1 in FIG. 35c) while the intervening oil stream split at the
junction. The splitting of the oil stream at the junction gives
rise to a shorter separation between plugs flowing along the
channel with pressure P.sub.1 compared to the separation between
plugs in the channel upstream of the branching point or
junction.
Example 7
Monitoring Autocatalytic Reactions Using a Microfluidic System
[0389] FIG. 37 illustrates the design of an experiment involving
chemical amplification in microfluidic devices according to the
invention that involves an investigation of a stochastic
autocatalytic reaction. This example illustrates how the devices of
the present invention can be used to study the acid-sensitive
autocatalytic reaction between NaClO.sub.2 and NaS.sub.2O.sub.3. On
the left side of the microfluidic network, a three-channel inlet
introduces an aqueous stream through channel 3702, an ester through
channel 3701, and an esterase through channel 3703. Oil flowed
through channels 3713, 3714. The reaction between ester and
esterase yield plugs 3704 that contain a small amount of acid. On
the right side of the microfluidic network, the five-channel inlet
introduces NaClO.sub.2 through inlet 3705, an aqueous stream
through inlet 3706, a pH indicator through inlet 3707, a second
aqueous stream through inlet 3708, and NaS.sub.2O.sub.3 through
channel 3709. A carrier fluid flows through channels 3713, 3714.
Unstirred mixtures of NaClO.sub.2 and NaS.sub.2O.sub.3 are highly
unstable and even a slight concentration fluctuation within that
mixture leads to rapid decomposition. Thus, the plugs 3710
containing NaClO.sub.2/NaS.sub.2O.sub.3 mixture must not only be
quickly mixed but also promptly used after formation. In this
proposed experiment, the curvy channels promote chaotic mixing.
When a slightly acidic plug of the ester-esterase reaction is
merged with a plug of an unstable NaClO.sub.2/NaS.sub.2O.sub.3
mixture at the contact region 3712, an autocatalytic reaction will
generally be triggered. Upon rapid mixing of these two plugs, the
resulting plugs 3711 become strongly acidic. The pH indicator
introduced in the five-channel inlet is used to visualize this
entire amplification process.
Example 8
Using Chemical Reactions as Highly Sensitive Autoamplifying
Detection Elements in Microfluidic Devices
[0390] In one aspect according to the invention, a sequential
amplification using controlled autocatalytic systems is used to
amplify samples that contain single molecules of autocatalysts into
samples containing a sufficiently high concentration of an
autocatalyst such that the amplified autocatalyst can be detected
with the naked eye can be detected with the naked eye. Although
systems displaying stochastic behavior are expected to display high
sensitivity and amplification, various autocatalytic systems can be
used in accordance with the invention. A sequential amplification
using the microfluidic devices according to the invention can be
illustrated using a reaction that has been characterized
analytically: the autocatalytic decomposition of violet
bis[2-(5-bromo-pyridylazo)-5-(N-propyl-N-sulfopropyl-amino-phenola-
to] cobaltate, (Co(III)-5-Br-PAPS), upon oxidation with potassium
peroxomonosulfate to produce colorless Co.sup.2+ ions. Here, the
Co.sup.2+ ions serve as the autocatalyst (the order of
autocatalysis, m, has not been established for this reaction).
Co(III)-[5-Br-PAPS]reduced+HSO.sub.5.sup.-.fwdarw.Co.sup.2++[5-Br-PAPS]o-
xidized+HSO.sub.4.sup.- (3)
[0391] Addition of small amounts of Co.sup.2+ to the violet mixture
of (Co(III)-5-Br-PAPS and peroxomonosulfate produces an abrupt loss
of color to give a colorless solution. The time delay before this
decomposition depends on the amount of the Co.sup.2+ added to the
solution. This reaction has been used to detect concentrations of
Co.sup.2+ as low as about 1.times.10.sup.-10 mole/L. The reaction
shows good selectivity in the presence of other ions (V(V),
Cr(III), Cr(VI), Mn(II), Fe(II), Ni(II), Cu(II) and Zn(II)).
[0392] To use this reaction for amplification, a microfluidic
network as shown in FIG. 38 is preferably used. An unstable
solution of Co(III)-[5-Br-PAPS].sub.reduced and peroxomonosulfate
at pH=7 buffer in large plugs are preferably formed in a channel.
These large plugs are preferably split in accordance with the
invention into three different sizes of plugs. Preferably, the plug
sizes are (1 .mu.m).sup.3=10.sup.-15 L in the first channel; (10
.mu.m).sup.3=10.sup.-12 L in the second channel; and (100
.mu.m).sup.3=10.sup.-9 L in the third channel. A three-step
photolithography is preferably used in the fabrication of masters
for these microfluidic channels.
Example 9
Multi-Stage Chemical Amplification in Microfluidic Devices for
Single Molecule Detection
[0393] FIG. 38 illustrates a method for a multi-stage chemical
amplification for single molecule detection using microfluidic
devices according to the invention. This example illustrates the
use of an autocatalytic reaction between Co(III)-5-Br-PAPS
(introduced through inlet 3803) and KHSO.sub.5 (introduced through
inlet 3801) in a pH=7 buffer (introduced through inlet 3802) that
is autocatalyzed by Co.sup.2+ ions. Oil streams are allowed to flow
through channels 3804, 3805. This reaction mixture (contained in
plugs 3811) is unstable and decomposes rapidly (shown in red) when
small amounts of Co.sup.2+ 3810 are added. Thus, this reaction
mixture is preferably mixed quickly and used immediately. The
reaction mixture is preferably transported through the network in
(1 .mu.m).sup.3, (10 .mu.m).sup.3, (100 .mu.m).sup.3 size plugs. On
the left side of the microfluidic network, the approximately 1
.mu.m.sup.3 plugs of the sample to be analyzed form at a junction
of two channels (shown in green). The merging of plugs containing
Co.sup.2+ ions and plugs containing the reaction mixture results in
a rapid autocatalytic reaction. By using an amplification cascade
in which larger and larger plugs of the reaction mixture are used
for amplification, each Co.sup.2+ ion in a plug can be amplified to
about 10.sup.10 Co.sup.2+ ions per plug. The result of
amplification is visually detectable.
[0394] The (10 .mu.m).sup.3 plugs are preferably merged with larger
(100 .mu.m).sup.3 plugs in the third channel to give approximately
4.times.10.sup.-8 mole/L solution of Co.sup.2+ ions. Autocatalytic
decomposition in the approximately 10.sup.-9 L plugs will produce
plugs 3809 with about 2.4.times.10.sup.10 Co.sup.2+ ions
(4.times.10.sup.-5 mole/L). The flow rates in this system are
preferably controlled carefully to control the time that plugs
spend in each branch. The time provided for amplification is
preferably long enough to allow amplification to substantially
reach completion, but short enough to prevent or minimize slow
decomposition.
[0395] Using different plug sizes is advantageous when merging
plugs. Plugs with a size of about (1 .mu.m).sup.3 are preferably
formed by flowing a sample containing about 3.times.10.sup.-9
mole/L Co.sup.2+ through channel 3806. This reaction can be used to
detect Co.sup.2+ at this, or lower, concentration (Endo et al.,
"Kinetic determination of trace cobalt(II) by visual autocatalytic
indication," Talanta, 1998, vol. 47, pp. 349-353; Endo et al.,
"Autocatalytic decomposition of cobalt complexes as an indicator
system for the determination of trace amounts of cobalt and
effectors," Analyst, 1996, vol. 121, pp. 391-394). These plugs have
a corresponding volume of about 10.sup.-15 L and carry just a few
cobalt ions, on average about 1.8 ions per plug (corresponding to a
Poisson distribution). These plugs 3810 are preferably merged with
the (1 .mu.m).sup.3 plugs 3811 containing the
Co(III)-5-Br-PAPS/peroxomonosulfate mixture (about
4.times.10.sup.-5 mole/L).
[0396] Upon autocatalytic decomposition of the complex, the number
of Co.sup.2+ ions in the merged plug 3807 will increase by a factor
of between about 10.sup.4 to 1.2.times.10.sup.4 Co.sup.2+ ions
(2.times.10.sup.-5 mole/L in 2 .mu.m.sup.3). These plugs 3807 are
preferably merged with the (10 .mu.m).sup.3 plugs 3811 containing
the unstable mixture (about 4.times.10.sup.-5 mole/L). The
concentration of Co.sup.2+ ions in these approximately 10.sup.-12 L
plugs is preferably about 2.times.10.sup.-8 mole/L, which is
sufficient to induce autocatalytic decomposition. The number of
Co.sup.2+ ions will increase by a factor of between about 10.sup.3
to about 2.4.times.10.sup.7 ions/plug in plugs 3808. The starting
solution is dark violet (.di-elect cons.=9.8.times.10.sup.4 L
mol.sup.-1cm.sup.-1 for Co(III)-5-Br-PAPS). Channels are preferably
designed to create an optical path through at least ten consecutive
100 .mu.m plugs. These plugs will provide an approximately 1-mm
long optical path, with absorbance of the starting
4.times.10.sup.-5 mole/L solution of about 0.4. This absorbance can
be detected by an on-chip photodetector or with the naked eye. If
Co.sup.2+ is present in the sample solution, an autocatalytic
cascade will result in the disappearance of the color of the
reaction mixture.
[0397] At low concentrations of Co.sup.2+ in the sample, the system
may show stochastic behavior, that is, not every Co.sup.2+ ion
would give rise to a decomposition cascade. However, the attractive
feature of this system is that thousands of tests can be carried
out in a matter of seconds, and statistics and averaging can be
performed. Preferably, a sequence of controlled autocatalytic
amplification reactions leads to a visual detection of single
ions.
Example 10
Enzyme Kinetics
[0398] A microfluidic chip according to the invention was used to
measure millisecond single-turnover kinetics of ribonuclease A
(RNase A; EC 3.1.27.5), a well-studied enzyme. Sub-microliter
sample consumption makes the microfluidic chip especially
attractive for performing such measurements because they require
high concentrations of both the enzyme and the substrate, with the
enzyme used in large excess.
[0399] The kinetic measurements were performed by monitoring the
steady-state fluorescence arising from the cleavage of a
fluorogenic substrate by RNase A as the reaction mixture flowed
down the channel (see FIG. 40(a)). In FIG. 40, a substrate, buffer,
and RNase A were introduced into inlets 401, 401, and 403,
respectively. A carrier fluid flowed through channel 404. The
amount of the product at a given reaction time t [s] was calculated
from the intensity of fluorescence at the corresponding distance
point d [m] (t=d/U where U=0.43 m/s is the velocity of the flow).
The channels were designed to wind so that rapid chaotic mixing was
induced, and were designed to fit within the field of view of the
microscope so that the entire reaction profile could be measured in
one spatially resolved image. Selwyn's test (Duggleby, R. G.,
Enzyme Kinetics and Mechanisms, Pt D; Academic Press: San Diego,
1995, vol. 249, pp. 61-90; Selwyn, M. J. Biochim. Biophys. Acta,
1965, vol. 105, pp. 193-195) was successfully performed in this
system to establish that there were no factors leading to product
inhibition or RNase A denaturation.
[0400] The flow rate of the stock solution of 150 .mu.M of RNase A
was kept constant to maintain 50 .mu.M of RNase A within the plugs.
By varying the flow rates of the buffer and substrate (see FIG.
45), progress curves were obtained for eight different substrate
concentrations. For [E].sub.o>>[S].sub.o, the simple reaction
equation is [P].sub.t=[S].sub.o(1-Exp(-kt)), where [E].sub.o is the
initial enzyme concentration, [S].sub.o is the initial substrate
concentration, [P].sub.t is the time-dependent product
concentration and k [s.sup.-1] is the single-turnover rate
constant. To obtain a more accurate fit to the data, the time delay
.DELTA.t.sub.n required to mix a fraction of the reaction mixture
f.sub.n was accounted for.
[0401] An attractive feature of the microfluidic system used is
that the reaction mixture can be observed at time t=0 (there is no
dead-time). This feature was used to determine .DELTA.t.sub.n and
f.sub.n in this device by obtaining a mixing curve using
fluo-4/Ca.sup.2+ system as previously described (Song et al.,
Angew. Chem. Int. Ed. 2002, vol. 42, pp.
[ P ] t = n f n [ S ] 0 ? - Exp ( - k ( t - .DELTA. t n ) ) ?
##EQU00001## ? indicates text missing or illegible when filed
##EQU00001.2##
768-772), and correcting for differences in diffusion constants
(Stroock et al., Science, 2002, vol. 295, pp. 647-651). All eight
progress curves gave a good fit with the same rate constant of
1100.+-.250 s.sup.-1. The simpler theoretical fits gave
indistinguishable rate constants. These results are in agreement
with previous studies, where cleavage rates of oligonucleotides by
ribonucleases were shown to be .about.10.sup.3 s.sup.-1.
[0402] Thus, this example demonstrates that millisecond kinetics
with millisecond resolution can be performed rapidly and
economically using a microchannel chip according to the invention.
Each fluorescence image was acquired for 2 s, and required less
than 70 nL of the reagent solutions. These experiments with
stopped-flow would require at least several hundreds of microliters
of solutions. Volumes of about 2 .mu.L are sufficient for .about.25
kinetic experiments over a range of concentrations. Fabrication of
these devices in PDMS is straightforward (McDonald, et al.,
Accounts Chem. Res. 2002, vol. 35, pp. 491-499) and no specialized
equipment except for a standard microscope with a CCD camera is
needed to run the experiments. This system could serve as an
inexpensive and economical complement to stopped-flow methods for a
broad range of kinetic experiments in chemistry and
biochemistry.
Example 11
Kinetics of RNA Folding
[0403] The systems and methods of the present invention are
preferably used to conduct kinetic measurements of, for example,
folding in the time range from tens of microseconds to hundreds of
seconds. The systems and methods according to the invention allow
kinetic measurements using only small amounts of sample so that the
folding of hundreds of different RNA mutants can be measured and
the effect of mutation on folding established. In one aspect
according to the invention, the kinetics of RNA folding is
preferably measured by adding Mg.sup.2+ to solutions of previously
synthesized unfolded RNA labeled with FRET pairs in different
positions. In accordance with the invention, the concentrations of
Mg.sup.2+ are preferably varied in the 0.04 to 0.4 .mu.M range by
varying the flow rates (see, for example, FIGS. 25a)-c)) to rapidly
determine the folding kinetics over a range of conditions. The
ability to integrate the signal over many seconds using the
steady-flow microfluidic devices according to the invention can
further improve sensitivity.
[0404] As shown in FIGS. 25a)-c), the concentrations of aqueous
solutions inside the plugs can be controlled by changing the flow
rates of the aqueous streams. In FIGS. 25a)-c), aqueous streams
were introduced into inlets 251-258 wherein flow rates of about 0.6
.mu.L/min for the two aqueous streams and 2.7 .mu.L/min was used
for the third stream. The stream with the 2.7 .mu.L/min volumetric
flow rate was introduced in the left, middle, and right inlet in
FIGS. 25a)-c), respectively. A carrier fluid in the form of
perfluorodecaline was introduced into channel 259, 260, 261. The
corresponding photographs on each of the right side of FIGS.
25a)-c) illustrate the formation of plugs with different
concentrations of the aqueous streams. The various shadings inside
the streams and plugs arise from the use of aqueous solutions of
food dyes (red/dark and green/light), which allowed visualization,
and water were used as the three streams, the darker shading
arising mainly from the red dye color while the lighter shading
arising mainly from the green dye color. The dark stream is more
viscous than the light stream, therefore it moves slower (in mm/s)
and occupies a larger fraction of the channel at a given volumetric
flow rate (in .mu.L/min).
Example 12
Nanoparticle Experiments with and without Plugs
[0405] FIG. 15 illustrates a technique for the synthesis of CdS
nanoparticles 155. In one experiment, nanoparticles were formed in
a microfluidic network. The channels of the microfluidic device had
50 .mu.m.times.50 .mu.m cross-sections. A fluorinated carrier-fluid
(10:1 v/v mixture of perfluorohexane and
1H,1H,2H,2H-perfluorooctanol) was flowed through the main channel
at 15 .mu.m min.sup.-1. An aqueous solution, pH=11.4, of 0.80 mM
CdCl.sub.2 and 0.80 mM 3-mercaptopropionic acid was flowed through
the left-most inlet channel 151 at 8 .mu.L min.sup.-1. An aqueous
solution of 0.80 mM polyphosphates Na(PO.sub.3).sub.n was flowed
through the central inlet channel 152 at 8 .mu.L min.sup.-1, and an
aqueous solution of 0.96 mM Na.sub.2S was flowed through the
right-most inlet channel 153 at 8 .mu.L min.sup.-1. To terminate
the growth of nanoparticles, an aqueous solution of 26.2 mM
3-mercaptopropionic acid, pH=12.1, was flowed through the bottom
inlet of the device 157 at 24 .mu.M min.sup.-1. FIG. 15 shows
various regions or points along the channel corresponding to
regions or points where nucleation 154, growth 158, and termination
156 occurs. Based on the UV-VIS spectrum, substantially
monodisperse nanoparticles formed in this experiment.
[0406] Nanoparticles were also formed without microfluidics.
Solutions of CdCl.sub.2, polyphosphates, Na.sub.2S, and
3-mercaptopropionic acid, identical to those used in the
microfluidics experiment, were used. 0.5 mL of the solution of
CdCl.sub.2 and 3-mercaptopropionic acid, 0.5 mL of polyphosphates
solution, and 0.5 mL of Na.sub.2S solution were combined in a
cuvette, and the cuvette was shaken by hand. Immediately after
mixing, 1.5 mL of 26.2 mM 3-mercaptopropionic acid was added to the
reaction mixture to terminate the reaction, and the cuvette was
again shaken by hand. Based on the UV-VIS spectrum, substantially
polydisperse nanoparticles formed in this experiment.
Example 13
Crystallization
[0407] Networks of microchannels were fabricated using rapid
prototyping in polydimethylsiloxane (PDMS). The PDMS was purchased
from Dow Corning Sylgard Brand 184 Silicone Elastomer. The PDMS
devices were sealed after plasma oxidation treatment in Plasma Prep
II (SPI Supplies). The devices were rendered hydrophobic by baking
the devices at 120.degree. C. for 2-4 hours. Microphotographs were
taken with a Leica MZ12.5 stereomicroscope and a SPOT Insight color
digital camera (Model#3.2.0, Diagnostic Instruments, Inc.).
Lighting was provided from a Machine Vision Strobe X-strobe X1200
(20 Hz, 12 .mu.F, 600V, Perkin Elmer Optoelectronics). To obtain an
image, the shutter of the camera was opened for 1 second and the
strobe light was flashed once with the duration of approximately 10
.mu.s.
[0408] Aqueous solutions were pumped using 10 .mu.l or 50 .mu.l
Hamilton Gastight syringes (1700 series). Carrier-fluid was pumped
using 50 .mu.l Hamilton Gastight syringes (1700 series). The
syringes were attached to microfluidic devices by means of Teflon
tubing (Weico Wire & Cable Inc., 30 gauge). Syringe pumps from
Harvard Apparatus (PHD 2000) were used to inject the liquids into
microchannels.
A. Microbatch Crystallization in a Microfluidic Channel
[0409] Microbatch crystallization conditions can be achieved. This
experiment shows that size of plugs can be maintained and
evaporation of water prevented. In this case, the PDMS device has
been soaked in water overnight before the experiment in order to
saturate PDMS with water. The device was kept under water during
the experiment. During the experiment, the flow rates of
carrier-fluid and NaCl solution were 2.7 .mu.L/min and 1.0
.mu.L/min, respectively. The flow was stopped by cutting off the
Teflon tubing of both carrier-fluid and NaCl solution.
[0410] FIG. 16 shows a schematic illustration of a microfluidic
device according to the invention and a microphotograph of plugs of
1M aqueous NaCl sustained in oil. The carrier-fluid is
perfluorodecaline with 2% 1H,1H,2H,2H-perfluorooctanol. Inside a
microchannel, plugs showed no appreciable change in size.
B. Vapor Diffusion Crystallization in Microchannels: Controlling
Evaporation of Water from Plugs
[0411] This experiment shows that evaporation of water from plugs
can be controlled by soaking devices in water for shorter amounts
of time or not soaking at all. The rate of evaporation can be also
controlled by the thickness of PDMS used in the fabrication of the
device. Evaporation rate can be increased by keeping the device in
a solution of salt or other substances instead of keeping the
device in pure water.
[0412] The plug traps are separated by narrow regions that help
force the plugs into the traps.
[0413] In this experiment, a composite glass/PDMS device was used.
PDMS layer had microchannel and a microscopy slide (Fisher,
35.times.50-1) was used as the substrate. Both the glass slide and
the PDMS were treated in plasma cleaner (Harrick) then sealed. The
device was made hydrophobic by first baking the device at
120.degree. C. for 2-4 hours then silanizing it by
(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (United
Chemical Technologies, Inc.).
[0414] During the experiment, a flow of carrier-fluid at 1.0
.mu.L/min was established, then flow of aqueous solution was
established at a total rate of 0.9 .mu.L/min. Plug formation was
observed inside the microchannel. The flow was stopped
approximately 5-10 minutes afterwards by applying a pressure from
the outlet and stopping the syringe pumps at the same time.
[0415] FIG. 41 shows a microphotograph (middle and right side) of
the water plugs region of the microfluidic network. FIG. 41(b)-(c)
show the plugs at time t=0 and t=2 hours, respectively. Red aqueous
solution is 50% waterman red ink in 0.5 M NaCl solution. Ink
streams were then introduced into inlets 411, 412, 413. An oil
stream flowed through channel 414. The carrier-fluid is FC-3283 (3M
Fluorinert Liquid) with 2% 1H,1H,2H,2H-perfluorodecanol. This
photograph demonstrates that the evaporation of water through PDMS
can be controlled, and thus the concentration of the contents
inside the drops can be increased (this is equivalent to microbatch
crystallization). FIG. 41(a) shows a diagram of the microfluidic
network.
C. Controlling Shape and Attachment of Water Plugs
[0416] During the experiment, a flow of carrier fluid at 1.0
.mu.L/min was established, then flow of aqueous solution was
established at a total rate of 2.1 .mu.L/min. Plug formation was
observed inside the microchannel. The flow was stopped
approximately 5-10 minutes afterwards by applying a pressure from
the outlet and stopping the syringe pumps at the same time.
[0417] FIG. 39 shows a diagram (left side) of a microfluidic
network according to the invention. Aqueous streams were introduced
into inlets 3901, 3902, 3903 while an oil stream flowed through
channel 3904. FIG. 39 also shows a microphotograph (right side) of
the water plug region of the microfluidic network. This image shows
water plugs attached to the PDMS wall. This attachment occurs when
low concentrations of surfactant, or less-effective surfactants are
used. In this case 1H,1H,2H,2H-perfluorooctanol is less effective
than 1H,1H,2H,2H-perfluorodecanol. In this experiment the oil is
FC-3283 (3M Fluorinert Liquid) with 2% 1H,1H,2H,2H-perfluorooctanol
as the surfactant.
D. Examples of Protein Crystallization
[0418] During the experiment, a flow of oil at 1.0 .mu.L/min was
established. Then the flow of water was established at 0.1
.mu.L/min. Finally flows of lysozyme and precipitant were
established at 0.2 .mu.L/min. Plug formation was observed inside
the microchannel. The flow of water was reduced to zero after the
flow inside the channel became stable. The flow was stopped
approximately 5-10 minutes afterwards by applying a pressure from
the outlet and stopping the syringe pumps at the same time.
[0419] FIG. 36 depicts lysozyme crystals grown in water plugs in
the wells of the microfluidic channel. Lysozyme crystals started to
appear inside aqueous plugs both inside and outside plug traps in
approximately 10 minutes. The image of the three crystals in FIG.
36 was taken 1 hour after the flow was stopped. Lysozyme crystals
appear colored because they were observed under polarized light.
This is common for protein crystals.
[0420] The left side of FIG. 36 is a diagram of a microfluidic
network according to the invention while the right side is
microphotograph of the crystals formed in plugs in the microfluidic
network. A precipitant, lysozyme, and water were introduced into
inlets 3601, 3602, and 3603, respectively. Oil was flowed through
channel 3604. The lysozyme solution contains 100 mg/ml lysozyme in
0.05 M sodium acetate (pH 4.7); the precipitant solution contains
30% w/v PEG (M.W. 5000), 1.0 M NaCl and 0.05 M sodium acetate (pH
4.7); The carrier-fluid is FC-3283 (3M Fluorinert Liquid) with 10%
1H,1H,2H,2H-perfluoro-octanol. The microchannel device was soaked
in FC-3283/H.sub.2O for one hour before experiment.
[0421] FIG. 32 shows that plug traps are not required for formation
of crystals in a microfluidic network. FIG. 32 shows a diagram
(left side) of the microfluidic network. A precipitant was
introduced into inlet 321, lysozyme was introduced into inlet 322,
and an aqueous stream was introduced into inlet 323. Oil was flowed
through channel 324. FIG. 32 also shows microphotographs (middle
and right side) of lysozyme crystals grown inside the microfluidic
channel. The experimental condition is same as in FIG. 36.
Example 14
Oil-Soluble Surfactants for Charged Surfaces
[0422] In accordance with the invention, neutral surfactants that
are soluble in perfluorinated phases are preferably used to create
positively and negatively-charged interfaces. To create charged
surfaces, neutral surfactants that can be charged by interactions
with water, e.g., by protonation of an amine or a guanidinium group
(FIG. 24B), or deprotonation of a carboxylic acid group (FIG. 24C),
are preferably used. Preferably, charged surfaces are used to
repel, immobilize, or stabilize charged biomolecules. Negatively
charged surfaces are useful for handling DNA and RNA without
surface adsorption. Preferably, both negatively and
positively-charged surfaces are used to control the nucleation of
protein crystals. Many neutral fluorinated surfactants with acidic
and basic groups (RfC(O)OH, Rf(CH.sub.2).sub.2NH.sub.2,
Rf(CH.sub.2).sub.2C(NH)NH.sub.2) are available commercially
(Lancaster, Fluorochem, Aldrich).
[0423] To synthesize oligoethylene-glycol terminated surfactants, a
modification and improvement of a procedure based on the synthesis
of perfluoro non-ionic surfactants is preferably used. In one
aspect, the synthesis relies on the higher acidity of the
fluorinated alcohol to prevent the polycondensation of the
oligoethylene glycol. The modified synthesis uses a selective
benzylation of one of the alcohol groups of oligoethylene glycol,
followed by activation of the other alcohol group as a tosylate. A
Williamson condensation is then performed under phase transfer
conditions followed by a final deprotection step via catalytic
hydrogenation using palladium on charcoal.
Example 15
Formation of Plugs in the Presence of Fluorinated Surfactants and
Surface Tension
[0424] The surface tension of the oil/water interface has to be
sufficiently high in order to maintain a low value of capillary
number, C.n. The fluorosurfactant/water interfaces for
water-insoluble fluorosurfactants have not been characterized, but
these surfactants are predicted to reduce surface tension similar
to that observed in a system involving Span on hexane/water
interface (about 20 mN/m). The surface tensions of the
aqueous/fluorous interfaces are preferably measured in the presence
of fluorosurfactants using the hanging drop method. A video
microscopy apparatus specifically constructed for performing these
measurements has been used to successfully characterize interfaces.
FIG. 24 illustrates the synthesis of fluorinated surfactants
containing perfluoroalkyl chains and an oligoethylene glycol head
group.
Example 16
Forming Gradients by Varying Flow Rates
[0425] FIG. 42 shows an experiment involving the formation of
gradients by varying the flow rates. In this experiment, networks
of microchannels were fabricated using rapid prototyping in
polydimethylsiloxane (PDMS). The width and height of the channel
were both 50 .mu.m. 10% 1H,1H,2H,2H-perfluorodecanol in
perfluoroperhydrophenanthrene was used as oil. Red aqueous solution
prepared from 50% waterman red ink in 0.5 M NaCl solution was
introduced into inlet 421. The oil flowed through channel 424 at
0.5 .mu.l/min. Aqueous streams were introduced into inlets 422,
423. To generate the gradient of ink in the channel, the total
water flow rate was gradually increased from 0.03 .mu.l/min to 0.23
.mu.l/min in 20 seconds at a ramp rate of 0.01 .mu.l/min per
second. At the same time, ink flow rate was gradually decreased
from 0.25 .mu.l/min to 0.05 .mu.l/min in 20 seconds at a ramp rate
of -0.01 .mu.l/min per second. The total flow rate was constant at
0.28 .mu.l/min. The established gradient of ink concentration
inside the plugs can be clearly seen from FIG. 42: the plugs
further from the inlet are darker since they were formed at a
higher ink flow rate.
Example 17
Lysozome Crystallization Using Gradients
[0426] FIG. 43 illustrates an experiment involving the formation of
lysozome crystals using gradients. The channel regions 435, 437
correspond to channel regions with very low precipitant
concentration while channel region 436 corresponds to optimal range
of precipitant concentration. In this experiment, networks of
microchannels were fabricated using rapid prototyping in
polydimethylsiloxane (PDMS). The width of the channel was 150 .mu.m
and the height was 100 .mu.m. 10% 1H,1H,2H,2H-perfluorodecanol in
perfluoroperhydrophenanthrene was used as oil.
[0427] During the experiment, a flow of oil through channel 434 at
1.0 .mu.l/min was established. Then the flow of water introduced
through inlet 432 was established at 0.2 .mu.l/min. The flows of
lysozyme introduced through inlet 431 and precipitant introduced
through inlet 433 were established at 0.2 .mu.l/min. Plugs formed
inside the channel. To create the gradient, water flow rate was
first gradually decreased from 0.35 .mu.l/min to 0.05 .mu.l/min
over 45 seconds at a ramp rate of (-0.01 .mu.l/min per 1.5
seconds), then increased back to 0.35 .mu.l/min in 45 seconds at a
ramp rate of (0.01 .mu.l/min per 1.5 seconds). At the same time,
precipitant flow rate was gradually increased from 0.05 .mu.l/min
to 0.35 .mu.l/min in 45 seconds at a ramp rate of (0.01 .mu.l/min
per 1.5 seconds), then decreased to 0.05 .mu.l/min in 45 seconds at
a ramp rate of (-0.01 .mu.l/min per 1.5 seconds). The flow was
stopped by pulling out the inlet tubing immediately after water and
precipitant flow rates returned to the starting values. The plugs
created in this way contained constant concentration of the protein
but variable concentration of the precipitant: the concentration of
the precipitant was lowest in the beginning and the end of the
channel, and it peaked in the middle of the channel (the center
row). Only the plugs in the middle of the channel have the optimal
concentration of precipitant for lysozyme crystallization, as
confirmed by observing lysozyme crystals inside plugs in the center
row. Visualization was performed under polarized light. Preferably,
all flow rates would be varied, not just the precipitant and
water.
Example 18
Lysozyme Crystallization in Capillaries Using the Microbatch
Analogue Method
[0428] To grow lysozyme crystal inside plugs within capillaries, a
10 .mu.l Hamilton syringe was filled with 100 mg/ml lysozyme in
0.05 M NaAc buffer (pH4.7) and another 10 .mu.l Hamilton syringe
was filled with 30% (w/v) MPEG 5000 with 2.0 M NaCl in 0.05 M NaAc
buffer (pH4.7) as precipitant. A 50 .mu.l Hamilton syringe filled
with PFP (10% PFO) was the oil supply. All three syringes were
attached to the PDMS/capillary device and driven by Harvard
Apparatus syringe pumps (PHD2000). The capillary has an inner
diameter of 0.18 mm and outer diameter of 0.20 mm. Oil flow rate
was 1.0 .mu.l/min and both lysozyme and precipitant solution were
at 0.3 .mu.l/min. The channel was filled with oil first. Protein
and precipitant streams converged immediately before entering the
channel to form plugs. After the capillary (Hampton Research) was
filled with the plugs containing lysozyme, the flows were stopped.
The capillary was disconnected from the PDMS device, sealed with
wax and stored in an incubator (18.degree. C.). A lysozyme crystal
appeared within an hour and was stable for at least 14 days without
change of size or shape (FIG. 47A).
Example 19
Thaumatin Crystallization in Capillaries Using the Microbatch
Analogue Method
[0429] Experiment 1. A 10 .mu.l Hamilton syringe was filled with 50
mg/ml thaumatin in 0.1 M ADA buffer (pH 6.5) and another 10 .mu.l
Hamilton syringe was filled with 1.5 M NaK Tatrate in 0.1 M HEPES
(pH 7.0). A 50 .mu.l Hamilton syringe filled with PFP (10% PFO) was
the oil supply. All three syringes were attached to the
PDMS/capillary device and driven by Harvard Apparatus syringe pumps
(PHD2000). The capillary has an inner diameter of 0.18 mm and outer
diameter of 0.20 mm. Oil flow rate was 1.0 .mu.l/min and both
thaumatin and precipitant solution were at 0.3 .mu.l/min. The
channel was filled with oil first. Protein and precipitant streams
were mixed immediately before entering the channel to form plugs.
After the capillary (Hampton Research) was filled with protein
plugs, the flows were stopped. The capillary was cut from the PDMS
device, sealed by wax and stored in an incubator (18.degree. C.).
The thaumatin crystal appeared in 2-3 days and was stable for at
least 45 days without size or shape change (FIG. 47B). Some
thaumatin crystals grew at the interface of protein solution and
oil, while others appeared to attach to the capillary wall.
[0430] Experiment 2. Thaumatin crystals were grown inside a
capillary tube using 50 mg/mL thaumatin in 0.1 M pH 6.5 ADA buffer
and a precipitant solution of 1 M Na/K tartrate in a 0.1M pH 7.5
HEPES buffer. Protein and precipitant solutions were mixed in a
1.4:1 protein:precipitant ratio. A fluorinated carrier fluid was a
saturated solution of FSN surfactant in FC3283. The capillary was
incubated at 18 degrees C. Tetragonal crystals appeared within 5
days (FIGS. 48A, B). X-ray diffraction was performed at BioCARS
station 14BM-C at the Advanced Photon Source at Argonne National
Laboratory. Beam wavelength was 0.9 A. The final length of a single
crystal was estimated at 100-150 microns.
[0431] Capillaries were cut to the appropriate length without
disturbing crystal-containing plugs, resealed using capillary waz,
and mounted on clay-tipped cryoloop holders at a distance of 12+/-5
mm from base to crystal. The holder was placed on the x-ray
goniometer. Crystals were centered on the beam. Snapshots were
taken using 10 second (thaumatin) exposures. Distance from sample
to detector was 150 mm. Diffraction to better than 2.2 A was
obtained.
Example 20
Vapor Diffusion Protein Crystallization in Capillaries by an
Alternating Droplet System
[0432] The principle of transferring water inside a capillary from
one set of plugs to another set of plugs is illustrated in FIG. 50.
Briefly, a 10 .mu.l Hamilton syringe was filled with 0.01
Fe(SCN).sub.3 and another 10 .mu.l Hamilton syringe was filled with
0.1 M Fe(SCN).sub.3 with 2.5 M KNO.sub.3. Two 50 .mu.l Hamilton
syringes were filled with FMS-121 (Gelest, Inc) (saturated with
PFO), which provided the oil supply. All four syringes were
attached to the PDMS/capillary device and driven by Harvard
Apparatus syringe pumps (PHD2000). The capillary has an inner
diameter of 0.18 mm and outer diameter of 0.20 mm. One of the oil
inlet channels was between the two aqueous inlets channels to
separate the two aqueous streams when forming the alternating
plugs. This oil inlet channel was vertical to the main channel and
had a flow rate of 2.0 .mu.l/min. The other oil inlet channel had a
flow rate of 1.0 .mu.l/min and was parallel to the main channel.
Both of the aqueous solutions had a flow rate of 0.5 .mu.l/min.
After establishing alternating aqueous droplet streams in the
capillary, the flows were stopped, and the capillary was
disconnected from the PDMS device, sealed with wax and stored in an
incubator at 18.degree. C. The size and color change of the plugs
were monitored with a Leica microscope (MZ125) having a color CCD
camera (SPOT Insight, Diagnostic Instruments, Inc.).
[0433] Following the stoppage of flow and sealing of the capillary
tube, plugs containing 0.01 M Fe(SCN).sub.3 in water were yellow,
while those containing 0.1 M Fe(SCN).sub.3 and 2.5 M KNO.sub.3 in
water were red (FIG. 50A). However, FIG. 50B shows that after 5
days, the yellow plugs were reduced in size and were more
concentrated, while the red plugs increased in size and were more
diluted. This demonstration reflects vapor diffusion conditions in
the capillary tube that are predicted to facilitate protein
crystallization. This technique can be further adapted to other
applications requiring concentration of reagents, such as
proteins.
[0434] Alternating plugs from two different aqueous solutions may
be generated in accordance with several representative geometries
as set forth in FIG. 51. In principle, the same oil or different
oils may be used in the two oil inlets. One scheme for generating
alternating plugs from two different aqueous solutions is depicted
in FIG. 51A. In this case, one 10 .mu.l Hamilton syringe was filled
with 0.1 Fe(SCN).sub.3, another with 1.5 M NaCl. Two 50 .mu.l
Hamilton syringes filled with PFP (with 10% PFO) provided the oil
supply. All four syringes were attached to the PDMS device and
driven by Harvard Apparatus syringe pumps (PHD2000). Alternatively,
multiple solutions can be co-introduced together in each of the two
aqueous channels as depicted in FIG. 51B. In each of these two
cases one of the oil inlet channels was between the two aqueous
inlet channels. This oil inlet channel was used to separate the two
aqueous streams into alternating plugs and was vertical to the main
channel, having a flow rate of 2.0 .mu.l/min. The other oil inlet
channel was parallel to the main channel and had a flow rate of 1.0
.mu.l/min. Each of the two aqueous solutions had flow rates of 0.5
.mu.l/min. Alternating plugs were found to form in the channel
(FIG. 51C).
[0435] FIG. 52 illustrates another example of generating
alternating plugs from two different aqueous solutions. In this
case, one 10 .mu.l Hamilton syringe was filled with 0.1
Fe(SCN).sub.3, the other with 1.5 M NaCl. Two 50 .mu.l Hamilton
syringes filled with FMS-121 (saturated with PFO) provided the oil
supply. All four syringes were attached to the device and driven by
Harvard Apparatus syringe pumps (PHD2000). One of the oil inlet
channels was between the two aqueous inlet channels and was used to
separate the two aqueous streams prior to formation of alternating
plugs (FIG. 52A). This oil inlet channel was vertical to the main
channel and had a flow rate of 1.5 .mu.l/min. The other oil stream
had a flow rate of 1.5 .mu.l/min and was parallel to the main
channel. Each of the two aqueous solutions had flow rates of 0.5
.mu.l/min. Alternating plugs were found to form in the channel
(FIG. 52B).
[0436] Other geometries that can support the formation of
alternating plugs are depicted in FIG. 53. Importantly, the flow
rates of solutions A and B may be changed in a correlated fashion
(FIG. 54). Thus, when the flow rate of solution A.sub.1 is
increased and solution A.sub.2 is decreased, the flow rate of
solutions B.sub.1 is also increased and solution B.sub.2 is also
decreased. This principle, depicted in FIG. 54, is useful for
maintaining a constant difference in salt concentration between the
plugs of stream A and stream B to ensure that transfer from all
plugs A to all plugs B occurs at a constant rate.
[0437] FIG. 54 provides a schematic illustration of a device for
preparing plugs of varying protein concentrations where the flow
rates of the A and B streams change in a correlated fashion. In
this example, A.sub.1 through A.sub.3 are for protein solution,
buffer and precipitants, such as PEG or salts. Highly concentrated
salt solutions are injected through B.sub.1.about.B.sub.3. The flow
rate ratio of inlet A.sub.1 to that of B.sub.i (i=1.about.3) is
maintained constant. Therefore all of the protein plugs will shrink
at a rate similar to the salt plugs.
[0438] FIG. 54 shows that if the flow rates of corresponding A and
B streams are changed in a correlative fashion, the composition of
plugs B will reflect the composition of plugs A. Therefore, one can
incorporate markers into the B stream plugs to serve as a code for
the plugs in the A stream. In other words, absorption/fluorescent
dyes or x-ray scattering/absorbing materials can be incorporated in
markers in the B streams to facilitate optical or x-ray-mediated
quantification so as to provide a read out of relative protein and
precipitant concentrations in the A streams. This approach can
provide a powerful means for optimizing crystallization conditions
for subsequent scale-up experiments.
* * * * *