U.S. patent application number 11/955902 was filed with the patent office on 2008-10-02 for apparatus and method for trapping bead based reagents within microfluidic analysis systems.
This patent application is currently assigned to The Governors of the University of Alberta. Invention is credited to D. Jed Harrison, Paul Li, Richard Oleschuk, Loranelle Shultz-Lockyear, Cameron Skinner.
Application Number | 20080237146 11/955902 |
Document ID | / |
Family ID | 4164711 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080237146 |
Kind Code |
A1 |
Harrison; D. Jed ; et
al. |
October 2, 2008 |
APPARATUS AND METHOD FOR TRAPPING BEAD BASED REAGENTS WITHIN
MICROFLUIDIC ANALYSIS SYSTEMS
Abstract
An on-chip packed reactor bed design is disclosed that allows
for an effective exchange of packing materials such as beads at a
miniaturized level. Also disclosed is a method of treating a sample
within a microfluidic analysis system, comprising: providing a main
channel having a trapping zone; providing a slurry of a reagent
treated packing material; inducing a flow of said packing material
into said trapping zone through a flow channel connected to said
trapping zone to load said trapping zone and form a packed bed of
said packing material; and flowing a sample containing analytes
through said packed bed, said reagent treating the sample. The
present invention extends the function of microfluidic analysis
systems to new applications including on-chip solid phase
extraction (SPE) and on-chip capillary electrochromatography (CEC).
The design can be further extended to include integrated packed bed
immuno- or enzyme reactors.
Inventors: |
Harrison; D. Jed; (Edmonton,
CA) ; Oleschuk; Richard; (Kingston, CA) ;
Shultz-Lockyear; Loranelle; (Durango, CO) ; Skinner;
Cameron; (Montreal, CA) ; Li; Paul; (Burnaby,
CA) |
Correspondence
Address: |
ECKERT SEAMANS CHERIN & MELLOTT
600 GRANT STREET, 44TH FLOOR
PITTSBURGH
PA
15219
US
|
Assignee: |
The Governors of the University of
Alberta
Edmonton
CA
|
Family ID: |
4164711 |
Appl. No.: |
11/955902 |
Filed: |
December 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10148425 |
Nov 4, 2002 |
7312611 |
|
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11955902 |
|
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PCT/CA00/01421 |
Nov 27, 2000 |
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10148425 |
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Current U.S.
Class: |
436/174 ;
210/749; 422/236 |
Current CPC
Class: |
G01N 30/56 20130101;
B01L 2300/0816 20130101; G01N 2030/565 20130101; Y10T 436/25
20150115; B01L 3/502761 20130101; B01L 2400/0487 20130101; G01N
2030/285 20130101; Y10T 436/25375 20150115; B01L 2400/043 20130101;
B01L 2200/0668 20130101; B01L 3/502707 20130101; G01N 1/40
20130101; G01N 1/405 20130101; G01N 30/6095 20130101; B01L
2400/0415 20130101; Y10T 436/2575 20150115; Y10T 436/255 20150115;
B01L 2300/0877 20130101 |
Class at
Publication: |
210/748 ;
210/749; 422/236 |
International
Class: |
B01J 8/00 20060101
B01J008/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 26, 1999 |
CA |
2,290,731 |
Claims
1. A method of treating a sample within a microfluidic analysis
system, comprising the steps of: a) providing a main channel having
"a trapping zone suitable for trapping packing material; b)
providing a slurry of a reagent treated packing material prepared
in a solution having a predetermined composition of a solvent; c)
inducing a flow of said packing" material into said trapping zone
through a flow channel connected to said trapping zone so as to
load said trapping zone and form a packed bed of said packing
material; d) flowing a sample containing analytes through said
packed bed, said reagent treating the sample, whereby. The sample
leaving the trapping zone has an altered analyte composition.
2. The method claimed in claim 1, further comprising the step of:
e) adjusting the composition of the solvent, so as to affect the
aggregation of said packing material and the stabilization of the
packed bed.
3. The method claimed in claim 1, wherein, step b) comprises
providing packing material comprising porous beads.
4. The method claimed in claim 3, wherein said porous beads are
selected to have a diameter in the range from about 0.7 to about
10.0 .mu.m.
5. (canceled)
6. The method claimed in claim 4, wherein said solvent is
acetonitrile, and step e) comprises adjusting the concentration
level to less than about 50% to stabilize the packed bed.
7. (canceled)
8. The method claimed in claim 6, further including the steps of
adjusting the concentration level to above 50% to destabilize the
packed bed, and reversing the flow in step c) so as to unload said
trapping zone.
9. (canceled)
10. The method claimed in claim 1, further comprising the steps of:
before step c), adding a neutral surfactant to said packing
material so as to inhibit aggregation; and after step c), removing
the neutral surfactant to promote aggregation.
11. The method claimed in claim 1, further comprising the steps of:
after step c) introducing a polymerizable agent into the flow
channel and polymerizing said agent, so as to stabilize the packed
bed.
12. The method claimed in claim 1, wherein step d) comprises
applying a fluid force to induce the flow of said packing
material.
13. The method claimed in claim 1, wherein said packing material
comprises at least some electrically charged particles and step d)
comprises applying a voltage potential to induce the flow of said
packing material.
14. (canceled)
15. (canceled)
16. A method of trapping bead based reagents within a microfluidic
analysis system, comprising the steps of: a) providing a main
channel having a trapping zone suitable for trapping bead based
packing material; b) providing a slurry of a reagent treated beads
prepared in a solution having a predetermined composition of a
solvent; c) inducing a flow of said beads into said, trapping zone
through a flow channel connected to said trapping zone so as to
load said trapping zone and form a packed bed of said beads; and d)
adjusting the composition of the solvent, so as to affect the
aggregation of. said packing material and the stabilization of the
packed bed.
17. A microfluidic analysis system, comprising: a) a substantially
planar substrate having an upper surface; b) at least one main
Channel formed into said upper surface, said main channel having
first and second ends and a defined direction of flow in use; c) a
cover plate arranged over said planar substrate, said cover plate
closing off said channel from above; and d) a first weir formed
across said main channel and between said first and second ends of
said channel, said first weir providing at least one flow gap to
allow, in use, at least some fluid to flow past said first weir
while trapping packing material having constituent particles that
are generally larger than said flow gap.
18. The microfluidic analysis system claimed in claim 17, further
comprising at least one side channel formed into the upper surface
of said planar substrate, said side channel being connected at a
first end to said main channel at a location upstream from said
first weir, and at a second end to a reservoir, said side channel
providing a higher flow resistance than said main channel.
19. The microfluidic analysis system claimed in claim 18, further
comprising a second weir located upstream from said connected first
end of said side channel, said first and second weirs forming a
chamber therebetween, said second weir providing at least one flow
gap to allow, in use, at least some fluid to flow past said second
weir while trapping said packing material within said chamber.
20. The microfluidic analysis system claimed in claim 19, wherein,
each side channel connection to said main channel is provided with
a hook structure curved to one side whereby, in use, packing
material is sprayed into said chamber to facilitate even
packing.
21.-28. (canceled)
29. A method of packing the chamber in the microfluidic analysis
system claimed in claim 19, said method comprising, providing a
nonconductive substrate and effecting an electrokinetic flow by
applying a relatively high voltage at said reservoir, said
reservoir containing packing material, and providing relatively low
voltages at said first and second ends of said main channel, so
that packing material flows from said reservoir into said chamber
and is trapped by said first and second weirs.
30. (canceled)
31. A method of packing the chamber in the microfluidic analysis
system claimed in claim 19, said method comprising, effecting a
pressure driven flow by providing a relatively high pressure at
said reservoir, said reservoir containing packing material, and
providing relatively low pressure at said first and second main
reservoirs, whereby, packing material flows from said packing
material reservoir into said chamber and is trapped by said first
and second weirs.
32. The method as claimed in claim 31, wherein, packing material
may be removed from the chamber by reversing said pressure driven
flow.
33. A method of packing the chamber in the microfluidic analysis
system claimed in claim 32, said method comprising, providing
magnetically charged packing material, and effecting a magnetically
driven flow by providing a magnetically attractive force in the
chamber, whereby, the packing material enters the chamber and is
trapped by said first and second weirs.
34. (canceled)
35. The method claimed in anyone of claims 25-34, wherein, said
packing material comprises porous beads.
36.-39. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of application Ser. No. 10/148,425
filed Nov. 4, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates generally to microfluidic
analysis systems, and more specifically to micro-Total Analysis
Systems (.mu.-TAS), for performing liquid phase analysis at a
miniaturized level.
BACKGROUND INFORMATION
[0003] Recent developments in the field of micro-Total Analysis
Systems (.mu.-TAS) have led to systems that perform chemical
reactions, separation and detection at a miniaturized level on a
single microchip [see, for example, Harrison, D. J.; Fluri, K.;
Seiler, K.; Fan, Z.; Effenhauser, C. S.; and Manz, A., Science
1993, 261, 895-897. Harrison, D. J.; and van den Berg, E.; Eds.,
Micro Total Analysis Systems '98, Proceedings of the .mu.TAS '98
Workshop (Kluwer: Dordrecht, 1998). Coyler, C. L.; Tang, T.; Chiem,
N.; and Harrison, D. J., Electrophoresis 1997, 18, 1733-1741].
[0004] Most prior art microfluidic devices are based on
conventional open tubular flow designs and solution phase reagents.
While the functionality of these devices has continued to increase,
one key feature that is presently lacking in these prior art
devices is the ability to effectively incorporate on-chip packed
reactor beds, for introduction of packing materials with
immobilized reagents or stationary phases. While a few attempts
have been made to employ packed reactor beds in some prior art
designs, the difficulty of packing portions of a complex
microfluidic manifold with packing material (such as microscopic
beads) has so far hindered the effective utilization of these
reagent delivery vehicles within microfluidic devices. (The
difficulty of packing has been well recognized by practitioners in
the field. See, for example, Ericson, C; Holm, J.; Ericson, T.; and
Hjerten, S., Analytical Chemistry.)
[0005] In one prior art example, a packed bed chromatographic
device with a bead trapping frit was fabricated in a silicon
substrate [Ocvirk, G., Verpoorte, E., Manz, A., Grasserbauer, M.,
and Widmer, H. M. Analytical Methods and Instrumentation 1995, 2,
74-82]. However, the packing material in this prior art design
could not be readily packed or exchanged, thus limiting its
utility.
[0006] Several authors have also described the difficulties
associated with reproducibly fabricating frits for retaining
packing material in conventional capillaries [Boughtflower, R. J.;
Underwood, T.; Paterson, C. J. Chromatographia 1995, 40, 329-335.
Van den Bosch, S. E.; Heemstra, S.; Kraak, J. C.; Poppe, H. J.
Chromatogr. A 1996, 755, 165-177. Colon, L. A.; Reynolds, K. J.;
Alicea-Maldonado, R.; Fermier, A. M. Electrophoresis 1997, 18,
2162-2174. Majors, R. E. LC-GC 1998, 16, 96-110.]. The frits used
in conventional systems are prepared using time and labor intensive
procedures, the most commonly used method involving the use of pure
silica gel, wetted down with aqueous sodium silicate. The frit is
made by first tapping a capillary end into a paste made from silica
and aqueous sodium silicate. The resulting plug of silica is then
heated to make a frit. Current construction methods do not produce
high yields of useable frits.
[0007] Furthermore, using frits produced by prior art methods of
construction often leads to the formation of undesirable bubbles.
[Altria, K. D.; Smith, N. W.; and Turnbull, C. H., Chromatographia,
46 (1997) 664. Majors, R. E., LC-GC, 16 (1998) 96.] Bubbles cause
discontinuity within a column, hindering solution flow and
ultimately preventing separation from occurring. The bubbles are
thought to arise from a change in electroosmotic flow (EOF)
velocity caused by moving from a bead trapping frit into an open
capillary. The formation of bubbles, which have been observed to
increase at higher voltages, also limits the amount of voltage that
can be applied across the capillary, thereby limiting column
length, separation efficiency, and speed of analysis.
[0008] Developing a functional on-chip packed reactor bed design
which overcomes the limitations in the prior art would
significantly enhance the range of the microfluidic toolbox and
extend the number of applications of such devices.
SUMMARY OF THE INVENTION
[0009] Generally, the present invention provides an on-chip packed
reactor bed design using one or more weir structures that allow for
an effective exchange of packing materials (beads for example) at a
miniaturized level. The present invention extends the function of
microfluidic analysis systems to new applications. For example, the
packed reactor bed formed according to the present invention allows
on chip solid phase extraction (SPE) and on-chip capillary
electrochromatography (CEC), as explained in detail further below.
The design can be further extended to include, for example,
integrated packed bed immuno or enzyme reactors.
[0010] In a first aspect, the present invention provides a method
of treating a sample within a microfluidic analysis system,
comprising the steps of: [0011] a) providing a main channel having
a trapping zone suitable for trapping packing material; [0012] b)
providing a slurry of a reagent treated packing material prepared
in a solution having a predetermined composition of a solvent;
[0013] c) inducing a flow of said packing material into said
trapping zone through a flow channel connected to said trapping
zone so as to load said trapping zone and form a packed bed of said
packing material; [0014] d) flowing a sample containing analytes
through said packed bed, said reagent treating the sample, whereby
the sample leaving the trapping zone has an altered analyte
composition. [0015] In one embodiment, the method further comprises
the step of: [0016] e) adjusting the composition of the solvent, so
as to affect the aggregation of said packing material and the
stabilization of the packed bed.
[0017] In another embodiment, step b) comprises providing packing
material comprising porous beads.
[0018] In another embodiment, said porous beads are selected to
have a diameter in the range from about 0.7 to about 10.0
.mu.m.
[0019] In yet another embodiment, said porous beads are selected to
have a diameter in the range from about 1.5 to about 4.0 .mu.m.
[0020] In another embodiment, said solvent is acetonitrile, and
step e) comprises adjusting the concentration level to less than
about 50% to stabilize the packed bed.
[0021] In another embodiment, said solvent is acetonitrile, and
step e) comprises adjusting the concentration level to less than
about 30% to stabilize the packed bed.
[0022] In another embodiment, the method further includes the steps
of adjusting the concentration level to above 50% to destabilize
the packed bed, and reversing the flow in step c) so as to unload
said trapping zone.
[0023] In another embodiment, the method further includes the step
of repeating step c) so as to reload said trapping zone, and
readjusting the concentration level to restabilize the packed bed.
[0024] In yet another embodiment, the method further comprises the
steps of: before step c), adding a neutral surfactant to said
packing material so as to inhibit aggregation; and after step c),
removing the neutral surfactant to promote aggregation. [0025] In
another embodiment, the method further comprising the steps of:
after step c) introducing a polymerizable agent into the flow
channel and polymerizing said agent, so as to stabilize the packed
bed.
[0026] In yet another embodiment, step d) comprises applying a
fluid force to induce the flow of said packing material.
[0027] In another embodiment, said packing material comprises at
least some electrically charged particles and step d) comprises
applying a voltage potential to induce the flow of said packing
material.
[0028] In another embodiment, said packing material comprises at
least some particles susceptible to a magnetic field and step d)
comprises applying a magnetic field to induce the flow of said
packing material.
[0029] In another embodiment, the method further includes the step
of providing a hook structure at the connection point between said
flow channel and said trapping zone, so as to prevent direct
line-of-sight entry of said packing material, thereby to promote
even packing.
[0030] In another aspect, the present invention provides a method
of trapping bead based reagents within a microfluidic analysis
system, comprising the steps of: [0031] a) providing a main channel
having a trapping zone suitable for trapping bead based packing
material; [0032] b) providing a slurry of a reagent treated beads
prepared in a solution having a predetermined composition of a
solvent; [0033] c) inducing a flow of said beads into said trapping
zone through a flow channel connected to said trapping zone so as
to load said trapping zone and form a packed bed of said beads; and
[0034] d) adjusting the composition of the solvent, so as to affect
the aggregation of said packing material and the stabilization of
the packed bed.
[0035] In a further aspect, the present invention provides a
microfluidic analysis system, comprising: [0036] a) a substantially
planar substrate having an upper surface; [0037] b) at least one
main channel formed into said upper surface, said main channel
having first and second ends and a defined direction of flow in
use; [0038] c) a cover plate arranged over said planar substrate,
said cover plate closing off said channel from above; and [0039] d)
a first weir formed across said main channel and between said first
and second ends of said channel, said first weir providing at least
one flow gap to allow, in use, at least some fluid to flow past
said first weir while trapping packing material having constituent
particles that are generally larger than said flow gap.
[0040] In one embodiment, the system further comprises at least one
side channel formed into the upper surface of said planar
substrate, said side channel being connected at a first end to said
main channel at a location upstream from said first weir, and at a
second end to a reservoir, said side channel providing a higher
flow resistance than said main channel.
[0041] In another embodiment, the system further comprises a second
weir located upstream from said connected first end of said side
channel, said first and second weirs forming a chamber
therebetween, said second weir providing at least one flow gap to
allow, in use, at least some fluid to flow past said second weir
while trapping said packing material within said chamber.
[0042] In another embodiment, each side channel connection to said
main channel is provided with a hook structure curved to one side
whereby, in use, packing material is sprayed into said chamber to
facilitate even packing.
direct line-of-sight entry of packing material from said side
channel into said chamber and forms a chamber mouth to one side of
said hook structure.
[0043] In another embodiment, said flow gaps comprise a generally
uniform gap between said cover plate and the top of said weirs.
[0044] In yet another embodiment, said flow gaps comprise a
plurality of substantially vertical gaps in said weirs.
[0045] In another embodiment, said system is formed entirely on a
single microfluidic chip.
[0046] In another embodiment, the present invention provides a
method comprising, providing a non-conductive substrate and
effecting an electrokinetic flow by applying a relatively high
voltage at said second end of said main channel and at said
reservoir, said reservoir containing packing material, and
providing a relatively low voltage at said first end of said main
channel, so that packing material flows from said reservoir into
said main channel and is trapped against said first weir.
[0047] In one embodiment, the packing material is removed from said
main channel by providing a relatively high voltage at said first
and second ends of said main channel while providing a relatively
low voltage at said reservoir.
[0048] In another aspect, the present invention provides a method
comprising, effecting a pressure driven flow by providing a
relatively high pressure at said second end of said main channel
and at said reservoir, said reservoir containing packing material,
and providing a relatively low pressure at said first end of said
main channel, so that packing material flows from said reservoir
into said main channel and is trapped against said first weir.
[0049] In one embodiment, the packing material is removed from the
said main channel by providing relatively high pressure at said
first and second ends of said main channel while providing
relatively low pressure at said reservoir.
[0050] In another embodiment, the method comprises providing a
non-conductive substrate and effecting an electrokinetic flow by
applying a relatively high voltage at said reservoir, said
reservoir containing packing material, and providing relatively low
voltages at said first and second ends of said main channel, so
that packing material flows from said reservoir into said chamber
and is trapped by said first and second weirs.
[0051] In another embodiment, the packing material is removed from
the chamber by reversing said electrokinetic flow.
[0052] In another aspect, the present invention provides a method
of packing the chamber in a microfluidic analysis system
comprising, effecting a pressure driven flow by providing a
relatively high pressure at said reservoir, said reservoir
containing packing material, and providing relatively low pressure
at said first and second main reservoirs, whereby, packing material
flows from said packing material reservoir into said chamber and is
trapped by said first and second weirs.
[0053] In another embodiment, the packing material may be removed
from the chamber by reversing said pressure driven flow.
[0054] In another embodiment, the method comprises providing
magnetically charged packing material, and effecting a magnetically
driven flow by providing a magnetically attractive force in the
chamber, whereby, the packing material enters the chamber and is
trapped by said first and second weirs.
[0055] In another embodiment, the packing material may be removed
from the chamber by reversing said magnetic force in said
chamber.
[0056] In any of the above embodiments, the packing material may
comprise porous beads.
[0057] In another embodiment, the beads may be generally
spheroid.
[0058] In another embodiment, the beads are initially suspended in
a buffer solution.
[0059] In another embodiment, the buffer solution is an organic
solvent miscible with water.
[0060] In another embodiment, the organic solvent is acetonitrile
with a concentration level of up to 50%.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] For a better understanding of the present invention, and by
way of example, reference will now be made to the accompanying
drawings, which show preferred embodiments of the present invention
in which:
[0062] FIG. 1A shows a top plan view of a microfluidic device
according to the present invention;
[0063] FIG. 1B shows an enlarged perspective view of a chamber in
which packing materials (such as beads) are trapped;
[0064] FIG. 2A shows a cross-sectional view of the chamber shown in
FIG. 1B taken along line A-A, and further shows packing material
(beads) which are packed into the chamber and which are retained by
a cover plate;
[0065] FIGS. 2B and 2C show a side view and end view, respectively,
of an alternative embodiment of a weir according to the present
invention;
[0066] FIG. 3A shows an initial stage of packing material (beads)
being packed into the chamber shown in FIGS. 1B and 2A;
[0067] FIG. 3B shows the chamber of FIG. 3A after it has been
completely filled with packing material (beads);
[0068] FIG. 4A shows an early stage of preconcentration of a 1.0 nM
BODIPY solution at the weir/bed interface near the top of FIG.
4A;
[0069] FIG. 4B shows a later stage of preconcentration of a 1.0 nM
BODIPY solution at the weir/bed interface near the top of FIG.
4B;
[0070] FIG. 5 shows a plot of fluorescence intensity vs. time,
showing fluorescence of a first 1.0 NM BODIPY sample during
loading, followed by a buffer flush, and then preconcentrated
BODIPY during elution with acetonitrile (ACN);
[0071] FIG. 6 shows an electrochromatogram of BODIPY and
fluorescein, showing different steps of the separation including
load, flush, and elution;
[0072] FIGS. 7A-7D show electrochromatograms of BODIPY and
fluorescein with different concentrations of acetonitrile in the
mobile phase, specifically at: (a) 30%; (b) 22%; (c) 15%; and (d)
10%;
[0073] FIG. 8A-8C show top plan views of alternative embodiments of
a microfluidic device according to the present invention;
[0074] FIG. 9 shows a top plan view of a microfluidic device
according to the present invention having multiple packed
chambers;
[0075] FIG. 10 shows a schematic view of a microfluidic device
according to the present invention being used in conjunction with a
mass spectrometer;
[0076] FIG. 11 shows a graph plotting the fluorescence intensity of
theophylline against time, as it saturates a packed bed;
[0077] FIG. 12 shows theophylline being eluted from packed bed in a
relatively narrow band; and
[0078] FIG. 13 shows each successive trial resulting in lower light
generated from the CL reaction.
DETAILED DESCRIPTION OF THE INVENTION
[0079] As explained above, the present invention is designed to
provide a convenient system and method of trapping packing
materials (such as beads) on-chip, and of effectively packing and
unpacking the trapping zones, to provide a functional on-chip
packed reactor bed which significantly extends the number of
applications of microfluidic analysis devices.
[0080] One such extended application facilitated by the present
invention is on-chip sample preconcentration by solid phase
extraction (SPE). In microfluidic analysis, SPE is often required
to overcome detection limit problems, or to eliminate a potential
interferent. To date, preconcentration within microchips has been
performed by sample stacking using "isoelectric focusing"
[Jacobson, S. C. and Ramsey, M. Electrophoresis 1995, 16, 481 486].
Advantageously, unlike sample stacking, SPE can be made selective
for a particular analyte and does not require precise control of
buffer concentrations. For SPE the amount of preconcentration is
limited by the preconcentration time, which makes it more flexible
than sample stacking. The SPE of an analyte can be beneficial not
only for analyte preconcentration, but also for removing other
impurities or changing solvent conditions. While the coupling of
SPE with microfluidic devices has been accomplished [Figeys, D. and
Aebersold, R. Anal. Chem. 1998, 70, 3721 3727], the SPE component
in these prior art devices have been made in a capillary or similar
cartridge external to the chip, thus resulting in a more complex
and more expensive system. The present invention is designed to
overcome this prior art limitation by facilitating an on-chip SPE
component.
[0081] As realized by the present inventors, an integrated, on chip
SPE component is ultimately easier to manufacture, does not require
low dead volume coupling to the chip, and eliminates sample
handling losses or contamination problems arising from the off chip
sample manipulation required in the prior art. It is anticipated
that routine incorporation of SPE onto a chip, as facilitated by
the present invention, will reduce problems with on chip detection
limits and will improve the range of sample preparation steps which
can be integrated.
[0082] Another extended application facilitated by the present
invention is on-chip capillary electrochromatography (CEC). CEC has
recently received significant attention due to the fact that it
combines the separation power of both liquid chromatography and
capillary electrophoresis. To date the difficulty associated with
packing chromatographic material within devices has focused most
previous chromatographic efforts upon prior art open channel
methods [Manz. A., Miyahara, Y., Miura, J., Watanabe, Y., Miyagi
and H. Sato, K., Sens. Actuators 1990, B1, 249 255; Jacobson, S.
C., Hergenroder, R., Koutny, L. B. and Ramsey, J. M. Anal. Chem.
1994, 66, 2369 2373; Kutter, J. P., Jacobson, S. C., Matsubara, N.
and Ramsey, J. M. Anal. Chem. 1998, 70, 3291 3297; He, B., Tait, N.
and Regnier, F. Anal. Chem. 1998, 70, 3790 3797]. In the prior art,
open channel method devices with channel widths of 2 .mu.m or less
were required to improve mobile phase transfer in open columns
leading to other practical considerations such as clogging and a
short path length for detection. There were also problems with the
reproducibility and the cost of stationary phase coating in such
structures.
[0083] As realized by the inventors, on-chip packed bed
chromatography according to the present invention has the benefit
of providing low mobile phase mass transfer, and makes available a
wide variety of stationary phases. In this case, the use of an off
chip prepared stationary phase offers the advantage that it
eliminates the need for coating the chip and allows for
optimization of the stationary phase preparation.
[0084] Yet another extended application facilitated by the present
invention is providing on-chip bead based immunoassay and enzyme
based assays. These applications are described further below.
EXAMPLE
[0085] To illustrate the present invention by way of example, the
inventors conducted a series of experiments, which are described
here.
Chip Design
[0086] FIGS. 1A and 1B show a microfluidic device 10 as used in
these experiments. The device 10 comprises a main channel 11 formed
into the top surface of a substrate 8, and the main channel 11 is
separated by a chamber 4, also formed into the substrate 8. Two
branches of the main channel 11, as separated by the chamber 4, are
further identified as main reservoirs 1 and 2. The chamber 4 is
connected to a packing material reservoir 3 by a narrow side
channel 5. The packing material reservoir and the narrow side
channel 5 are also formed into the substrate 8. FIG. 1B shows an
enlarged image of the chamber 4 obtained with a scanning electron
microscope (Jeol X Vision JSM6301FXV, Peabody, Mass.). The chamber
4 is formed by providing two weirs 6, 7 formed across the main
channel 11 at a relatively narrow portion of the main channel 11
(FIG. 1A). As can be seen from FIG. 1B, the weirs 6, 7 are not as
high as the main channel 11 is deep, so that some fluid is allowed
to flow over the weirs 6, 7 as explained below. The device 10 was
prepared in Corning 0211 glass by the Alberta Microelectronic
Corporation (Edmonton, AB), using known chemical etching procedures
[Fan, Z. H.; Harrison, D. J. Anal. Chem. 1994, 66, 177 184]. It is
noted that this substrate material is non-conductive, but if other
than electrokinetic forces are being used (as detailed further
below), then the substrate material may be semiconducting or
conducting. Two photomasks were required to create device 10: a
first photomask was used to etch the tops of the weirs 6, 7 to a
depth of approximately 1 .mu.m; and a second photomask was used to
etch the channels 5, 11 to a depth of approximately 10 .mu.m.
[0087] FIG. 2A shows a cross-sectional view of the weirs 6, 7 which
are not as high as the channel 11 (main reservoirs 1, 2) is deep,
and thus small flow gaps 14, 15 are provided between the top of the
weirs 6, 7 and a cover plate 9 (not shown in FIG. 1A or 1B) which
is placed on top of the substrate 8, thereby closing off the
chamber 4, channels 5, 11 and reservoirs 1, 2, 3. As can be seen in
FIG. 2A, the beads 12 are generally larger than the flow gaps 14,
15 and therefore cannot escape from the chamber 4.
[0088] FIGS. 2B and 2C show a side view and an end view,
respectively, of an alternative embodiment of a weir 6' in which
substantially vertical notches 6'' are provided so that the weir 6'
provides less flow impedance. The vertical notches 6'' should be
narrow enough that no beads can pass through them (i.e. they should
be at least about 10% smaller than the smallest bead diameter).
Solutions and Reagents
[0089] Various solutions and reagents were used in these
experiments. Acetonitrile (BDH, Toronto, ON) was filtered through a
0.45 .mu.m Nylon 6,6 filter (Altech, Deerfield, Ill.) prior to use.
Otherwise, the acetonitrile was used as received, with no added
electrolyte. Also, 50 mM potassium phosphate (pH 7.0) and ammonium
acetate (pH 8.5) buffers were prepared in ultra-pure water
(Millipore Canada, Mississauga, ON). A 1:1 (v/v) mixture of
acetonitrile and buffer was prepared. A stock solution of 0.10 mM,
4,4 difluoro 1,3,5,7,8 penta methyl-4-bora-3a,4a-diaza-s-indacene,
BODIPY 493/503 (Molecular Probes, Eugene, Oreg.) was prepared in
HPLC grade methanol (Fisher, Fair Lawn, N.J.). A 1 mM stock
solution of fluorescein di sodium salt (Sigma) was prepared in
phosphate buffer. Both stock solutions were then diluted in the 50
mM phosphate and 50 mM ammonium acetate buffers to give 1.0 .mu.M
solutions, which were then diluted to 1.0 nM. This 1.0 nM solution
served as the sample for preconcentration and
electrochromatography. All aqueous (buffer and sample) solutions
were filtered through a cellulose acetate syringe filter (0.2 .mu.m
pore size) (Nalgene, Rochester, N.Y.) prior to use.
Packing Material
[0090] One suitable packing material used in these experiments
comprised a reverse phase chromatographic stationary resin. The
resin was Spherisorb ODS1 (Phase Separations, Flintshire, UK), a
porous C-18 resin whose particles ranged from 1.5 to 4.0 .mu.m in
diameter, as determined by scanning electron microscopy (ODS beads
12). A slurry of approximately 0.003 g/mL of ODS1 was prepared in
acetonitrile. This slurry was used to supply the packing material
reservoir 3, to subsequently pack the chamber 4.
[0091] Certain solvent and additive combinations were found to help
the packing material stay in the packed chambers. For example, if
ODS beads are introduced in acetonitrile they flow readily, while
subsequently switching to an aqueous or predominately aqueous
solvent causes the beads to aggregate and become trapped within the
chamber. With ODS beads up to 30% acetonitrile could be present in
the aqueous solution without disrupting the aggregation observed to
the point of destabilizing the packed bed. Up to 50% acetonitrile
could be present with only modest loss in aggregation and weak
destabilization of the bed. As another example protein G or protein
A coated beads formed aggregates in aqueous solution, which made it
hard to introduce them into the trapping zone. However, the
addition of a neutral surfactant such as Tween 20 or Brij 35 (both
are trademarks) prevented such aggregation and allowed the beads to
be introduced. Conversely, subsequent removal of the surfactant
form the aqueous solvent resulted in aggregation and enhanced
stability of the trapped bed. The following trend was observed:
when using non polar or partially non polar bead phases (for
example, ODS and protein coated beads) lowering the surface tension
of the solvent from that of water or buffered water, by the
addition of organic additives such as organic solvents or
surfactants, reduced the tendency to aggregate. Conversely reducing
or eliminating materials with lower surface tension from aqueous
solution increased the tendency to lock the beads in place on the
bed, creating a "solvent lock" method to enhance bead trapping
within these devices. Other organic solvents other than
acetonitrile, miscible with water may also be used for these
purposes, such as methanol, ethanol, dimethylsulfoxide, propylene
carbonate, etc. Charged surfactants may also be used instead of
neutral surfactants, so long as they are compatible with the
proteins that may be present on the beads or in the sample.
[0092] Magnetic beads used for magnetic packing may comprise
Abebaw-protein "A" coated beads: composition 36-40% magnetite
dispersed within a copolymer matrix consisting of styrene and
divinyl benzene (Prozyme, Calif.) Also, Guifeng-oligo (dT)25 coated
beads may be used for the isolation of mRNA. The beads have an even
dispersion of magnetic material (Fe2 O3 and Fe3O4) through out the
bead. The beads are coated with a polystyrene which encases the
magnetic material (Dynal, Oslo, Norway).
Instrumentation
[0093] Various instruments were used in conducting the present
experiments. As these instruments and their operation are well
known to those skilled in the art, only a brief description is
provided, and the instruments are not shown in the figures.
[0094] A power supply and relay system used to control the
electrophoretic voltages necessary for bead packing and all liquid
handling on chip has been described previously [Fluri, K.,
Fitzpatrick, G., Chiem, N. and Harrison, D. J. Anal. Chem. 1996,
68, 4285-4290]. LabVIEW programs (National Instruments, Austin,
Tex.), were written for computer control of the voltage system and
for data acquisition.
[0095] A laser induced fluorescence detection system used in this
experiment consisted of a 488 nm argon ion laser (Uniphase, San
Jose, Calif.), operated at 4.0 mW, and associated focusing optics
[Manz. A., Miyahara, Y., Miura, J., Watanabe, Y., Miyagi, H. and
Sato, K. Sens. Actuators 1990, B1, 249 255] (Melles Griot, Irvine,
Calif.). Fluorescence emitted from the BODIPY sample (as described
above) was collected by a 25.times., 0.35 NA microscope objective
(Leitz Wetzlar, Germany). The images were observed with a SONY CCD
IRIS camera. Alternatively a 530 nm emission filter and a photo
multiplier tube (PMT) (R1477, Hamamatsu, Bridgewater, N.J.) were
used as a detector positioned so that the narrow channel 5 between
the chamber 4 and packing material reservoir 3 could be monitored.
Data was collected from the section of main channel 11 just next to
the chamber 4. The weir 6 was just out of the field of view. The
PMT was biased at 530 V while the PMT signal was amplified,
filtered (25 Hz Butterworth) and sampled at a frequency of 50
Hz.
[0096] The fluorescence of the buffer, acetonitrile, and 1.0 nM
BODIPY in both buffer and acetonitrile was measured using a
Shimadzu RF 5301PC Spectrofluorophotometer.
[0097] While specific models and manufacturers have been provided
for various instrumentation described above, it will be understood
by those skilled in the art that any suitable, functional
equivalent may be used.
Chip Operation
[0098] Referring back to FIGS. 1A and 1B, the narrow side channel 5
leading into the chamber 4 from packing material reservoir 3 was
used to direct stationary phase packing material into the chamber 4
using electrokinetic pumping [Yan, C., U.S. Pat. No. 5,453,163,
1995; Knox, J. H. and Grant, I. H. Chromatographia 1991, 32, 317
328]. As mentioned above, the substrate 8 is non-conductive, which
allows packing of the beads 12 using the electrokinetic pumping
method.
[0099] The device 10 was not conditioned with any aqueous solutions
prior to use. The chamber 4, channels 5, 11, and reservoirs 1, 2, 3
were first filled with acetonitrile. The chamber 4 was packed with
ODS beads 12 (FIG. 2) by replacing the acetonitrile in packing
material reservoir 3 with the ODS/acetonitrile slurry (described
above), then applying positive high voltage at packing material
reservoir 3 while holding main reservoirs 1 and 2 at ground. The
voltage applied at packing material reservoir 3 was ramped from 200
V to 800 V over approximately 5 min to effect packing of chamber
4.
[0100] Once the chamber 4 was packed, a step gradient was performed
to introduce aqueous solution to the main channel 11 and the ODS
beads 12 in the chamber 4. A 1:1 (v/v) mixture of acetonitrile and
buffer was placed in reservoirs 1 and 2. Acetonitrile replaced the
slurry in packing material reservoir 3. A voltage was then applied
to main reservoir 1 and was ramped from 200 V to 800 V, with
packing material reservoir 3 biased at 400 V and main reservoir 2
grounded. After 2 to 5 min at 800 V, the acetonitrile/buffer
mixture in reservoirs 1 and 2 was replaced with buffer, and the
same voltage program repeated. The chamber 4 was monitored visually
to ensure that the acetonitrile was completely replaced by buffer
and that the packing material (beads 12) did not shift or unpack
during this procedure. (The beads 12 could be seen to agglomerate
as the acetonitrile was expelled, and the index of refraction
change at the water/acetonitrile interface was clearly visible.)
The experiments conducted are described in further detail
below.
Experimental Results and Discussion
[0101] In order to conduct the experiments, it was necessary to
pack the chamber 4 with packing material (beads 12), as shown in
FIG. 2A.
[0102] The narrow side channel 5 shown in FIGS. 1A and 1B was made
to be about 30 .mu.m wide to supply packing material (beads 12) to
the chamber 4. A sample could then be delivered from reservoir 2
(the inlet channel), across the chamber 4 and on towards main
reservoir 1 (the outlet channel). The volume of the chamber 4 was
330 pL, while the volume of the outlet and inlet channels was
1.5.times.10 7 L and 4.1.times.10 8 L, respectively. The main
channel 11 had much lower flow resistance than the side channel 5,
in spite of the weirs 6, 7, given their relatively wide widths (580
.mu.m, tapering to 300 .mu.m at the weirs) in comparison to the
width of the narrow channel 5 (30 .mu.m). The relative flow
resistance in the device 10 was manipulated by the selection of the
width dimensions for these channels 5, 11 in order to encourage
flow between main reservoirs 1 and 2, rather than into the narrow
bead introduction side channel 5 during sample loading and
elution.
[0103] Reverse phase ODS beads 12 (as described previously) were
used in the SPE device because of their extensive use for the
chromatography of proteins, peptides and tryptic digests [Seifar,
R. M.; Kok, W. T.; Kraak, J. C.; and Poppe, H. Chromatographia,
1997, 46, 131 136. Yan, C.; Dadoo, R.; Zhao, H.; Zare, R. N.; and
Rakestraw, D. J,. Anal Chem. 1995, 67, 2026 2029.] as well as other
applications of SPE and CEC [Nielsen, R. G.; Riggin, R. M.;
Rickard, E. C. J. Chromatogr. 1989, 480, 393 401. Hancock, W. S.;
Chloupek, R. C.; Kirkland, J. J.; Snyder, L. R. J. Chromatogr. A
1994, 686, 31 43.]. Electrokinetic packing of conventional
capillaries has been described previously, [Yan, C.; U.S. Pat. No.
5,453,163, 1995. Knox, J. H.; Grant, I. H. Chromatographia 1991,
32, 317 328.], and the inventors have adapted the method for the
present invention.
[0104] As briefly explained earlier, the packing procedure involved
applying a positive voltage (ramped from 200 800 V) to the packing
material reservoir 3, while grounding main reservoirs 1 and 2. The
applied voltage induced EOF to flow down the bead channel, carrying
the beads into the cavity. An organic solvent was required to
suspend the chromatographic beads 12 to prevent them from
aggregating and plugging the narrow side channel 5. Studies have
shown that capillaries filled with acetonitrile exhibit substantial
electroosmostic flow [Wright, P. B.; Lister, A. S.; Dorsey, J. G.
Anal Chem. 1997, 69, 3251 3259. Lister, A. S.; Dorsey, J. G.;
Burton, D. E. J. High Resol. Chromatogr. 1997, 20, 523 528. Schwer,
C.; Kenndler, E. Anal. Chem. 1991, 63, 1801 1807. Salimi Moosavi,
H.; Cassidy, R. M. Anal. Chem. 1995, 67, 1067 1073.].
[0105] As shown in FIG. 3A, at the early stages of packing, the
beads 12 entering the chamber 4 contacted the weirs 6, 7 on either
side of the chamber 4. As explained earlier, the beads 12 are
unable to traverse the weirs 6, 7 because the distance from the top
of the weirs 6, 7 to the bottom of the cover plate 9 (approximately
1.0 .mu.m) is less than the diameter of the individual particles of
the ODS beads 12 (approximately 1.5-4.0 .mu.m).
[0106] As shown in FIG. 3B, the chamber 4 continued to pack until
it was entirely filled with chromatographic material. As discussed
earlier, the difficulties associated with reproducibly fabricating
frits for retaining packing material is well known. Importantly,
the weir design used in the present invention circumvented this
problem, and the electrokinetic packing of the beads provided an
even distribution of beads throughout the chamber with no
observable voids. In fact, the use of weir structures may
ultimately eliminate the need for on column frit fabrication.
[0107] The weir design of the present invention allows electric
fields to be applied across the trapping zone formed by two weirs,
when filled with beads, in a range as high as 20,000 to 80,000 V/cm
without bubble formation at the weir. Separations performed in
devices with these weirs can use electric fields at least as high
as 15,000 V/cm. The power dissipated across a weir can be as high
as 3-7 W/m without the formation of bubbles. In contrast frits
formed in conventional columns have at the best been reported to
form bubbles at power dissipations above 0.6 W/m, and electric
fields in the range of 150-600 V/cm are the best that have been
reported without bubble formation.
[0108] It is possible to couple an external capillary to a chip and
allow the weir to be used as the trapping element for the beads
packed within the external electrochromatography capillary. This
can be accomplished using a low dead volume coupling, such as
described by Bings et al. (N. H. Bings, C. Wang, C. D. Skinner, C.
L. Colyer, P. Thibeault, D. J. Harrison, Anal. Chem. 71 (1999) 3292
3296.) In this way the chip based weir can replace the frits
normally formed within external capillaries, and allow higher
electric fields to be used, improving speed and separation
efficiency.
[0109] (It is noted here that it was also possible to pack the
cavity by applying a vacuum at main reservoirs 1 and 2, although
this was less convenient when electrokinetic flow was used for
sample loading and elution.)
[0110] If for some reason the beads 12 did not pack as tightly as
was desirable (as shown in FIGS. 2 and 3B) they were removed from
the chamber 4 by simply reversing the voltages, and the packing
procedure was then repeated. It is noted that once an aqueous
solution was introduced to the chamber 4, the reverse phase beads
12 tended to aggregate and were more difficult to remove. However,
subsequent removal was accomplished by flushing the aqueous
solution out with acetonitrile, using either EOF or vacuum, or a
combination of the two. Advantageously, the ability to effectively
remove the beads 12 from chamber 4 allowed used chromatographic
beads to be refreshed, or a more applicable material to be
substituted.
[0111] Significantly, a design utilizing a hook structure 13 at the
chamber entrance (FIGS. 1B and 3A) yielded the most favorable
results in packing, enabling the chamber 4 to be packed and remain
so after removal or alteration of voltages or vacuum. As seen from
the figures, the side channel 5 connects to the chamber 4 via a
chamber mouth 4A in an asymmetric fashion, relative to the weirs 6,
7. Also, the hook structure 13 preferably obstructs direct
line-of-sight entry of packing material from the side channel 5
into the chamber 4. Rather, the hook structure 13 forces packing
material to enter the chamber 4 indirectly via the chamber mouth
4A.
[0112] As explained earlier, during the packing step, the packing
material reservoir 3 has a positive bias applied with reservoirs 1
and 2 grounded. The inventors believe that the hooked structure 13
causes electric field lines to follow a curved pathway into the
cavity. Consequently, as the chromatographic beads 12 follow the
electric field lines into the chamber mouth 4A they appear to be
"sprayed" as if from a snow blower (FIG. 3A), to become uniformly
packed.
[0113] During the packing procedure the chamber 4 filled only to
the beginning of the hook structure 13 (see FIG. 3B). Once filled,
the beads were observed to flow down the sides and up the middle of
the narrow side channel 5 (toward packing material reservoir 3)
mimicking the solvent back flow generated in a closed
electrophoretic system [Shaw, D. J. Introduction to Colloid and
Surface Chemistry, 3.sup.rd ed. Butterworths: London, 1980.]. In
such a closed system, EOF is directed along the walls until it
reaches the end of the chamber, where pressure causes the solution
to reverse direction and flow back up the center of the bead
introduction channel.
[0114] A key aspect of the hooked structure as shown is the
asymmetric entrance into the trapping zone, which allows for better
packing. A symmetric entrance means the entering beads can go to
both weirs equally, which tends to lead to uneven or difficult
packing. An asymmetric structure allows the beads to pack
preferentially at one end of the trapping zone first and then build
up in one direction from that location. The key role of the hook
structure is to prevent line of sight outflow from the trapping
zone during use of the packed bed.
[0115] Chambers constructed without an asymmetry in the entrance
were not observed to pack as well as asymmetric entry designs. In
these cases, packing material tended to fill the corners furthest
from the entrance, but no additional material would enter the
chamber. The inventors believe that, due to its symmetric design,
this type of chamber exhibits solvent back flow, after it has
filled to a certain extent. That is, the partially filled chamber
may resemble a closed or restricted system. Such an occurrence
would preclude the filling of the symmetric chamber with beads and
is consistent with previously observed behavior, as explained by
Shaw. Such behavior may account for the ability to fill symmetric
structures on some occasions but less readily on others. In
contrast, an asymmetric design, with or without a hook structure 13
guarding the entrance is less likely to experience back flow
directly into the narrow bead introduction channel 5.
Solid Phase Extraction (SPE) On-Chip
[0116] As explained earlier, the present invention allows
applications of microfluidic analysis systems to be extended. One
such extension is facilitating SPE directly on-chip.
Preconcentration is a valuable tool that can be used to enhance the
sensitivity of microfluidic devices. To determine the ability of a
packed SPE bed constructed on a microchip to preconcentrate an
analyte, the inventors concentrated a 1.0 nM solution of BODIPY
reagent from 50 mM phosphate buffer. Solution conditions utilized
were similar to those used for protein and peptide analysis in HPLC
CE systems. [Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62,
978 984. Castagnola, M.; Cassiano, L.; Rabino, R.; Rossetti, D. V.
J. Chromatogr. 1991, 572, 51 58.] The BODIPY reagent, when diluted
in aqueous buffer, exhibits a high affinity for ODS material and is
an excellent fluorophore. The preconcentration and elution of the
BODIPY reagent was carried out in four steps: equilibration of the
SPE bed with buffer; sample introduction; buffer flush; and elution
of analyte. Following rinsing of the packed bed with phosphate
buffer, a solution of 1.0 nM BODIPY was placed in main reservoir 1,
and +200 V was applied for 2 minutes, with main reservoir 2
grounded. The EOF (0.2 mm/sec, 1.2.times.10.sup.-9 L/sec) flowed
towards reservoir 2, carrying the BODIPY onto the SPE bed during
the loading step. As shown in FIG. 4A, fluorescence of the absorbed
BODIPY occurred initially at the first few layers of beads 12 only
(near the top of the Figure). FIG. 4B shows the SPE bed after 1.5
minutes, with a total of 1.4.times.10.sup.-16 moles of BODIPY
reagent loaded on the bed (assuming complete capture of the dye).
No sample breakthrough was observed with BODIPY, due to its high
affinity for the ODS material. In fact, visual observation
indicated that after concentrating 1.0 n M BODIPY solution for two
minutes only 5% of the physical volume of the SPE bed was utilized
suggesting that the capacity of the 330 pL bed was about
2.8.times.10.sup.-15 moles of analyte. A buffer wash step was used
after loading to wash sample remaining within the channel 11 onto
the bed (in chamber 4). The solutions in reservoirs 1 and 2 were
then replaced with acetonitrile, and the dye was eluted with
solvent moving in the same direction as the initial loading step
(or by reversal of the potential gradient during the elution step,
it could be directed back towards the original sample reservoir).
Both procedures work well, but the latter was more convenient for
our testing. FIG. 5 shows graphically the 3-step preconcentration
experiment for a 1.0 nM BODIPY sample following bed equilibration.
The 90-second loading step showed an increase in signal as the
fluorescent sample passed by the detector positioned as shown in
FIG. 1A. This was followed by a 60-second rinse step. Acetonitrile
was then used to elute the BODIPY reagent off the bed in the
opposite direction to which it was loaded, eliminating the need for
detector repositioning. The BODIPY reagent eluted in a relatively
narrow 3 second band following a 90 second preconcentration step
exhibiting a many fold concentration increase compared to the
original sample. The fluorescence of the BODIPY (1.0 nM) reagent
was tested in both buffer and acetonitrile and did not show a
significant difference in intensity for either of the solvents. The
preconcentration factor (P.F.) can be estimated using equation
(1):
P . F . = V i V i = t pre f buff t elute f elute ( 1 )
##EQU00001##
where Vi is the volume of buffer containing analyte and V f is the
volume of acetonitrile containing analyte. The volume Vi is the
product of the preconcentration time (t.sub.pre, sec.) and the
electroosmotic flow of the sample being concentrated (f.sub.buff,
L/sec.) while Vf is the product of width of the eluted analyte peak
(t.sub.elute, sec.) and the flow rate of the eluting solvent
(f.sub.elute, L/sec). For this case, the analyte was
preconcentrated by a factor of at least 100 times. After sufficient
concentration the BODIPY is easily observed visually on the SPE
bed.
[0117] Different sample loading times were utilized to increase the
amount of preconcentration. In the experiments, preconcentration
times ranging from 120-532 seconds were studied yielding
preconcentration factors of 80-500. Peak area (rsd 3-11%) plotted
versus preconcentration time yielded a linear relationship
(r2=0.9993) over the studied conditions.
Capillary Electrochromatography (CEC) On Chip
[0118] As explained earlier, another application facilitated by the
present invention is on-chip capillary electrochromatography (CEC).
Reversed phase mode CEC was performed on a chamber 4 packed with
octadecyl silane beads 12 equilibrated with buffer. Due to the lack
of an injector within the chip design, the samples were loaded onto
the front of the chromatographic bed in 50 mM ammonium acetate
buffer, pH 8.5 (see "Solutions and Reagents," above). Both
compounds were totally retained under these conditions, as
indicated by a lack of analyte signal in the loading and flush
steps. The loading step functioned to both introduce the sample and
preconcentrate the retained analytes at the front of the bed
[Swartz, M. E.; Merion, M.; J. Chromatogr, 1993, 632, 209 213.]
FIG. 6 shows the three steps involved in the CEC separation of
BODIPY and fluorescein with a mobile phase composition of 30%
acetonitrile/70% aqueous 50 mM ammonium acetate. Once the mixed
mobile phase reaches the bed, both compounds begin to undergo
chromatography and are eluted from the bed. The compounds are
completely eluted and separated in less than 20 sec on less than
200 .mu.m of chromatographic bed, yielding a plate height of 2
.mu.m (N=100 plates or 500 000 plates/m) for the fluorescein peak.
Under these conditions, the fluorescein is eluted prior to the
BODIPY reagent. Peaks were identified by comparing retention times
of the standards with those of the mixture. At pH 8.5 fluorescein
possesses a net (-2) charge while BODIPY is neutral. In a normal
CZE separation the electrophoretic mobility of fluorescein would
oppose the EOF, causing the BODIPY to elute prior to fluorescein.
In this case the elution order of the two components is reversed,
indicating an interaction between the analytes and the stationary
phase. The BODIPY being more hydrophobic has a higher affinity for
the chromatographic material than does fluorescein causing the
BODIPY to be retained more and eluted later.
[0119] Finally, FIGS. 7A-7D shows the CEC separation of BODIPY and
fluorescein utilizing mobile phases with different concentrations
of acetonitrile. It was observed that the increased acetonitrile
concentration lowers the polarity of the mobile phase, decreasing
the amount of time required for the BODIPY to elute. The elution
time for fluorescein does not change, indicating little to no
chromatographic retention except at low % acetonitrile. Decreasing
the acetonitrile concentration provides baseline resolution, but
leads to more extensive band broadening.
[0120] Our present results are comparable to that reported for open
tubular CEC on a chip [Jacobson, S. C., Hergenroder, R., Koutny, L.
B., Ramsey, J. M. Anal. Chem. 1994, 66, 2369 2373. Kutter, J. P.;
Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70,
3291 3297. He, B., Tait, N., Regnier, F. Anal. Chem. 1998, 70, 3790
3797.].
[0121] Immunoassay Using Bead Based Reagents
[0122] Immunoassay on beads, or immunosorbent assays involves
placing either an antibody or antigen on the surface of the bead.
As a solution containing an antigen passes over the beads, the
antigen specifically binds the antibody. In this way the
specificity of the antigen for the antibody is utilized to separate
it from other species in solution. Later the solution conditions
are changed so that the antibody or antigen is eluted from the
beads and is detected as either complex or the free antibody. The
development of immunosorbent assays on chip is attractive because
of the small amounts of reagents that are consumed. In addition
microchips offer very fast analysis times compared to conventional
methods performed in micro titer plates or in syringes packed with
immuno beads. Immunosorbent assays on chip also provide lower
concentration detection limits than solution phase immunoassays on
chip, making the development of bead based immunoassay on chip
important.
[0123] Beads that have specific enzymes linked to them are packed
into the chamber created by the two weirs. The use of beads is
preferential because of the increased surface area of the beads as
opposed surface area of the channel walls. The higher surface area
leads to a greater capacity and more efficient trapping of the
analyte. The weirs form a well defined chamber for the immunoassay
beads to pack. The inventors have demonstrated bead based
immunoassay on chip for the enzyme theophylline. In the experiment
magnetic beads coated with protein A are packed within the chamber
of the chip. Later the antibody (antitheophylline) is flowed across
the bed in a 1 mM tricine buffer pH 8.0. When the antitheophylline
flows through the packed bed the antibody binds to the protein A.
The antitheophylline was passed over the bed for several minute to
ensure that the bed is saturated with antibody. A buffer washing
step was then utilized to remove the remaining unbound antibody
from the chamber and channels. The bed was then saturated with
fluorescently labelled theophylline (diluted from a kit) by flowing
it through the bed where it binds to the antitheophylline. The
point at which the bed was saturated was determined by monitoring
fluorescence below the bed and determining the point where the
breakthrough curve (FIG. 1) plateaus. Following breakthrough the
theophylline solution is washed from the device using a buffer
flush step. A chaotropic agent is then added to elute the
theophylline from the bed as either free protein or
theophylline/antibody complex. Chaotropic agents can be of various
types, however in this example a mixture of 90% acetone/10% tricine
buffer was used. Once the chaotropic agent reaches the packed bed
the theophylline is eluted in a relatively narrow band (FIG.
2).
[0124] Although normally under these circumstances a competitive
assay would be performed the direct assay demonstrates the ability
of the chamber formed by the weirs to act as an immunoassay
bed.
Enzyme Reactor Beds
[0125] There have been several methods developed for immobilizing
enzymes onto solid supports like beads. Once immobilized the enzyme
beads can be packed into beds to perform chemical reactions on
solutions as they are flowed through them. Normally a solution
containing a substrate is passed through the bed. When the
substrate comes in contact with the enzyme the enzyme reacts with
the substrate to yield a product. The product resulting from the
reaction of the immobilized enzyme and substrate can be later used
as a method of detection or in other synthetic processes. This
example illustrates the use of the immobilized enzyme horse radish
peroxidase(HRP) and xanthine oxidase(XO) on porous silica beads (5
.mu.m diameter). These results show that enzymes, once immobilized
onto beads, can be trapped/packed into the weir device, where they
are still active and can be used as an enzyme reactor bed.
[0126] XOD and HRP were immobilized onto Nucleosil 1000 5 silica
beads (Machrey Nagel, Germany) that had been silanized with
3-aminopropyltriethoxysilane, by crosslinking with gluteraldehyde
(Sigma). The immobilization of enzymes on glass beads has been
described previously and is known by practitioners of the art. All
studies were performed using 50 mM boric acid adjusted with 1 M
NaOH to pH 9.
The immobilization of HRP and XOD was performed to demonstrate two
principals. First was the ability to pack the enzyme immobilized
beads within the weir device and then second was to demonstrate
that the enzyme was still active and could be utilized to catalyze
reactions once packed. To show each of these principals a
chemiluminescent reaction was performed using the weir device. The
ability to pack immobilized enzymes allows different methods of
detection to be used for certain analytes. For example the luminol
chemiluminescence (CL) reaction can be used for very sensitive
determinations when only small amounts of analyte are available or
when labeling reactions are otherwise difficult to perform. CL
reactions are unique in that they do not require a light source
simplifying the detection scheme. The chemiluminescence reaction
catalyzed by HRP is shown below.
Luminol+H.sub.2O.sub.2+HRP(Light(425 nm)+other products
Beads immobilized with HRP were packed into the weir device and a
solution containing the reagents for the reaction passed through
the bed. The immobilized HRP was found to catalyze the
chemiluminescent reaction when a solution of H.sub.2O.sub.2 (100
(M) and luminol (10 mM) was flowed over a bed that had been packed
with beads containing immobilized HRP. Light generated from the
reaction was detected downstream from the enzyme bed.
[0127] However, it was noticed that with each successive trial the
light generated from the CL reaction was lower than in the previous
trial FIG. 1. This is probably caused by a decrease in the activity
of the enzyme with each successive run. These results evidence the
advantage of a method of removing the exhausted beads and replacing
them with fresh ones, such as discussed for the replacement of ODS
beads within the weir device.
ALTERNATIVE EMBODIMENTS
[0128] While a two weir embodiment of the design according to the
present invention has been described above, other embodiments are
also possible. For example, it is possible to implement a single
weir design to form an on-chip reactor bead (i.e. not having a
second weir 6 located upstream in the main channel 11).
Specifically, by providing a downstream weir 7 formed across the
main channel and providing pressure only in a downstream direction
(i.e. from main reservoir 2 and side channel 3 to main reservoir
1), it has been observed that packing can be achieved against the
downstream weir 7. However, it is noted that a single weir design
may result in the formation of a ragged leading edge for the packed
bed that reduces separation efficiency when used for SPE or CEC.
Additionally, the high back pressure associated with a long bed of
small beads limited the length of the pack to about 4-6 mm. A high
pressure fitting for the microchip would allow high pressure
pumping and allow somewhat greater lengths.
[0129] Other types of forces may also be used to create a packed
bed using a single weir design. For example, it was also possible
to achieve a limited degree of packing (to a length of a few
millimeters) using electrokinetic forces, directed only in a
downstream direction (i.e. from main reservoir 2 and packing
material reservoir 3, to main reservoir 1).
[0130] In addition to varying the number of weirs, it is also
possible to provide more than one inlet or outlet to a chamber, as
shown in alternative embodiments of the present invention in FIGS.
8A-8C.
[0131] In FIG. 8A, a chamber 4 is formed between two weirs 6, 7.
Two side channels 5a, 5b are provided to serve as an inlet or
outlet to the chamber 4. As shown in FIG. 6, the side channels 5a,
5b may be offset relative to each other to better facilitate
packing of the chamber. A second side channel is added to allow the
beads to be flushed out to waste at the other end of the trapping
zone, or to allow the flushing agent to be delivered from an
alternate reservoir. The latter design can prevent used beads from
contaminating the fresh bead stream, and/or prevent sample and
sample waste solutions from being directed into the trapping zone
during flushing.
[0132] As shown in FIG. 8B the side channel in this design may have
one or more optional branches 5c, to allow the side channel 5b to
be flushed of beads, or to allow beads being flushed out of the
trapping zone to be directed, for example, into a waste reservoir
instead of into the packing material reservoir 3 (not shown).
[0133] Another embodiment is shown in FIG. 8C, in which a side
channel weir 16 is provided near the entrance of a third side
channel 5d to the chamber 4, to allow fluid flow without passage of
beads. This "weired" side channel 5d may be used, for example, to
release pressure build up in the chamber 4 during loading of the
beads, particularly when the length of the chamber 4 (as measured
between the weirs 6, 7) is greater than 4-6 mm.
[0134] In all three embodiments shown in FIGS. 8A-8C, the side
channel entrance into the chamber 4 may be modified to include a
hook or similar shape, as described earlier, in order to prevent
direct "line of sight" flow from a side channel into the chamber 4,
or vice versa. As explained earlier, this entrance modification
serves to spray the beads into the trapping zone in order to assist
packing, and to reduce the tendency of the beads to exit from the
chamber 4 during later use.
[0135] Loading of beads with more than one side channel, as shown
in FIGS. 8A-8C, is performed in a manner similar to that for a
single side channel, two weir design, (as described above) except
that a potential must also be applied to the additional side
channels to prevent flow into those side channels when using
electrokinetic loading. During removal of the beads a voltage may
be applied to a second side channel (e.g. side channel 5b in FIG.
8A) to drive beads out of the trapping zone or chamber 4, applying
voltage potentials such as those used with the single side channel
design but adjusted for the potential drop in the additional side
channel. As will be appreciated, the direction of flow during the
flushing step can be controlled by the polarity of the applied
voltage.
[0136] When using pressure driven flow to load beads, a back
pressure must be applied to the additional side channels during
loading, or else the reservoirs attached to the additional side
channels may be temporarily sealed. When flushing the beads from
the chamber 4, a pressure may be applied to the bead supply channel
5a to flush beads out of one or more additional side channels.
[0137] When performing SPE or CEC using a multiple side channel
design and electrokinetic forces, a voltage may be applied to the
additional side channels to prevent leakage of sample or beads out
of the trapping zone and into the side channels, substantially in
the same manner as described for a single side channel in the
trapping zone. When using pressure driven pumping, the side
channels may a have enough positive pressure applied to eliminate
flow into the side channel, or else the reservoirs attached to the
respective side channels can be temporarily sealed.
Immobilizing the Packed ODS Beads with Polymer
[0138] After packing the chromatographic bed with beads using
electrokinetic, magnetic or pressure packing techniques, a
reasonable portion of the bead introduction channel was then packed
with the beads using one of these packing techniques. Excess beads
in the reservoir were then removed. Then about 40 ml of a monomer
mixture was pipeted into the bead reservoir and delivered by
pressure or electrokinetic flow along the bead introduction
channel, towards the beads. During this step, a change in
refractive index in a region within the bead introduction channel
was used to monitor the position of the monomer solution, and flow
was stopped well before the monomer reached the packed bead
bed.
[0139] A typical monomer solution may be prepared as follows: to a
vial that contained 800 ml of a ternary solvent mixture that
contained 10 wt % H.sub.2O, 40 wt % 1,4-butanediol and 50 wt %
1-propanol, 200 ml of a mixture of 2,2'-azobisisobutyronitrile
(AIBN, 2 wt %) and ethylene dimethacryllate (EDMA) was added. (C.
Peters et al, Anal. Chem. 1997, 69, 3646-3649.) This monomer
solution was then purged with N.sub.2 for 15 min to remove
dissolved oxygen. Other polymerizable solution of monomer may also
be used.
[0140] The device was then kept in an oven at 60.degree. C. for
24-48 hr. The device was taken out from the oven and cooled down to
room temperature, with all reservoirs covered to prevent
evaporation. Alternatively, photo-initiated polymerization with
AIBN or other initiator may be used to polymerize the monomer
solution, without a need for extended heating of the device.
Following polymerization the device was rinsed with acetonitrile
then with buffer. Mobile phase compositions of up to 100%
acetonitrile could be used in such devices without destabilizing
the bead bed.
Dimension Guidelines
[0141] While the theoretical limits of various dimensions of a
microfluidic device designed according to the present invention are
not known, the inventors have adopted some general guidelines for
practical purposes, which are discussed below.
[0142] It is thought that the length of the trapping zone may range
anywhere from about 10 .mu.m up to about 200 cm (using a coiled or
serpentine path if necessary to allow for incorporation of such a
length within the confines of a single device wafer). The trapping
zone length required will be dependent upon the application and
will also be limited by the forces which may be applied to achieve
packing and unpacking. For example, on-chip CEC would require
relatively long trapping zones, with a preferred upper limit of
about 5 cm.
[0143] As to the depth of the trapping zone, sample and waste
channels, a practical range is estimated to be about 400 .mu.m to
0.25 .mu.m. More preferably, the upper limit should be about 100
.mu.m and the lower limit should be about 10% larger than the
particle depth at a minimum.
[0144] Also, in order to reduce the likelihood of clogging, the
bead delivery and bead waste channels (side channels 5, 5a-5d)
preferably should be at least about 3 times deeper and three times
wider than the bead diameter.
[0145] The maximum dimensions of the side channels 5, 5a-5d are
also dependant upon the relative flow resistances required (i.e.
the flow resistance of the side channel versus the main channel and
the weirs, so as to minimize side channel backflow during use).
Generally speaking, the flow resistance of the side channels should
be higher than the flow resistance of weirs to minimize the
backflow problem.
[0146] The accompanying tables provide information on the
calculated effect of channel and weir dimensions on the volumetric
flow rates out of the trapping zone, as a function of flow channel
depth, weir depth and side channel length using pressure driven
flow.
[0147] In the tables below, what is called channel W is element 1
in FIG. 1A; what is called channel C is called element 5 in FIG.
1A; and what is called channel C' is element 3 in FIG. 1A
TABLE-US-00001 Correlation to FIG. 1A Width Length 20 .mu.m Deep
Element 1 Channel W 600 6,500 Weir 280 variable Element 5 Channel C
50 variable Element 3 Channel C' 600 3,500 10 .mu.m Deep Element 1
Channel W 580 6,500 Weir 280 variable
The volumetric flow rates were estimated using the Navier Stokes
equation for a rectangular channel cross section and Perry's
tabulated values of the effect of channel shape. The flow
resistance of a channel with half width a and half depth b is given
by equation 2:
.DELTA.P/U=hL/abN (2)
where .DELTA.P is the pressure drop along a channel segment of
length L, U is the average linear flow velocity, h is the
viscosity, and N is a form factor dependent upon the cross
sectional ratio b/a (b<a). The factor N may be estimated from
solutions to the Navier Stokes equation for pressure driven,
parabolic flow, and was tabulated by Perry in Chemical Engineer's
Handbook, (3rd edition, 1950) pp 387. The goal in device design is
to make the resistance of the side channel, C in the Tables, higher
than the resistance of the weir and the following flow channel W,
so that flow across the weir is favoured. When flow elements are in
series the fluid resistance given by the right hand side of
equation 1 for each segment can be added in the manner that the
resistance of series electrical impedances can be added. When fluid
elements are in parallel the inverse of their fluid resistance can
be added to obtain the inverse of the total impedance, as is done
for parallel electrical resistances. The volumetric flow rate, Q,
through a channel or a combination of channels is then given by
equation 3.
Q=ab.DELTA.P/Rf (3)
Where Rf is the resistance to fluid flow defined by the right hand
side of equation 1, combined together for all channel segments as
discussed above. The ratio, r, of volumetric flow rate across the
weir, Q.sub.w versus into the side channel, Q.sub.C,
r=Q.sub.w/Q.sub.C, should be large to ensure the percent of
solution flowing across the weir, % Q.sub.w=1/(1+r), is high. This
can be accomplished by using a long narrow side channel compared to
a wide main channel, by increasing the depth of the weir relative
to the depth of the other channels, by decreasing the depth of the
side channel relative to the main channel, etc, as indicated by
several calculations presented in the Tables.
TABLE-US-00002 TABLE Volumetric Flow Ratios for 10 and 20 .mu.m
Deep Designs Channel Channel C Weir Weir Volumetric depth Length
Depth Length ratio r % Qw 20 .mu.m 15,000 3 20 12.58 92.6 25,000
20.85 95.4 15,000 10 18.75 94.9 20 .mu.m 15,000 1 20 0.687 41.1
25,000 1.16 53.6 15,000 10 1.37 57.8 10 .mu.m 15,000 3 40 38.9 97.5
25,000 63.7 98.5 15,000 30 41.7 97.7 10 .mu.m 15,000 1 40 4.14 80.5
25,000 6.87 87.3 15,000 30 5.83 84.3
TABLE-US-00003 TABLE Fixed Device Dimensions for Calculations with
a Given Etch Depth Width Length 20 .mu.m Deep Channel W 600 6,500
Weir 280 variable Channel C 50 Variable Channel C' 600 3,500 10
.mu.m Deep Channel W 580 6,500 Weir 280 variable Channel C 30
Variable Channel C' 580 3,500
Integrated Analytical Procedures
[0148] It will be appreciated that the various features of the
present invention as described above may be utilized in a more
complex microfluidic design.
[0149] FIG. 9 shows a multiple weir and multiple side channel
design, generally referred to by reference numeral 20, in which
several trapping zones are integrated, each serving a different
function.
[0150] As an illustrative example, in a first trapping zone 25,
formed between weirs 6a and 6b, beads loaded with an antibody to a
specific protein are introduced via side channel 25 (and exit via
side channel 26). A cell lysate or serum sample or other protein
source is directed from a sample reservoir (not shown) and loaded
into the chip via sample inlet 21 and entrance channel 38 (the
sample is removed at sample outlet 22 and an eluent inlet 23 is
also provided at the entrance channel. The sample is then passed
into the antibody bead bed in trapping zone 25 to isolate a
specific protein, while the effluent is directed towards waste
outlet 27.
[0151] A chaotropic elution agent, such as an acetonitrile, water
mix, is then introduced (eluent inlet 23) to elute the protein from
the column and deliver it to the next trapping zone 30 (formed
between weirs 6c and 6d) where it is digested by a protease enzyme
immobilized on beads loaded into the zone 30 (via side channels 29,
31). The effluent at this stage would be directed towards waste
outlet 32. After sufficient reaction time, a buffer is delivered
(elution inlet 28, running buffer 28a, waste from bed 25) to flush
the protein digest from the bed and into the next trapping zone 35
(formed between weirs 6e and 6f) with effluent delivered to waste
outlet 39.
[0152] The third trapping zone 35 contains a solid phase extraction
material (packed and unpacked via side channels 34, 35), allowing
concentration of the digest peptides onto the bed in zone 35. An
elution solvent, such as a methanol/aqueous mixture or
acetonitrile/aqueous mixture is then introduced (elution inlet 33,
running buffer 33a) to deliver (exit channel 37, waste 39, or
collection 40) a concentrated protein digest to another location on
the chip for final analysis.
Packed Bed Chip to Electrospray Mass Spectrometry Interface
[0153] Packed bed flow channels according to the present invention
may be interfaced to a mass spectrometer via an electrospray
coupler 41, as illustrated in FIG. 10. The packed bed 4 may perform
an enzyme digestion of a protein, affinity purification and pre
concentration of a specific chemical or protein, solid phase
extraction concentration enhancement, or capillary
electrochromatographic separation, or any combination of these and
other steps, prior to electrospray introduction in to a mass
spectrometer. The chip to electrospray interface may be made using
any method that provides a less than 100 mL dead volume, preferably
less than 1 mL and most preferably less than 100 pL dead volume at
the coupling region. A method such as that described by Wang et
al., or Karger can be used to create the interface [Bings, N. H.;
Wang, C.; Skinner, C. D.; Colyer, C. L.; Thibeault, P.; Harrison,
D. J. Anal. Chem. 71 (1999) 3292 3296. Zhang, B.; Liu, H.; Karger,
B. L.; Foret, F. Anal. Chem. 71 (1999) 3258 3264].
[0154] While the present invention has been described by reference
to various preferred embodiments, it will be understood that
obvious changes may be made and equivalents substituted without
departing from the true spirit and scope of the invention which is
set out in the following claims.
* * * * *