U.S. patent application number 11/102063 was filed with the patent office on 2005-09-01 for apparatus and method for small-volume fluid manipulation and transportation.
Invention is credited to Liu, Shaorong, Lu, Juan.
Application Number | 20050189225 11/102063 |
Document ID | / |
Family ID | 27372823 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050189225 |
Kind Code |
A1 |
Liu, Shaorong ; et
al. |
September 1, 2005 |
Apparatus and method for small-volume fluid manipulation and
transportation
Abstract
A microfluidic system has an electroosmotic flow pumping means
for propelling fluids through a series of microchannels and
selection valves. Pump channels are configured in groups which may
be fabricated singly or in multiple groups onto a substrate. A tube
filled with an immobilized polymer provides a means to apply
voltages across pump channels, while prevents passages of fluids
through it. It also avoids electrolysis and bubble formation in or
close to the microfluidic channels. The selection valves provide
for routing functions within the microfluidic system and can also
be configured to route fluids outside the system. A rate monitoring
system is provided for determining and compensating for flow rates.
In one application the microfluidic system may be configured to
operate as a small volume pipettor or other fluid transport or
analysis device. A micro-dialysis jacket is additionally provided
to permit desalting, pH adjustment, concentration adjustment, and
other functions.
Inventors: |
Liu, Shaorong; (Lubbock,
TX) ; Lu, Juan; (Lubbock, TX) |
Correspondence
Address: |
LIU , SHAORONG
5504 71st STREET
LUBBOCK
TX
79424
US
|
Family ID: |
27372823 |
Appl. No.: |
11/102063 |
Filed: |
April 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11102063 |
Apr 8, 2005 |
|
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|
10076170 |
Feb 11, 2002 |
|
|
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60267474 |
Feb 9, 2001 |
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60278508 |
Mar 23, 2001 |
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Current U.S.
Class: |
204/600 ;
204/280 |
Current CPC
Class: |
G01N 2035/00158
20130101; B01D 61/28 20130101; G01N 27/44743 20130101; G01N 35/1016
20130101; B01L 3/0293 20130101; G01N 2001/4016 20130101; B01D 61/56
20130101; B01L 3/50273 20130101; B01L 2400/0415 20130101; G01N
2035/1032 20130101; G01N 2035/00247 20130101; G01N 27/44791
20130101; B01L 3/0268 20130101; B01L 3/0241 20130101; B01L 13/02
20190801; B01L 3/502715 20130101 |
Class at
Publication: |
204/600 ;
204/280 |
International
Class: |
C25D 017/10 |
Claims
We claim:
1: A microfluidic system, comprising: a substrate; a fluid network
disposed on the substrate for transporting fluids, said fluid
network comprising a first segment and a second segment in fluid
communication; and an electrical source coupled across the first
segment of the fluid network to apply an electric potential to
induce electroosmotic flow in the first segment; a coupler filled
with a chemically immobilized polymer medium inside for introducing
electric potential from the electrical source to the first segment
and directing flow between the first segment and second segment of
the fluid network to cause a flow in the second segment in the
presence of electroosmotic flow in the first segment.
2: A microfluidic system as in claim 1, wherein the fluid network
comprises at least one fluid channel.
3: A microfluidic system as in claim 1, wherein the first segment
of the fluid network comprises a plurality of fluid channels
operatively connected on both ends to a single fluid channel.
4: A microfluidic system as in claim 1, wherein the electrical
source includes a first electrode reservoir and a second electrode
reservoir, operatively connected to a first end and a second end,
respectively, of the first segment of the fluid network, wherein
the second end of the first segment is fluid coupled to the second
segment.
5: A microfluidic system as in claim 4, wherein the second
electrode reservoir is electrically connected to the second end of
the first segment through the immobilized polymer medium inside the
coupler to apply electric potential to the first segment without
inducing electrolysis in the fluid network.
6: A microfluidic system as in claim 1, further comprising a valve
and a second segment that has a plurality of output channels, where
the valve routes fluid from the first segment to one of the
plurality of output channels of the second segment.
7: A microfluidic system as in claim 1, further comprising a valve
and a first segment that has a plurality of fluid pumping units,
wherein the valve routes fluid from the second segment to one of
the plurality of the pumping units of the first segment.
8: A microfluidic system as in claim 1, wherein the second segment
comprises an isolation channel preventing contamination of fluids
between the first and second segment.
9: A microfluidic system as in claim 1, further comprising a
pipette discharge.
10: A microfluidic system as in claim 1, wherein the second segment
comprises at least one double-T injector.
11: A device as in claim 10, further comprising at least one HPLC
column.
12: A device as in claim 10, further comprising at least one
freeze/thaw valve.
13: A device as in claim 10, further comprising at least one
detection system of a ultraviolet-visible absorbance detector, a
fluorescence detector, a laser-induced fluorescence detector, a
mass spectrometer, an electrochemical detector, a conductivity
detector, a scintillation counter, a radioactive particle intensity
detector, a Raman spectrometer, and a light scattering
detector.
14: An electrolysis isolation electrode comprises: a reservoir
containing an electrolyte solution inside; a protective housing; a
polymer medium that is chemically immobilized inside the said
protective housing, wherein the polymer medium being such that
fluids cannot be transferred through it neither by an electrical
means nor by pressure while electrolytes can be transferred through
it by an electrical means; and a metal electrode that is
electrically connected to the said polymer medium via the said
electrolyte solution inside the said reservoir.
15: A device as in claim 14, wherein the protective housing
comprises a flexible tubing.
16: A device as in claim 14, wherein the polymer medium comprises
at least one of an agarose gel with a concentration of greater than
0.5% (w/w), a polyacrylamide gel with a concentration of greater
than 1% (w/w), sol-gel monoliths, and acrylate polymer monoliths.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. patent application Ser. No. 10/076,170 filed Feb. 11, 2002,
now abandoned, which claimed the benefit under 35 U.S.C. .sctn.
119(e) of U.S. provisional patent application Ser. No. 60/267,474,
filed on Feb. 9, 2001, and Ser. No. 60/278,508, filed on Mar. 23,
2001.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention generally concerns miniature
instrumentation for the facilitation of chemical reactions and the
analytical separation of chemical solutions. More specifically,
this invention concerns the manipulation of fluids in microfluidic
chips and transportation of fluids between external devices and
microfluidic chips for the facilitation of chemical reactions and
the analytical separation of chemical solutions. In particular this
invention provides a reliable and functionally versatile
microfabricated electroosmotic flow pump with integrated
microfluidic conduits on a single chip.
[0004] 2. Description of Related Art
[0005] The field of microfluidics utilizes fabrication techniques
borrowed from the semiconductor industry to cost effectively
miniaturize and mass-produce extremely complex fluid systems. These
microfluidic systems take advantage of the physical properties and
flow characteristics of fluids within channels or capillaries to
perform transportive and analytical functions on aqueous chemical
solutions. Common applications of microfluidics include
micro-pipetting, microarray spotting, sample deposition for
MALDI-MS, as well as integrated microfluidic systems for chemical
analysis and sensing, and analytical separation techniques such as
capillary electrophoresis, capillary electrochromatography,
microcolumn liquid chromatography, and flow injection analysis.
[0006] One of the main principles incorporated in microfluidic
chips to facilitate the transportation or pumping of fluids is
called electroosmotic flow or (EOF). EOF principles have been known
for nearly two centuries, but only in the most recent decades has
it been practiced on a microscopic level. To explain EOF in brief,
the surface of many solids carries a net charge when in contact
with an aqueous solution due to chemical associations or
dissociations, physical adsorption on, or desorption from the solid
surface. For example, at mildly acidic to alkaline pH, surfaces of
quartz, ceramics, clay, sand, etc. are negatively charged. The
charged surface attracts oppositely charged counterions present in
the aqueous solution. As a result, a higher concentration of the
counterions builds near the surface and thermodynamic processes
forces these counterions to diffuse back into the bulk solution. At
equilibrium, the two processes balance each other, and the
counterions form a diffuse double layer. This diffuse double layer
is often called the Guoy-Chapman layer. Application of an external
electric field results in a net migration of these counterions in
the diffuse double layer towards the oppositely charged electrodes.
Due to viscous drag, the whole solution contained in microporous or
capillary structures moves with the counterions. This flow is
called electroosmotic flow.
[0007] EOF as well as other fluid propulsion methods has been
utilized in prior art microfluidic systems, but all lack the level
of sophistication, functionality, and ease of production inherent
in the current invention. For example, the inventor considered the
EOF fluid propulsion means described in U.S. Pat. No. 5,573,651 for
flow injection analysis (FIA). Capillary tubes are used to generate
EOF by connecting the pump capillary tubes and FIA conduits through
an ion exchangeable membrane tube that maintains hydraulic
connectivity between the pump capillary tubes and FIA conduits
while also serving as an electric grounding point for the system.
The grounding point provides for the elimination of electric fields
in the FIA reaction zone. To increase the fluid flow rate, multiple
capillaries are used, but in practice, connecting the FIA conduits
and pump capillaries via the ion exchangeable membrane tube becomes
tedious and commercially impracticable.
[0008] Additionally, when many capillary tubes are desired to
generate sufficient flow rate and pressure, it is very difficult to
arrange all the capillary tubes tightly to occupy a very small
space. In order to increase EOF pressure, the bore size of the pump
capillary tube may be reduced, which will then decrease the flow
rate. To compensate for this flow rate reduction, the number of
pump capillary tubes must then be increased.
[0009] Furthermore, when fluids in the FIA system need to be merged
and/or split with zero-dead volume, it is impossible to form
zero-dead volume connectors using conventional capillary tubes.
Fluid merging/splitting is a common event in FIA systems. For
example, as described in Analytical Chemistry, 1994, 66, 1792-1798,
a small dead volume T-joint was made from a segment of an
experimental double bore Polytetrafluoroethylene (PTFE) tubing
product that has two separate parallel channels. An oblique hole
was manually punctured between the two parallel channels using a
needle to make a connection between the two conduits. Three of the
four ends of the two parallel channels of PTFE tubing were
connected to three capillary tubes while the remaining end was
blocked. A dead volume of greater than one microliter was still
found to be present in the joint.
[0010] Finally, it is very difficult to construct a compact system
with multiple pumps using the configuration disclosed in U.S. Pat.
No. 5,573,651, especially when configuring parallel FIA systems.
Parallel units are commonly integrated into one system to enhance
the sample throughput. In Analytical Chemistry, 1994, 66,
1792-1798, two EOF pumps were used in a two-line FIA system to
facilitate reliable measurements of chloride, but the pump
electrolyte solution containers and pump capillaries made the
system bulky compared to the volumes of solutions handled.
[0011] Another EOF based pumping mechanism described in Analytical
Chemistry, 1997, 69, 1174-1178 for microchip ESI-MS detection was
considered by the inventor. In this chip, a T-shaped channel is
disclosed with three ends, only two of which are connected to
electrodes. When a voltage is applied, EOF goes from one electrode
to the other and the net flow in the third channel is zero. In
order to create a net flow in the third channel, the channel
connected to the cathode was coated with linear polyacrylamide.
This coating substantially suppressed the EOF in this channel. When
a voltage is applied, EOF generated in the anode channel goes
directly to the third channel and ESI-MS detection may be
performed. This pump however would be insufficient to propel fluids
on chips because any backpressure from the system will make the
fluid flow to the cathode channel.
[0012] In addition to the aforementioned difficulties, previous
microchip EOF pump implementations have proven unsatisfactory
because of variations introduced by electric fields, which are
generally applied across the entire system to balance fluids moving
in the conduit network. It is extremely challenging, if not
impossible, to balance the fluid flow reliably when the ionic
strength of a sample changes, adsorption of substances occurs on
the channel surfaces, the solution pH varies, or the system
temperature shifts.
[0013] Other pumping mechanisms were considered by the inventor for
microfabricated devices. In Micro Total Analysis Systems 2001,
401-402, a squeezing micropump is described for elastomer
microchips. In squeezing micropumps, a deformable channel is formed
inside the elastomeric substrate. A roller, actuated by a motorized
x-translator, is then rolled along the channel to squeeze the fluid
inside the channel forward. The same principle is used in a
peristaltic pump, which takes advantage of the flexibility of pump
tubing. This roller based method is not practical for
microfabricated systems, especially when complicated fluid
movements are required.
[0014] Another type of squeezing pump is described in Science 2000,
288, 113-116 wherein a pump channel is sandwiched by two pieces of
elastomer sheets. On top of this assembly, a third sheet with
multiple parallel channels is attached with the parallel channels
aligned perpendicular to the pump channel underneath. Pressurized
air is then introduced into one of the parallel channels. The air
pressure is sufficiently high so that it squeezes and blocks the
fluid inside the pump channel. The air pressure is then introduced
into the next parallel channel while maintaining the pressure level
in the first channel. Because the pressure in the first parallel
channel blocks the pump channel such that fluid cannot flow
backwards, the fluid inside the pump channel is squeezed forward.
Pressure is similarly introduced into subsequent channels to
squeeze the fluid further forward. Depending on the application,
pressure in the first channel may be released to allow more fluid
to enter the channel. Using this method however, it is very
difficult to control the fluid flow in complex devices.
[0015] It is therefore desirable to design a small, robust, and
easily producible fluid pump which overcomes the deficiencies in
the prior art and can be adapted to fully exploit the benefits of
microfabricated devices.
SUMMARY OF THE INVENTION
[0016] The present invention overcomes shortcomings of prior art
microfluidic pumping systems and enables new applications in the
field of microfluidics. In one aspect of the present invention, a
microfabricated EOF pump on a microchip is disclosed which utilizes
a microfabricated channel or channels to generate EOF as its
pumping means. The present invention also utilizes a bubble-free
electric connection joint on the chip to separate the
microfabricated pump channel(s) from the chemical assay conduit(s)
while maintaining hydraulic connectivity between these two parts.
The present invention also permits many pump channels to be
constructed for a single pump to generate sufficient flow rate with
sufficient pumping power and multiple pumps to be constructed on a
single chip to facilitate high throughput assays and complicated
fluid manipulations and transportations. It also permits zero
dead-volume connections between microfluidic channels.
[0017] One object of the present invention is to provide a method
and apparatus in which microfabricated channels are utilized to
construct an EOF pump on a microfabricated device;
[0018] Another object of the present invention is to provide a
method and apparatus that utilizes a bubble-free electric
connection joint to separate the pump channels from the rest of the
microfluidic conduits such that the connection joint is
electrically grounded and allows the microfabricated pump to
manipulate fluids in microfluidic devices but prevents the electric
field on the pump from interfering with the rest of the
microfluidic conduits on the chip. It also permits application of
an electric potential to the microfluidic conduits when needed;
[0019] Another object of the present invention is to provide a
method and apparatus that utilizes an isolation channel to separate
the pump channels from the rest of the microfluidic network such
that the isolation channel maintains hydraulic connectivity between
the pump channels and the rest of the microfluidic network but
prevents the fluids in the microfluidic network from contaminating
the pump channels and pump solution;
[0020] Another object of the present invention is to provide a
bubble-free electrode that permits application of an external
voltage/current source to a microfluidic channel but prevent
bubbles from forming in the microfluidic channel;
[0021] Yet another object of the present invention is to provide a
method and apparatus that utilizes a selection valve to direct
fluids to different channels in a microfluidic device;
[0022] Yet another object of the present invention is to provide a
method and apparatus that utilizes an air bubble or oil droplet as
a marker to monitor the flow rate in a microfluidic device;
[0023] Still another object of the present invention is to provide
a method and apparatus that utilizes a microfabricated EOF pump to
construct a pipetting device to transport small volumes of fluids
between external sample and/or reagent holders and microfluidic
devices;
[0024] Another object of the present invention is to provide a
method and apparatus that utilizes a membrane jacket on the
pipettor to perform sample treatment such as desalting, pH
adjustment, concentration and dilution;
[0025] Another object of the present invention is to provide a
method and apparatus that integrates a membrane jacket to a
microfluidic device to perform sample treatment such as desalting,
pH adjustment, concentration and dilution.
[0026] Another object of the present invention is to provide a
method and apparatus that integrates an EOF pump with an HPLC (high
performance liquid chromatograph) system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a schematic representation of a microfluidic chip
containing a microfabricated EOF pump and microfluidic conduits
that are connected to the selection valve, in accordance with one
embodiment of the present invention;
[0028] FIG. 2a is a side-view schematic representation of an
alternative bubble-free electric connection joint; FIG. 2b is a
side view representation of another alternative bubble-free
electric connection joint; FIG. 2c is a top-view schematic
representation of the alternative bubble-free electric connection
joint of FIG. 2b;
[0029] FIG. 3a is a schematic representation of an alternative
configuration of a microfabricated EOF pump containing a
bubble-free electrode; FIG. 3b is a side view schematic of the
bubble-free electrode of FIG. 3a;
[0030] FIG. 4 is an exploded view schematic representation of a
microfluidic selection valve;
[0031] FIG. 5a is a schematic representation of a multiple-tip
small volume pipettor based on EOF pumping; FIG. 5b is a magnified
view of a single small volume pipettor of FIG. 5a;
[0032] FIG. 6a is a schematic representation of a cleaning device
for the EOF pumped pipettor; FIG. 6b is a scheme to release a small
volume of fluid to a targeted location;
[0033] FIG. 7a is a magnified portion of the flow rate monitoring
assembly for the microfluidic device including an air bubble and
two photodiode pairs; FIG. 7b is a schematic representation of the
construction of one on-chip photodiode/LED pair of FIG. 7a; FIG. 7c
is a schematic representation of a flow rate monitoring assembly
for the microfluidic device; and
[0034] FIG. 8a is a schematic representation of the construction of
a membrane jacket on a small volume pipettor; FIG. 8b is a
sectional view of a chip with two access holes for integration of a
membrane into a microfluidic chip; FIG. 8c is a sectional view of
the chip in FIG. 8b after a groove is made between the two access
holes; FIG. 8d is a top-view of FIG. 8c; FIG. 8e is a sectional
view of the completed membrane-integrated microfluidic chip.
[0035] FIG. 9a is a schematic representation of the construction of
an EOF pumped HPLC system.
DETAILED DESCRIPTION OF THE INVENTION
[0036] This invention is described below in reference to various
embodiments and drawings. While this invention is described in
terms of the best presently contemplated mode of carrying out the
invention, it will be appreciated by those skilled in the art that
variations and improvements may be accomplished in view of these
teachings without deviating from the scope and spirit of the
invention. This description is made for the purpose of illustrating
the general principles of the invention and should not be taken in
a limiting sense. The scope of the invention is best determined by
reference to the appended claims.
[0037] Referring now to FIG. 1, chip 1 comprises a microfabricated
EOF pump 2, a selection valve 11 and microfluidic conduits 3
(partially shown). Multiple units of this design may be integrated
onto a single chip as desired. In this illustrated embodiment, chip
1 is a glass substrate and fabrication of the microfluidic system
components is performed using standard photolithographic
techniques. Preferably, a sacrificial mask of Cr/Au is used, the
Chromium layer (approximately 100 to 500 angstroms thick) being
present solely to enhance the adhesion between the substrate and
gold layer. HF is the preferred etchant and can be prepared in
various solutions including HF/NH4F, HF/HNO3, HF/H3PO4, and
concentrated HF. Pump 2 comprises multiple pump-channels 4, a high
voltage electrode reservoir 5, a bubble-free electric connection
joint 6, and an isolation channel 9. Bubble-free electric
connection joint 6 functions as the ground electrode reservoir for
the system most of the time, but may be used to apply an electric
potential to the fluidic system as desired. The dimensions of pump
channels are normally between 0.1 .mu.m to 500 .mu.m, preferably 1
.mu.m to 200 .mu.m, and more preferably, 5 .mu.m to 50 .mu.m.
Multiple channels are often desirable on one chip, as the flow rate
of the system is proportional to the number of pump channels.
Systems may be thus tailored for a desired flow rate by adjusting
the size and number of channels. In general it would be possible to
fabricate approximately 1000 pump channels which are approximately
100 .mu.m from center to center on a 10 centimeter wide
substrate.
[0038] FIG. 2a shows the schematic assembly of bubble-free electric
connection joint 6 (also the ground electrode reservoir). Ion
exchangeable membrane 20 is fixed over access hole 19 and small
bottomless container 21 is sealed on top of the membrane 20 and
secured in position using adhesive 22 (preferably epoxy). Membrane
20 is preferably a flat Nafion membrane sheet, but may be any ion
exchangeable membrane. Access hole 19 is preferably fabricated to
be smaller than the space occupied by membrane 20 and branches off
in a T joint fashion to channels 17 and 18. The access
hole/membrane assembly should be carefully fabricated so that the
membrane 20 seals access hole 19 so that no fluids are able to pass
through. A buffer electrolyte solution 23 is introduced into
container 21. The ion exchangeable membrane 20 in this assembly
allows ions to pass through such that bubble-free electrode 8 and
the solutions in the access hole 19 are electrically connected, but
fluids cannot pass across the membrane 20. In FIGS. 2b and 2c, two
blocks 24 and 25 on the opposite sides of the chip 16 are held
together through four screws 27. The top block 24 will press an
O-ring 26 on the membrane 20 against the shoulder of the access
hole 19, to prevent fluids from leaking across the membrane 20.
FIG. 2c shows a top-view of the bubble-free electric connection
joint assembly.
[0039] Referring back to FIG. 1, when a voltage is applied across
the pump-channels 4 through two bubble-free electrodes 7 and 8, EOF
is created in the pump channels 4. Because the membrane 20 in the
bubble-free electric connection joint 6 prevents fluids from moving
across the membrane, the EOF can thus be used to drive the solution
in isolation channel 9 and hence the fluids in the rest of
microfluidic conduits 3. Isolation channel 9 is used to prevent
fluids in the microfluidic conduits 3 from contaminating the pump
channels 4 and pump solution 23 in the bubble-free electric
connection joint 6 and high voltage reservoir 5. In another
embodiment of this invention, the isolation channel is used to hold
an air bubble 15 or an oil droplet as a marker for monitoring the
pumping flow rate.
[0040] Selection valve 11 in FIG. 1 is used to direct the pump to
various channels of conduit networks. The common port 12 of the
selection valve 11 is normally directly connected to the pump part
of the chip 2. Selection valve 11 allows connection of the common
port 12 to any but one of the of the selection ports. For example,
when the common port 12 is connected to selection port 13, the pump
assembly 2 will be able to drive fluids in channel 14 and the down
stream conduits.
[0041] In another embodiment, the pump element 2 of FIG. 1 may be
reconfigured as illustrated in FIG. 3a. Multiple groups of channels
are connected in series or channels may be curved (not shown) to
form pump channels 4 in order to create higher pump pressures than
are possible from a single group of channels. Ideally, every single
pump channel experiences equal electric field strength. The high
voltage electrode reservoir 37 and ground electrode reservoir 36
are moved outside the chip body 52. A tube, preferably a capillary
tube, 28 is used to connect one end of the pump channels 4 to the
high voltage electrode reservoir 37. A bubble-free electrode
(referring to FIG. 3b) is connected to the other end 32 of the pump
channels 4 and sealed using adhesive 33 (preferably epoxy). This
configuration allows reservoirs of large volumes to be used, which
is important for stable pumping rate because electrolysis changes
the pH of the pump solution, which in turn changes the pump flow
rate. Regular metal electrodes 34 and 35 (preferably though not
necessarily platinum or gold wires) may be used directly in the
high voltage electrode reservoir 37 and ground electrode reservoir
36. The volume of the large containers can be several liters if
need be.
[0042] In this embodiment, a bubble-free electrode is employed to
prevent electrolysis and bubble formation in or close to the
microfluidic channels. Referring to FIG. 3b, one particular element
of a bubble-free electrode is a piece of tube 29 filled with high
viscous media. Tube 29 may be loaded with a viscous polymer
solution or packed with porous media. The viscous polymer solution
may be agarose gel with a concentration of greater than 0.5% (w/w),
polyacrylamide gel with a concentration of greater than 1% (w/w),
or other polymer gel solutions. In one embodiment, the viscous
polymer is chemically bonded to the inner wall of tube 29. In a
preferred embodiment, the viscous polymer solution is
polyacrylamide gel with a weight concentration of 2-10%.
Polyacrylamide may be either a linear or cross-linked polymer. In
additional embodiments, the polymerization reaction is performed
in-situ in tube 29. Tube 29 may also be packed with porous media
such as micro beads of smaller than 10 .mu.m in diameter, more
preferably between 0.1 .mu.m to 3 .mu.m in diameter. In-situ
prepared polymeric monoliths such as sol-gel monoliths and acrylate
polymer monoliths may also be used to prepare tube 29. When tube 29
is packed with porous media, an electrolyte solution is flushed
through and filled the pores in tube 29. The flow resistance in
tube 29 is very high when filled with such high viscous media. Tune
29 should normally be less than 1 m, preferably less than 10 cm,
more preferably less than 3 cm in order to reduce the voltage drop
across it. The diameter of tube 29 should normally be within 2
.mu.m to 2 mm, more preferably within 25 .mu.m to 250 .mu.m.
[0043] The bubble-free electrode of FIG. 3b comprises a large
container 36, a platinum or gold electrode 34, and a tube 29 filled
with high viscous media. When tube 29 is short, another tube 31
filled with an electrolyte solution may be used to connect tube 29
through a joint 30 to the solution in the large container 36. The
joint 30 is preferably a piece of silicone tubing that tightly fit
to tube 29 and 31. Referring to FIG. 3a, as a potential is applied
between electrodes 35 and 34, EOF is generated in pump channels 4.
Because EOF in tube 29 is zero if polymer gel is fixed in the tube,
or very small if tube 29 is packed with micro-porous media, the EOF
generated in the pump channels 4 will drive fluids in isolation
channel 9 and subsequently the fluids in microfluidic channel
connected to the isolation channel 9. Electrolysis occurs and
bubble forms only in the large volume reservoir 36, not at the tip
of tube 29. The assembly shown in FIG. 3b is referred to as a
bubble-free electrode in the present invention. Bubble-free
electrodes can be used inside a microfluidic channel, or in small
volume buffer electrolyte reservoirs (such as in electrodes 7 and 8
in FIG. 1). Such electrodes are bubble-free, and even more
precisely, electrolysis-free. Because no electrolysis occurs at the
tip of tube 29, the solution pH is maintained during operation in
the microfluidic channel, or small volume buffer reservoirs
connected to the bubble-free electrode.
[0044] FIG. 4 shows an exploded view of a selection valve (such as
selection valve 11 from FIG. 1) integrated onto a microchip 38. In
this example, channels 14 connect microfluidic conduits to the
selection ports and a connection channel 10 connects a pump to the
common port of the selection valve. All these ports are normal
access holes with their openings facing down. The diameters of
these access holes should be less than 2 mm, preferably less than 1
mm, more preferably less than 500 .mu.m, more preferably less than
200 .mu.m, more preferably less than 100 .mu.m, to reduce the
connection dead volumes. A rotor 42 has a groove 43 on the top and
a recessed structure 44 on the bottom. The groove 43 is used to
make connections between the common port 12 to any one of the
selection ports of the selection valve. Two blocks 40 and 45 are
used to hold the rotor 42 tightly to the chip 38 through screws 51
and threads 41. Four through holes 39 on the chip 38 allow the
screws 51 to go through. There is a three tiered recessed structure
formed in the bottom block 45. The diameter of the first tier
portion 46 of the recessed structure matches the diameter of the
rotor 42 and its depth is slightly smaller than the height of the
rotor 42. This permits the rotor 42 to be held tightly to the chip
38 when the two blocks 40 and 45 are tightened together by the
screws 51. The diameter of the second tier portion 47 of the
recessed structure matches the diameter of the larger portion 124
of a transmission rod 48. The diameter of the third tier portion
123 of the recessed structure matches the smaller portion 125 of
the transmission rod 48. The raised structure 49 on this rod 48
matches the recessed structure 44 on the rotor. When all the pieces
are placed tightly together, an external force is applied to the
rod 48 through structure 50 to rotate the rotor 42 to a desired
position so that the common port 12 is connected to a desired
selection port of the selection valve. Rotation and positioning of
the transmission rod 48 may be automatically operated through a
step motor (not shown).
[0045] FIG. 5a shows a small volume pipettor constructed utilizing
a microfabricated EOF pump 53. The pump portions may be
conceptually similar to those detailed in FIG. 3a. The pipettor tip
54 is a piece of capillary tube such as glass capillary tube,
stainless steel capillary tube or other polymeric tubing. The
diameter of the pipettor tip may vary with the desired pipetting
volume. It normally ranges from 5 .mu.m to 1 mm, preferably between
25 to 250 .mu.m. When the pump channels are narrow, for example
less than 10 .mu.m, a stable pumping rate of a few nanoliters per
minute may be reliably created. Using a few seconds pipetting time,
fluids of sub-nanoliter volumes may be reliably picked or
delivered.
[0046] When handling fluids in these small volumes, it may be
challenging to prevent solvent evaporation or cross contamination
between samples. FIG. 5b shows one pipettor embodiment wherein a
non-interfering fluid 55 is picked up in the pipettor tip, followed
by target fluid 56, and then an additional segment of the
non-interfering fluid 57. The target fluid 56 is sandwiched between
two non-interfering fluid segments 55 and 57 so as to prevent
evaporation of the target fluid 56. To deliver this small volume of
fluid, the non-interfering fluid segments 55 and 57 are delivered
with the target fluid 56. When fluid segment 55 is delivered it
washes the residual of the target fluid 56, which facilitates
complete and accurate delivery of the target fluid 56.
[0047] In another pipettor embodiment, referring to FIG. 6a, the
outside and the end of pipetter tip is washed with a
non-interfering fluid 61. The washing device 58 has a large guiding
opening 62 that permits the pipettor tip 54 entering the washing
chamber 122 easily. The non-interfering washing fluid is introduced
using tubing 60 through a couple of small openings 66 on the
opposite sides of the washing chamber 122. The openings 66 are
preferably located on the top portion of the washing chamber.
Tubing 60 is inserted all the way to the bottom of hole 65. An
O-ring 67 is squeezed by a hollow screw 59 to seal the tubing 60
and secure it in position.
[0048] Generally, it will be desirable to have receiving fluid to
accept the target fluid when very small volumes of fluids are
transferred. This ensures that the target fluid is fully released
and little hangs on the end of the pipettor tip. Sometimes,
however, it is required to deliver small volumes of solutions to
dry surfaces. In the embodiment shown in FIG. 6b, a potential may
be applied through the bubble-free electric connection joint 6
(referring to the pump configuration of FIG. 1) or the bubble-free
electrode 29 (referring to the pump configuration of FIG. 3a) to
the target fluid 68 to make its surface 70 charged, which reduces
the surface tension of the target fluid 68 and hence becomes more
easily released to a dry surface 69. Appropriate potential may also
be applied to the dry surface 69 to create charge 71 opposite to
that on the droplet 68. The local electric field will direct the
target fluid 68 to a desired position 71 on the dry surface 69.
This method may also be used to release a target fluid to a liquid
surface to avoid contact between the pipettor tip 54 and receiving
solution.
[0049] In another pipettor embodiment, referring to FIG. 8a, a
micro-dialysis jacket is attached to a small volume pippetor tip to
permit desalting, pH adjustment, concentration, and other such
functions requiring dialysis-type mechanisms. A tubular membrane 82
such as porous cellulose, porous PTFE or Nafion (or any other ion
exchangeable membrane) is used to connect a pipettor tip 84 to a
connection tube 117. The other end of tube 117 is connected to a
microfabricated EOF pump. A jacket 79 surrounding the tubular
membrane is secured and sealed to the pipettor tip 84 and
connection tube 117. As a proper external solution goes into the
jacket through opening 83, passes across the outside of the tubular
membrane 82 and exits through the other opening 80, the salt
concentration of the solution inside the tubular membrane 82 may be
reduce and the pH of the solution may be adjusted.
[0050] In an additional aspect of this embodiment, a porous
cellulose membrane combined with an aqueous solution containing low
or not salt as an external solution is used for desalting; a Nafion
(or any other ion exchangeable) membrane combined with a certain pH
buffer solution as an external solution is used for pH adjustment;
and a porous PTFE membrane combined with dry air as an external
fluid is used for concentration.
[0051] Normally, the external solution is constantly flowing across
the outside of the tubular membrane 82. By using this particular
pipettor configuration to pick up a sample solution, allowing the
solution to pass across the tubular membrane, and then delivering
the solution to a target location (for example a sample reservoir
85 on a microchip), the delivered sample may have already been
desalted and/or its pH adjusted.
[0052] In another embodiment shown in FIG. 8e, the membrane 94 such
as porous cellulose, porous PTFE or Nafion (or any other ion
exchangeable membrane) is directly integrated into a chip system.
To construct this system, traditional chip 86 as shown in FIG. 8b
(a cross-section side-view) can first be fabricated. Channels 87
and 88 are connected to a pump and a microfluidic network. The
diameter of the two access holes 89 and 90 are preferably less than
1 mm, more preferably less than 500 .mu.m, and even more preferably
less than 100 .mu.m, in order to reduce the dead volume. A groove
91 is then created on the top of the chip between the two access
holes 89 and 90. A cross-section side-view is presented in FIG. 8c.
FIG. 8d shows a top-view of the chip after groove 91 has been
fabricated. Then a sheet membrane (such as porous cellulose, porus
PTFE, Nafion, or any other ion exchangeable membrane) 94 is
employed to cover the groove and access holes (89 and 90). Another
chip having a similar groove is then used to enclose the membrane
and secure it in position as illustrated in FIG. 8e. Screws may be
used to tighten these two chips together. The groove on the second
chip forms channel 92 and the groove on the first chip forms
channel 93. To illustrate, when water enters channel 118, passes
through channel 92 and exits channel 120 and a sample solution,
preferably prepared on chip, enters channel 121, passes through
channel 93 and exits channel 119, the sample has already been
desalted as it leaves channel 93. The two solutions above and below
the membrane 94 may flow in the same direction, but material
transferring across the membrane is more efficient when they flow
counter-currently. Adjustment of sample pH and concentration of a
sample may also be performed using this device.
[0053] FIG. 7a shows an on chip system for flow rate monitoring.
Air bubble 15 is introduced into isolation channel 9.
LED/photodiode pairs 74/72 and 75/73 are mounted operatively on
both sides of the isolation channel 9. FIG. 7b presents a schematic
diagram of the LED/photodiode 74/72 assembly on the chip. LED's and
photodiodes are glued in position using adhesive 76 (preferably
epoxy resin). On both sides of the isolation channel 9, a Cr layer
78 is sputtered to block the environmental light and other
scattered light. An opening 77 is fabricated for LED light to pass
through the channel and reach the photodiode on the opposite side
of the channel. Both the LED and photodiode are switched on at all
times such that the photodiode is constantly detecting an optical
signal from the LED. As the air bubble 15 passes through the
assembly, a large signal change is detected by the photodiode
presumably due to an optical focusing effect of the meniscus of the
air bubble. If the bubble is large, two separate strong signals,
one for each meniscus, may be detected. Generally only one, more
often the rising signal, is selected to record the position of the
air bubble. The moving velocity of the air bubble 15 is calculated
based on the distance of two LED/photodiode pairs and the time for
the air bubble 15 to move from one LED/photodiode pair 74/72 to the
other 75/73. Any variation of the pump flow rate will be detected
by monitoring the velocity change of the air bubble. Once a
velocity change is detected, the pump voltage may be adjusted
properly to resume the same pump rate.
[0054] In another rate monitoring embodiment, now referring to FIG.
7c, flow rate monitoring channels are separated from the main
conduits. Two selection valves 114 and 115 are used in this
assembly. Channel 112 connects the pump to the common port 101 of
selection valve 114 and channel 113 connects the common port 98 of
selection valve 115 to the rest of the microfluidic conduits.
During normal operation, selection valve 114 connects common port
101 to selection port 100 and selection valve 115 connects common
port 98 to the selection port 95. An air bubble is pre-introduced
into channel 107 between two T-connectors 103 and 104. To measure
the flow rate, selection valve 114 connects the common port 101 to
selection port 99 and selection valve 115 connects the common port
98 to selection port 97 if the air bubble is close to T-connector
103. Alternatively, if the air bubble is close to T connector 104,
selection valve 114 connects the common port 101 to selection port
102 and selection valve 115 connects the common port 98 to
selection port 96. Multiple LED/photodiode pairs are used to
measure the velocity of the air bubble. The total distance of
channels 109, 107 and 111 should be the same as that of channels
110, 107 and 108, and equal to that of channel 116. When all
channel dimensions are the same, this ensures the same flow
resistance whether the system is in normal operation or in flow
rate measurements.
[0055] FIG. 9 presents a schematic diagram of an EOF pumped HPLC
system. A sample solution is pressurized (or vacuumed) into the
system from a sample vial 127, through the sample injector 132 (the
structure inside the dashed box), to waste 126. Because the flow
resistances from the HPLC column 135 and the shallow pump-channels
125 are high, little sample solution will go to the HPLC column or
the EO pump. After the channel from positions 128 to 129 is filled
with the sample completely, the freeze/thaw valves 130 and 131 will
be actuated to freeze the solutions in the sample inlet and outlet
capillaries. Then, a voltage will be applied across the pump
channels 125 to drive the eluent from the eluent reservoir 124,
carrying the sample (between positions 128 and 129 in the sample
injector 132), to the HPLC column 135 for separation. The separated
compounds can then be on-column or off-column detected, or fraction
collected.
[0056] The working principle of a freeze/thaw valve has been
described in Analytical Chemistry, 1999, 71, 1138-1145). Liquid
nitrogen was used to actuate the valve actions. Fact actuations
were accomplished with this approach, but it will be inconvenient
to use liquid nitrogen for many applications such as the field
analyses and point-of-care measurements. A solid-state cold plate
will be more practical to implement the freeze/thaw valve
actuations for these applications. The solid-state cold plate
should be powerful enough to actuate the freeze/thaw valve rapidly.
Note: There are no moving parts in this microfluidic HPLC
system.
EXAMPLES
[0057] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Microfabrication of Glass Chips
[0058] Schematic diagrams showing preferred embodiments of the
small volume fluid manipulation and transportation devices of the
present invention are provided in FIGS. 1 through 8. A variety of
methods known in the art may be used to make and use the claimed
fixed-volume-injectors. For example, the chip microfabrication
protocols disclosed in Analytical Chemistry 71 (1999) 566-573, or
their equivalents known in the art are readily be adapted to
produce the chip component of the hybrid apparatus of the present
invention.
[0059] Alternative methods known in the art may be employed within
the scope of the present invention. For example, for
photolithograpy a thin sacrificial layer of Cr/Au (300 .ANG. Cr and
0.5 .mu.m Au) may be deposited onto a glass wafer, followed by
photoresist coating (Shipley photoresist 1818). After soft baking
at 80 C.degree., the photoresist may be exposed to UV radiation
through a mask. The mask pattern will be transferred to the wafer
after the photoresist is developed. After the exposed Cr/Au is
etched off using gold and chromium etchants, the channel pattern is
chemically etched into the glass. We have been using concentrated
HF as the chemical etchant with an etching rate of ca. 7 .mu.m per
minute at 21 C.degree. for borofloat glass. After etching, the
residual photoresist and Cr/Au may be stripped and access holes
were drilled. The etched wafer may be thermally bonded with another
wafer to enclose the grooves and form channels.
[0060] The bonded chips are then taken to a dicing saw and diced to
form the three-piece and two-piece fixed-volume-injectors.
Example 2
Preparation of an Immobilized Polymer Coupler
[0061] A piece of bare capillary (150-.mu.m-id, 375-.mu.m-od and
50-cm-long) is flushed with 1 M NaOH for 45 min, ultra-pure water
and acetonitrile for 15 min each, and then dried with helium. A
solution of 0.4% (vol/vol) of 3-(trimethoxysilyl) propyl
methacrylate and 0.2% (vol/vol) acetic acid in acetonitrile is
flushed into the dried capillary with a syringe pump at 50
.mu.l/min for 1 h. The capillary is then rinsed with acetonitrile
and dried with helium. The bi-functionalized capillary is cut into
desired lengths (e.g. 5 cm) for the immobilization of
polyacrylamide.
[0062] 5 mL solution containing 4% (w/v) of acrylamide and a small
amount (e.g. 0.003%) of N, N'-methylene-bis-acrylamide (Bis) is
purged with helium for about 30 min. Then, 2 .mu.L of 10% APS and 2
.mu.L of TEMED are added to the solution using micro-syringes.
Mixing is carried out automatically as the purging helium bubbled
up. Promptly (<5 s) after the APS and TEMED are mixed with
acrylamide, the polymerizing solution is introduced into the above
bi-functionalized capillary and allowed to polymerize inside the
capillary. After about 30 min, the capillary is taken out with both
ends cleaned. The capillary is stored (immersed) in a buffer
solution before use.
[0063] All of the methods and apparatus disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the invention has been
described with respect to the described embodiments in accordance
therewith, it will be apparent to those skilled in the art that
various modifications and improvements may be made without
departing from the scope and spirit of the invention. For example,
it will be apparent to those of skill in the art that variations
may be applied to the methods and apparatus and in the steps or in
the sequence of steps of the methods described herein without
departing from the concept, spirit and scope of the invention. It
also will be apparent that certain substance such as polymeric and
ceramic materials may be substituted for the glass materials
described herein to achieve the same, similar or improved results.
By way of example and not limitation, the EOF pump concepts of the
present invention is described in connection with micro-channels in
a microfabricated chip. It is understood that the present invention
is applicable to integrated microfluidic systems for chemical
analysis and sensing, and analytical separation techniques such as
capillary electrophoresis, capillary electrochromatography,
microcolumn liquid chromatography, flow injection analysis, and
field-flow fractionation. It is also applicable to microarray
spotting and MALDI-MS sample deposition. Furthermore, while the
separation channels in the described embodiments are defined by
micro-separation channels etched in a substrate (micro-fluidics
type devices or bio-chips), it is understood that the concepts of
the present invention is equally applicable to columns or tubes
defining the micro-channels.
[0064] All such similar substitutes and modifications apparent to
those skilled in the art are deemed to be within the spirit, scope
and concept of the invention as defined by the appended claims.
Accordingly, it is to be understood that the invention is not to be
limited by the specific illustrated embodiments, but only by the
scope of the appended claims.
* * * * *