U.S. patent application number 10/426818 was filed with the patent office on 2003-12-11 for apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis.
Invention is credited to Ramsey, J. Michael.
Application Number | 20030226753 10/426818 |
Document ID | / |
Family ID | 23087469 |
Filed Date | 2003-12-11 |
United States Patent
Application |
20030226753 |
Kind Code |
A1 |
Ramsey, J. Michael |
December 11, 2003 |
Apparatus and method for performing microfluidic manipulations for
chemical analysis and synthesis
Abstract
A microchip laboratory system and method provide fluid
manipulations for a variety of applications, including sample
injection for microchip chemical separations. The microchip is
fabricated using standard photolithographic procedures and chemical
wet etching, with the substrate and cover plate joined using direct
bonding. Capillary, electrophoresis and electrochromatography are
performed in channels formed in the substrate. Analytes are loaded
into a four-way intersection of channels by electrokinetically
pumping the analyte through the intersection, followed by switching
of the potentials to force an analyte plug into the separation
channel.
Inventors: |
Ramsey, J. Michael;
(Knoxville, TN) |
Correspondence
Address: |
DANN, DORFMAN, HERRELL & SKILLMAN
1601 MARKET STREET
SUITE 2400
PHILADELPHIA
PA
19103-2307
US
|
Family ID: |
23087469 |
Appl. No.: |
10/426818 |
Filed: |
April 30, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
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10426818 |
Apr 30, 2003 |
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10262533 |
Oct 1, 2002 |
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10262533 |
Oct 1, 2002 |
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09477585 |
Jan 4, 2000 |
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6475363 |
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09477585 |
Jan 4, 2000 |
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09153470 |
Sep 15, 1998 |
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6033546 |
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09153470 |
Sep 15, 1998 |
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08776645 |
Feb 3, 1997 |
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5858195 |
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08776645 |
Feb 3, 1997 |
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08283769 |
Aug 1, 1994 |
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6001229 |
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Current U.S.
Class: |
204/450 ;
204/452 |
Current CPC
Class: |
B01F 33/3011 20220101;
B01J 2219/00891 20130101; B01L 2300/0883 20130101; B01J 2219/0097
20130101; G01N 2030/285 20130101; Y10S 366/01 20130101; B01J
2219/00853 20130101; B01J 2219/00826 20130101; B01L 2400/0415
20130101; B01L 3/502715 20130101; B29C 66/54 20130101; B01J
2219/00783 20130101; B01J 2219/00912 20130101; B29C 66/026
20130101; B01F 33/3031 20220101; B01J 2219/00831 20130101; B01L
2400/0418 20130101; G01N 27/44743 20130101; G01N 30/02 20130101;
B01J 2219/00828 20130101; B01L 3/502753 20130101; G01N 2030/027
20130101; B01L 2300/0816 20130101; B29C 66/034 20130101; G01N
2030/8435 20130101; B01L 3/502776 20130101; B01L 2200/0605
20130101; B01J 2219/00889 20130101; B01J 2219/00916 20130101; B01L
2300/0867 20130101; G01N 2030/383 20130101; G01N 2030/162 20130101;
B01J 2219/00952 20130101; B01L 3/50273 20130101; G01N 30/16
20130101; B01J 19/0093 20130101; B01L 3/502784 20130101; G01N
30/6095 20130101; B01L 3/5027 20130101; B01F 33/30 20220101; B01J
2219/00995 20130101; B01L 3/502738 20130101; B29C 65/4895 20130101;
G01N 27/44791 20130101; B01L 2400/0421 20130101; G01N 30/02
20130101; B01D 15/3842 20130101 |
Class at
Publication: |
204/450 ;
204/452 |
International
Class: |
G01N 027/27 |
Goverment Interests
[0001] This invention was made with Government support under
contract DE-AC05-84OR21400 awarded by the U.S. Department of Energy
to Martin Marietta Energy Systems, Inc. and the Government has
certain rights in this invention.
Claims
What is claimed is:
1. A method of controllably moving material in a microfluidic
device, comprising: a. providing a microfluidic device that
includes a body having at least first, second, third and fourth
channels disposed therein, the body comprising a cover plate
covering the first, second, third and fourth channels, wherein the
first, second, third and fourth channels communicate at a first
intersection, the first channel connecting at least a first
material source to the first intersection; b. applying a first
voltage difference between the first material source and the second
channel to move the first material from the first channel, through
the intersection and into the second channel; c. applying a second
voltage difference between the third channel and the first
intersection, and a third voltage difference between the fourth
channel and the first intersection simultaneously with the applying
of the first voltage difference, to direct movement of the first
material through the intersection into the second channel.
2. The method of claim 1, wherein the step of applying the first
voltage difference comprises applying a first voltage to the first
channel, and maintaining a second voltage level different from the
first voltage at a reservoir that is fluidly connected to the
second channel.
3. The method of claim 1, wherein the step of applying the first
voltage difference comprises applying a first voltage to the first
channel and grounding a reservoir that is fluidly connected to the
second channel.
4. The method of claim 1, wherein the step of applying the first
voltage difference comprises applying a first voltage to the first
channel and a second voltage lower than the first voltage, to the
second channel.
5. The method of claim 1, wherein the step of applying the first
voltage difference comprises applying a first voltage to the second
channel and a second voltage lower than the first voltage, to the
first channel.
6. The method of claim 1, wherein the step of simultaneously
applying the second and third voltage differences comprises
applying a third voltage to the third channel and a fourth voltage
to the fourth channel, each of the third and fourth voltages being
less than a voltage applied to the first channel, and greater than
a voltage level maintained at a reservoir fluidly connected to the
second channel.
7. The method of claim 1, wherein the first channel communicates
with the first intersection between the third and fourth channels,
and the third channel communicates with the first intersection
between the first and second channels, and wherein the second and
third voltage differences move at least a second material from the
third and fourth channels, respectively, into the second
channel.
8. The method of claim 7 further comprising: a. removing the second
and third voltage differences; and b. applying a fourth voltage
difference between the third and fourth channels through the first
intersection to move the first material in the intersection, into
the fourth channel.
9. The method of claim 8 further comprising applying a fifth
voltage difference between the first channel and the first
intersection, and a sixth voltage difference between the second
channel and the intersection to move the first material in the
first and second channels away from the intersection concurrently
with the step of applying the fourth voltage difference.
10. The method of claim 1, wherein the first channel communicates
with the first intersection between the fourth channel and the
second channel and the second channel communicates with the first
intersection between the first channel and the third channel, and
wherein the second and third voltage differences move at least a
second material from the fourth channel into the third channel.
11. The method of claim 10, wherein the first, second and third
voltage differences move at least a second material from the fourth
channel into both the second and third channels.
12. The method of claim 10, further comprising: a. removing the
third voltage difference; and b. applying a fourth voltage
difference between the first and third channels through the first
intersection, to move the first material in the first channel into
the third channel.
13. The method of claim 1, wherein the first material comprises
charged species that are moved by electrophoresis.
14. The method of claim 1, wherein the material is moved by
electroosmosis.
15. The method of claim 1, wherein the first material is moved by a
combination of electrophoresis and electroosmosis.
16. The method of claim 1, wherein the channels of the device
provided in the providing step have first and second ends, the
second ends being in communication at the first intersection, and
wherein the applying steps comprise applying electrical potentials
to the first ends of the channels.
17. The method of claim 1, wherein the first material comprises
nucleic acids.
18. The method of claim 17, wherein the nucleic acids comprise
DNA.
19. The method of claim 18, wherein the DNA comprises restriction
enzyme fragments of DNA.
20. The method of claim 1, wherein the first material comprises
proteins.
21. The method of claim 20, wherein the first material further
comprises a micellar material.
22. The method of claim 21, wherein the micellar material is sodium
dodecyl sulfate.
23. The method of claim 1, wherein at least a portion of the first
material is fluorescently labeled.
24. The method of claim 23, wherein the fluorescently labeled
material is labeled with a fluorescein dye.
25. The method of claim 23, wherein the fluorescently labeled
material is labeled with a rhodamine dye.
26. The method of claim 8, wherein the first material comprises a
fluorescent label, and further comprising detecting the
fluorescently labeled material in the fourth channel.
27. The method of claim 26, wherein the detecting step comprises
exciting the fluorescent label, and detecting emitted
fluorescence.
28. The method of claim 27, wherein the step of detecting emitted
fluorescence comprises imaging a portion of the fourth channel with
an array detector.
29. The method of claim 27, wherein the step of detecting emitted
fluorescence comprises imaging a portion of the fourth channel with
a charge coupled device (CCD).
30. The method of claim 27, wherein the step of detecting emitted
fluorescence comprises detecting the emitted fluorescence with a
PMT.
31. The method of claim 27, wherein the fluorescent label is
excited using a laser.
32. The method of claim 27, wherein the fluorescent label is
excited using a laser diode.
33. The method of claim 27, wherein the fluorescent label is
excited using a LED.
34. A method of controllably moving material through a microfluidic
device, comprising: a. providing a body having at least first,
second, third and fourth channels disposed therein, the body
comprising a cover plate covering the first, second, third and
fourth channels, wherein each channel has a first and second end,
the first ends of the first, second, third and fourth channels
being in communication at a first intersection, the second ends of
the first, second, third and fourth channels respectively
communicating with first, second, third and fourth reservoirs; b.
applying a first voltage difference between the first end of the
first channel and the first end of the second channel to move the
first material from the first channel, through the intersection and
into the second channel; and c. applying a second voltage
difference between the first end of the third channel and the first
intersection, and a third voltage difference between the first end
of the fourth channel and the first intersection simultaneously
with the applying of the first voltage difference, to control
movement of the first material through the intersection into the
second channel.
35. A method of controllably moving material through a covered
microfluidic channel network, comprising: a. electrokinetically
moving a first material from a first channel through an
intersection of the first channel with second, third and fourth
channels, into the second channel; and b. electrokinetically
transporting material from at least one of the third and fourth
channels into the intersection, simultaneously with the
electrokinetic moving of the first material from the first channel,
to control movement of the first material within the
intersection.
36. A method of controllably moving material, comprising: a.
providing a microchip that includes a substrate having at least
first, second, third and fourth channels disposed therein, a cover
plate bonded to the substrate that covers the first, second, third
and fourth channels, wherein the first, second, third and fourth
channels communicate at a first intersection; b. transporting a
first material from the first channel, through the intersection and
into the second channel; and c. concurrently transporting at least
a second material from at least one of the third and fourth
channels into the intersection, to direct movement of the first
material through the intersection into the second channel.
37. The method of claim 36, wherein the first channel communicates
with the first intersection between the third and fourth channels,
and the third channel communicates with the first intersection
between the first and second channels, and wherein the at least
second material is concurrently transported from the third and
fourth channels into the second channel.
38. The method of claim 36, wherein the first channel communicates
with the first intersection between the fourth channel and the
second channel and the second channel communicates with the first
intersection between the first channel and the third channel, and
wherein the at least second material is concurrently transported
from the fourth channel into the third channel.
39. The method of claim 37 further comprising transporting the
first material in the intersection, into the fourth channel
subsequent to the transport of the first material from the first
channel into the intersection.
40. The method of claim 38, wherein the step of transporting the
first material into the third channel is carried out for a selected
period of time, a volume of the first material transported into the
fourth channel being a function of the selected period of time.
41. The method of claim 38 further comprising transporting the
first material in the intersection into the fourth channel
subsequent to the transport of the first material from the first
channel into the intersection.
42. The method of claim 41 further comprising transporting the
first material in the first and second channels away from the
intersection concurrently with the transport of the first material
in the intersection into the fourth channel.
43. The method of claim 38 further comprising transporting the
first material in the intersection into the third channel
subsequent to the step of transporting the second material from the
fourth channel into the third channel.
44. The method of claim 36, wherein the first material comprises
charged species that are transported by electrophoresis.
45. The method of claim 36, wherein the first material is
transported by electroosmosis.
46. The method of claim 36, wherein the first material is
transported by a combination of electrophoresis and
electroosmosis.
47. The method of claim 36, wherein the first material comprises
nucleic acids.
48. The method of claim 47, wherein the nucleic acids comprise
DNA.
49. The method of claim 48, wherein the DNA comprises restriction
enzyme fragments of DNA.
50. The method of claim 36, wherein the first material comprises
proteins.
51. The method of claim 50, wherein the first material further
comprises a micellar material.
52. The method of claim 51, wherein the micellar material is sodium
dodecyl sulfate.
53. The method of claim 36, wherein at least a portion of the first
material is fluorescently labeled.
54. The method of claim 53, wherein the fluorescently labeled
material is labeled with a fluorescein dye.
55. The method of claim 54, wherein the fluorescently labeled
material is labeled with a rhordamine dye.
56. The method of claim 40, wherein the first material comprises a
fluorescently labeled material, and further comprising detecting
the fluorescently labeled material in the fourth channel.
57. The method of claim 56, wherein the detecting step comprises
exciting the fluorescently labeled material, and detecting emitted
fluorescence.
58. The method of claim 57, wherein the step of detecting emitted
fluorescence comprises imaging a portion of the fourth channel with
an array detector.
59. The method of claim 57, wherein the step of detecting emitted
fluorescence comprises imaging a portion of the fourth channel with
a charge coupled device (CCD).
60. The method of claim 57, wherein the step of detecting emitted
fluorescence comprises detecting the emitted fluorescence with a
PMT.
61. The method of claim 57, wherein the fluorescently labeled
material is excited using a laser.
62. The method of claim 57, wherein the fluorescently labeled
material is excited using a laser diode.
63. The method of claim 57, wherein the fluorescently labeled
material is excited using a LED.
Description
FIELD OF THE INVENTION
[0002] The present invention relates generally to miniature
instrumentation for chemical analysis, chemical sensing and
synthesis and, more specifically, to electrically controlled
manipulations of fluids in micromachined channels. These
manipulations can be used in a variety of applications, including
the electrically controlled manipulation of fluid for capillary
electrophoresis, liquid chromatography, flow injection analysis,
and chemical reaction and synthesis.
BACKGROUND OF THE INVENTION
[0003] Laboratory analysis is a cumbersome process. Acquisition of
chemical and biochemical information requires expensive equipment,
specialized labs and highly trained personnel. For this reason,
laboratory testing is done in only a fraction of circumstances
where acquisition of chemical information would be useful. A large
proportion of testing in both research and clinical situations is
done with crude manual methods that are characterized by high labor
costs, high reagent consumption, long turnaround times, relative
imprecision and poor reproducibility. The practice of techniques
such as electrophoresis that are in widespread use in biology and
medical laboratories have not changed significantly in thirty
years.
[0004] Operations that are performed in typical laboratory
processes include specimen preparation, chemical/biochemical
conversions, sample fractionation, signal detection and data
processing. To accomplish these tasks, liquids are often measured
and dispensed with volumetric accuracy, mixed together, and
subjected to one or several different physical or chemical
environments that accomplish conversion or fractionation. In
research, diagnostic, or development situations these operations
are carried out on a macroscopic scale using fluid volumes in the
range of a few microliters to several liters at a time. Individual
operations are performed in series, often using different
specialized equipment and instruments for separate steps in the
process. Complications, difficulty and expense are often the result
of operations involving multiple laboratory processing steps.
[0005] Many workers have attempted to solve these problems by
creating integrated laboratory systems. Conventional robotic
devices have been adapted to perform pipetting, specimen handling,
solution mixing, as well as some fractionation and detection
operations. However, these devices are highly complicated, very
expensive and their operation requires so much training that their
use has been restricted to a relatively small number of research
and development programs. More successful have been automated
clinical diagnostic systems for rapidly and inexpensively
performing a small number of applications such as clinical
chemistry tests for blood levels of glucose, electrolytes and
gases. Unfortunately due to their complexity, large size and great
cost, such equipment, is limited in its application to a small
number of diagnostic circumstances.
[0006] The desirability of exploiting the advantages of integrated
systems in a broader context of laboratory applications has led to
proposals that such systems be miniaturized. In the 1980's,
considerable research and development effort was put into an
exploration of the concept of biosensors with the hope they might
fill the need. Such devices make use of selective chemical systems
or biomolecules that are coupled to new methods of detection such
as electrochemistry and optics to transduce chemical signals to
electrical ones that can be interpreted by computers and other
signal processing units. Unfortunately, biosensors have been a
commercial disappointment. Fewer than 20 commercialized products
were available in 1993, accounting for revenues in the U.S. of less
than $100 million. Most observers agree that this failure is
primarily technological rather than reflecting a misinterpretation
of market potential. In fact, many situations such as massive
screening for new drugs, highly parallel genetic research and
testing, micro-chemistry to minimize costly reagent consumption and
waste generation, and bedside or doctor's office diagnostics would
greatly benefit from miniature integrated laboratory systems.
[0007] In the early 1990's, people began to discuss the possibility
of creating miniature versions of conventional technology. Andreas
Manz was one of the first to articulate the idea in the scientific
press. Calling them "miniaturized total, analysis systems," or
".mu.-TAS," he predicted that it would be possible to integrate
into single units microscopic versions of the various elements
necessary to process chemical or biochemical samples, thereby
achieving automated experimentation. Since that time, miniature
components have appeared, particularly molecular separation methods
and microvalves. However, attempts to combine these systems into
completely integrated systems have not met with success. This is
primarily because precis Population of tiny fluid volumes in
extremely narrow channels has proven to be a difficult
technological hurdle.
[0008] One prominent field susceptible to miniaturization is
capillary electrophoresis. Capillary electrophoresis has become a
popular technique for separating charged molecular species in
solution. The technique is performed in small capillary tubes to
reduce band broadening effects due to thermal convection and hence
improve resolving power. The small tubes imply that minute volumes
of materials, on the order of nanoliters, must be handled to inject
the sample into the separation capillary tube.
[0009] Current techniques for injection include electromigration
and siphoning of sample from a container into a continuous
separation tube. Both of these techniques suffer from relatively
poor reproducibility, and electromigration additionally suffers
from electrophoretic mobility-based bias. For both sampling
techniques the input end of the analysis capillary tube must be
transferred from a buffer reservoir to a reservoir holding the
sample. Thus, a mechanical manipulation is involved. For the
siphoning injection, the sample reservoir is raised above the
buffer reservoir holding the exit end of the capillary for a fixed
length of time.
[0010] An electromigration injection is effected by applying an
appropriately polarized electrical potential across the capillary
tube for a given duration while the entrance end of the capillary
is in the sample reservoir. This can lead to sampling bias because
a disproportionately larger quantity of the species with higher
electrophoretic mobilities migrate into the tube. The capillary is
removed from the sample reservoir and replaced into the entrance
buffer reservoir after the injection duration for both
techniques.
[0011] A continuing need exists for methods and apparatuses which
lead to improved electrophoretic resolution and improved injection
stability.
SUMMARY OF THE INVENTION
[0012] The present invention provides microchip laboratory systems
and methods that allow complex biochemical and chemical procedures
to be conducted on a microchip under electronic control The
microchip laboratory systems comprises a material handling
apparatus that transports materials through a system of
interconnected, integrated channels on a microchip. The movement of
the materials is precisely directed by controlling the electric
fields produced in the integrated channels The precise control of
the movement of such materials enables precise mixing, separation,
and reaction as needed to implement a desired biochemical or
chemical procedure.
[0013] The microchip laboratory system of the present invention
analyzes and/or synthesizes chemical materials in a precise and
reproducible manner. The system includes a body having integrated
channels connecting a plurality of reservoirs that store the
chemical materials used in the chemical analysis or synthesis
performed by the system. In one aspect, at least five of the
reservoirs simultaneously have a controlled electrical potential,
such that material from at least one of the reservoirs is
transported through the channels toward at least one of the other
reservoirs. The transportation of the material through the channels
provides exposure to one or more selected chemical or physical
environments, thereby resulting in the synthesis or analysis of the
chemical material,
[0014] The microchip laboratory system preferably also includes one
or more intersections of integrated channels connecting three or
more of the reservoirs. The laboratory system controls the electric
fields produced in the channels in a manner that controls which
materials in the reservoirs are transported through the
intersection(s). In one embodiment, the microchip laboratory system
acts as a mixer or diluter that combines materials in the
intersection(s) by producing an electrical potential in the
intersection that is less than the electrical potential at each of
the two reservoirs from which the materials to be mixed originate.
Alternatively, the laboratory system can act as a dispenser that
electrokinetically injects precise, controlled amounts of material
through the intersection(s).
[0015] By simultaneously applying an electrical potential at each
of at least five reservoirs, the microchip laboratory system can
act as a complete system for performing an entire chemical analysis
or synthesis. The five or more reservoirs can be configured in a
manner that enables the electrokinetic separation of a sample to be
analyzed ("the analyte") which is then mixed with a reagent from a
reagent reservoir. Alternatively, a chemical reaction of an analyte
and a solvent can be performed first, and then the material
resulting from the reaction can be electrokinetically separated. As
such, the use of five or more reservoirs provides an integrated
laboratory system that can perform virtually any chemical analysis
or synthesis.
[0016] In yet another aspect of the invention, the microchip
laboratory system includes a double intersection formed by channels
interconnecting at least six reservoirs. The first intersection can
be used to inject a precisely sized analyte plug into a separation
channel toward a waste reservoir. The electrical potential at the
second intersection can be selected in a manner that provides
additional control over the size of the analyte plug. In addition,
the electrical potentials can be controlled in a manner that
transports materials from the fifth and sixth reservoirs through
the second intersection toward the few intersection and toward the
fourth reservoir after a selected volume of material from the first
intersection is transported through the second intersection toward
the fourth reservoir. Such control can be used to push the analyte
plug further down the separation channel while enabling a second
analyte plug to be injected through the first intersection.
[0017] In another aspect, the microchip laboratory system acts as a
microchip flow control system to control the flow of material
through an intersection formed by integrated channels connecting at
least four reservoirs The microchip flow control system
simultaneously applies a controlled electrical potential to at
least three of the reservoirs such that the volume of material
transported from the first reservoir to a second reservoir through
the intersection is selectively controlled solely by the movement
of a material from a third reservoir through the intersection.
Preferably, the material moved through the third reservoir to
selectively control the material transported from the first
reservoir is directed toward the same second reservoir Vs the
material from the first reservoir. As such, the microchip flow
control system acts as a valve or a gate that selectively controls
the volume of material transported through the intersection. The
microchip flow control system can also be configured to act as a
dispenser that prevents the first material from moving through the
intersection toward the second reservoir after a selected volume of
the first material has passed through the intersection.
Alternatively) the microchip flow control system can be configured
to act as a diluter that mixes the fat and second materials in the
intersection in a manner that simultaneously transports the first
and second materials from the intersection toward the second
reservoir.
[0018] Other objects, advantages and salient features of the
invention will become apparent from the following detailed
description, which taken in conjunction with the annexed drawings,
discloses preferred embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a schematic view of a preferred embodiment of the
present invention;
[0020] FIG. 2 is an enlarged, vertical sectional view of a channel
shown;
[0021] FIG. 3 is a schematic, top view of a microchip according to
a second preferred embodiment of the present invention;
[0022] FIG. 4 is an enlarged view of the intersection region of
FIG. 3;
[0023] FIG. 5 are CCD images of a plug of analyte moving through
the intersection of the FIG. 30 embodiment;
[0024] FIG. 6 is a schematic top view of a microchip laboratory
system according to a third preferred embodiment of a microchip
according to the present invention;
[0025] FIG. 7 is a CCD image of "sample loading mode for rhodamine
B" (shaded area);
[0026] FIG. 8(a) is a schematic view of the intersection area of
the microchip of FIG. 6, prior to analyte injection;
[0027] FIG. 8(b) is a CCD fluorescence image taken of the same area
depicted in FIG. 8(a), after sample loading in the pinched
mode,
[0028] FIG. 8(c) is a photomicrograph taken of the same area
depicted in FIG. 8(a), after sample loading in the floating
mode;
[0029] FIG. 9 shows integrated fluorescence signals for injected
volume plotted versus time for pinched and floating injections;
[0030] FIG. 10 is a schematic, top view of a microchip according to
a fourth preferred embodiment of thc present invention;
[0031] FIG. 11 is an enlarged view of the intersection region of
FIG. 10;
[0032] FIG. 12 is a schematic top view of a microchip laboratory
system according to a fifth preferred embodiment according to the
present invention;
[0033] FIG. 13(a) is a schematic view of a CCD camera view of the
intersection area of the microchip laboratory system of FIG.
12;
[0034] FIG. 13(b) is a CCD fluorescence image taken of the same
area depicted in FIG. 13(a), after sample loading in the pinched
made;
[0035] FIGS. 13(c)-13(e) are CCD fluorescence images taken of the
same area depicted in FIG. 13(a), sequentially showing a plug of
analyte moving away from the channel intersection at 1, 2, and 3
seconds, respectively, after switching to the run mode;
[0036] FIG. 14 shows two injection profiles for didansyl-lysine
injected for 2 s with .gamma. equal to 0.97 and 9.7;
[0037] FIG. 15 are electropherograms taken at (a) 3.3 cm, (b) 9.9
cm, and (c) 16.5 cm from the point of injection for rhodamine B
(less retained) and sulforhodamine (more retained);
[0038] FIG. 16 is a plot of the efficiency data generated from the
electropherograms of FIG. 15, showing variation of the plate number
with channel length for rhodamine B (square with plus) and
sulforhodamine (square with plus) and sulforhodamine (square with
dot) with best linear fit (solid lines) for each analyte;
[0039] FIG. 17(a) is an electropherogram of rhodamine B and
fluorescein with a separation field strength of 1.5 kV/cm and a
separation length of 0.9 mm;
[0040] FIG. 17(b) is an electropherogram of rhodamine B and
fluorescein with a separation field strength of 1.5 kW/cm and a
separation length of 1.6 mm;
[0041] FIG. 17(c) is an electropherogram of rhodamine B and
fluorescein with a separation field strength of 1.5 kV/cm and a
separation length of 11.1 mm;
[0042] FIG. 18 is a graph showing variation of the number of plates
per unit time as a function of the electric field strength for
rhodamine B at separation lengths of 1.6 mm (circle) and 11.1 mm
(square) and for fluorescein at separation lengths of 1.6 mm
(diamond) and 11.1 mm (triangle),
[0043] FIG. 19 shows a chromatogram of coumarins analyzed by
electrochromatography using the system of FIG. 12;
[0044] FIG. 20 shows a chromatogram of coumarins resulting from
micellar electrokinetic capillary chromatography using the system
of FIG. 12;
[0045] FIGS. 21(a) and 21(b) show the separation of three metal
ions using the system of FIG. 12,
[0046] FIG. 22 is a schematic, top plan view of a microchip
according to the FIG. 3 embodiment, additionally including a
reagent reservoir and ration channel;
[0047] FIG. 23 is a schematic view of the embodiment of FIG. 20,
showing applied voltages;
[0048] FIG. 24 shows two electropherograms produced using the FIG.
22 embodiment;
[0049] FIG. 25 is a schematic view of a microchip laboratory system
according to a sixth preferred embodiment of the present
invention;
[0050] FIG. 26 shows the reproducibility of the amount injected for
arginine and glycine using the system of FIG. 25;
[0051] FIG. 27 shows the overlay of three electrophoretic
separations using the system of FIG. 25;
[0052] FIG. 28 shows a plot of amounts injected versus reaction
tine using the system of FIG. 25;
[0053] FIG. 29 shows an electropherogram of restriction fragments
produced using the system of FIG. 25;
[0054] FIG. 30 is a schematic view of a microchip laboratory system
according to a seventh preferred embodiment of the present
invention.
[0055] FIG. 31 is a schematic view of the apparatus of FIG. 21,
showing sequential applications of voltages to effect desired
fluidic manipulations; and
[0056] FIG. 32 is a graph showing the different voltages applied to
effect the fluidic manipulations of FIG. 23.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Integrated, micro-laboratory systems for analyzing or
synthesizing chemicals require a precise way of manipulating fluids
and fluid-borne material and subjecting the fluids to selected
chemical or physical environments that produce desired conversions
or partitioning. Given the concentration of analytes that produces
chemical conversion in reasonable time scales, the nature of
molecular detection, diffusion times and manufacturing methods for
creating devices on a microscopic scale, miniature integrated
micro-laboratory systems lend themselves to channels having
dimensions on the order of 1 to 100 micrometers in diameter. Within
this context, electrokinetic pumping has proven to be versatile and
effective in transporting materials in microfabricated laboratory
systems.
[0058] The present invention provides the tools necessary to make
use of electrokinetic pumping not only in separations, but also to
perform liquid handling that accomplishes other important sample
processing steps, such as chemical conversions or sample
partitioning. By simultaneously controlling voltage at a plurality
of ports connected by channels in a microchip structure, it is
possible to measure and dispense fluids with great precision, mix
reagents, incubate reaction components, direct the components
towards sites of physical or biochemical partition and subject the
components to detector systems. By combining these capabilities on
a single microchip, one is able to create complete, miniature,
integrated automated laboratory systems for analyzing or
synthesizing chemicals.
[0059] Such integrated micro-laboratory systems can be made up of
several component elements. Component elements can include liquid
dispersing systems, liquid mixing systems, molecular partition
systems, detector sights, etc. For example, as described herein,
one can construct a relatively complete system for the
identification of restriction endonuclease sites in a DNA molecule.
This single microfabricated device thus includes in a single system
the functions that are traditionally performed by a technician
employing pipettors, incubators, gel electrophoresis systems, and
data acquisition systems. In this system, DNA is mixed with an
enzyme, the mixture is incubated, and a selected volume of the
reaction mixture is dispensed into a separation channel.
Electrophoresis is conducted concurrent with fluorescent labeling
of the DNA.
[0060] Shown in FIG. 1 is an example of a microchip laboratory
system 10 configured to implement an entire chemical analysis or
synthesis. The laboratory system 10 includes six reservoirs 12, 14,
16, 18, 20, and 22 connected to each other by a system of channels
24 micromachined into a substrate or base member (not shown in FIG.
1), as discussed in more detail below. Each reservoir 12-22 is in
fluid communication with a corresponding channel 26, 28, 30, 32,
34, 36, and 38 of the channel system 24. The first channel 26
leading from the first reservoir 12 is connected to the second
channel 28 leading from the second reservoir 14 at a first
intersection 38. Likewise, the third channel 30 from the third
reservoir 16 is connected to the fourth channel 32 at a second
intersection 40. The first intersection 38 is connected to the
second intersection 40 by a reaction chamber or channel 42. The
fifth channel 34 from the fifth reservoir 20 is also connected to
the second intersection 40 such that the second intersection 40 is
a four-way intersection of channels 30, 32, 34, and 42. The fifth
channel 34 also intersects the sixth channel 36 from the sixth
reservoir 22 at a third intersection 44.
[0061] The materials stored in the reservoirs preferably are
transported electrokinetically through the channel system 24 in
order to implement the desired analysis or synthesis. To provide
such electrokinetic transport, the laboratory system 10 includes a
voltage controller 46 capable of applying selectable voltage
levels, including ground. Such a voltage controller can be
implemented using multiple voltage dividers and multiple relays to
obtain the selectable voltage levels. The voltage controller is
connected to an electrode positioned in each of the six reservoirs
12-22 by voltage lines V1-V6 in order to apply the desired voltages
to the materials in the reservoirs. Preferably, the voltage
controller also includes sensor channels S1, S2, and S3 connected
to the first, second, and third intersections 38, 40, 44,
respectively, in order to sense the voltages present at those
intersections.
[0062] The use of electrokinetic transport on microminiaturized
planar liquid phase separation devices, described above, is a
viable approach for sample manipulation and as a pumping mechanism
for liquid chromatography. The present invention also entails the
use of electroosmotic flow to mix various fluids in a controlled
and reproducible fashion. When an appropriate fluid is placed in a
tube made of a correspondingly appropriate material, functional
groups at the surface of the tube can ionize. In the case of tubing
materials that are terminated in hydroxyl groups, protons will
leave the surface and enter an aqueous solvent. Under such
conditions the surface will have a net negative charge and the
solvent will have an excess of positive charges, mostly in the
charged double layer at the surface. With the application of an
electric field across the tube, the excess cations in solution will
be attracted to the cathode, or negative electrode. The movement of
these positive charges through the tube will drag the solvent with
them. The steady state velocity is given by equation 1, 1 v = E 4 (
1 )
[0063] where v is the solvent velocity, .di-elect cons. is the
dielectric constant of the fluid, .xi. is the zeta potential of the
surface, E is the electric field strength, and .pi. is the solvent
viscosity. From equation 1 it is obvious that the fluid flow
velocity or flow rate can be controlled through the electric field
strength. Thus, electroosmosis can be used as a programmable
pumping mechanism.
[0064] The laboratory microchip system 10 shown in FIG. 1 could be
used for performing numerous types of laboratory analysis or
synthesis, such as DNA sequencing or analysis,
electrochromatography, micellar electrokinetic capillary
chromatography (MECC), inorganic ion analysis, and gradient elution
liquid chromatography, as discussed in more detail below. The fifth
channel 34 typically is used for electrophoretic or
electrochromatographic separations and thus may be referred to in
certain embodiments as a separation channel or column. The reaction
chamber 42 can bc used to mix any two chemicals stored in the first
and second reservoirs 12, 14. For example, DNA from the first
reservoir 12 could be mixed with an enzyme from the second
reservoir 14 in the first intersection 38 and the mixture could be
incubated in the reaction chamber 42. The incubated mixture could
then be transported through the second intersection 40 into the
separation column 34 for separation. The sixth reservoir 22 can be
used to store a fluorescent label that is mixed in the third
intersection 44 with the materials separated in the separation
column 34. An appropriate detector (D) could then be employed to
analyze the labeled materials between the third intersection 44 and
the fifth reservoir 20. By providing for a pre-separation column
reaction in the first intersection 38 and reaction chamber 42 and a
post-separation column reaction in the third intersection 44, the
laboratory system 10 can be used to implement many standard
laboratory techniques normally implemented manually in a
conventional laboratory. In addition, the elements of the
laboratory system 10 could be used to build a more complex system
to solve more complex laboratory procedures.
[0065] The laboratory microchip system 10 includes a substrate or
base member (not shown in FIG. 1) which can be an approximately two
inch by one inch piece of microscope slide (Corning, Inc. #2947).
While glass is a preferred material, other similar materials may be
used, such as fused silica, crystalline quartz, fused quartz,
plastics, and silicon (if the surface is treated sufficiently to
alter its resistivity). Preferably, a non-conductive material such
as glass or fused quartz is used to allow relatively high electric
fields to be applied to electrokinetically transport materials
through channels in the microchip. Semiconducting materials such as
silicon could also be used, but the electric field applied would
normally need to be kept to a minimum (approximately less than 300
volts per centimeter using present techniques of providing
insulating layers), which may provide insufficient electrokinetic
movement.
[0066] The channel pattern 24 is formed in a planar surface of the
substrate using standard photolithographic procedures followed by
chemical wet etching. The channel pattern may be transferred onto
the substrate with a positive photoresist (Shipley 1811) and an
e-beam written chrome mask (Institute of Advanced Manufacturing
Sciences, Inc.). The pattern may be chemically etched using
HF/NH.sub.4F solution
[0067] After forming the channel pattern, a cover plate may then be
bonded to the substrate using a direct bonding technique whereby
the substrate and the cover plate surfaces are first hydrolyzed in
a dilute NH.sub.4OH/H.sub.2O.sub.2 solution and then joined. The
assembly is then annealed at about 500.degree. C. in order to
insure proper adhesion of the cover plate to the substrate.
[0068] Following bonding of the cover plate, the reservoirs are
affixed to the substrate, with portions of the cover plate
sandwiched therebetween, using epoxy or other suitable means. The
reservoirs can be cylindrical with open opposite axial ends.
Typically, electrical contact is made by placing a platinum wire
electrode in each reservoirs. The electrodes are connected to a
voltage controller 46 which applies a desired potential to select
electrodes, in a manner described in more detail below.
[0069] A cross section of the first channel is shown in FIG. 2 and
is identical to the cross section of each of the other integrated
channels. When using a non-crystalline material (such as glass) for
the substrate, and when the channels are chemically wet etched, an
isotropic etch occurs, i.e., the glass etches uniformly in all
directions, and the resulting channel geometry is trapezoidal. The
trapezoidal cross section is due to "undercutting" by the chemical
etching process at the edge of the photoresist. In one embodiment,
the channel cross scion of the illustrated embodiment has
dimensions of 5.2 .mu.m in depth, 57 .mu.m in width at the top and
45 .mu.m in width at the bottom. In another embodiment, the channel
has a depth "d" of 10 .mu.m, an upper width "w1" of 90 .mu.m, and a
lower width "w2" of 70 .mu.m.
[0070] An important aspect of the present invention is the
controlled electrokinetic transportation of materials through the
channel system 24. Such controlled electrokinetic transport can be
used to dispense a selected amount of material from one of the
reservoirs through one or more intersections of the channel
structure 24. Alternatively, as noted above, selected amounts of
materials from two reservoirs can be transported to an intersection
where the materials can be mixed in desired concentrations.
[0071] Gated Dispenser
[0072] Shown in FIG. 3 is a laboratory component 10A that can be
used to implement a preferred method of transporting materials
through a channel structure 24A The A following each number in FIG.
3 indicates that it corresponds to an analogous clement of FIG. 1
of the same number without the A. For simplicity, the electrodes
and the connections to the voltage controller that controls the
transport of materials through the channel system 24A are not shown
in FIG. 3.
[0073] The microchip laboratory system 10A shown in FIG. 3 controls
the amount of material from the first reservoir 12A transported
through the intersection 40A toward the fourth reservoir 20A by
electrokinetically opening and closing access to the intersection
40A from the first channel 26A. As such, the laboratory microchip
system 10A essentially implements a controlled electrokinetic
valve. Such an electrokinetic valve can be used as a dispenser to
dispense selected volumes of a single material or as a mixer to mix
selected volumes of plural materials in the intersection 40A. In
general, electro-osmosis is used to transport "fluid materials" and
electrophoresis is used to transport ions without transporting the
fluid material surrounding the ions. Accordingly, as used herein,
the term "material" is used broadly to cover any form of material,
including fluids and ions.
[0074] The laboratory system 10A provides a continuous
unidirectional flow of fluid through the separation channel 34A.
This injection or dispensing scheme only requires that the voltage
be changed or removed from one (or two) reservoirs and allows the
fourth reservoir 20A to remain at ground potential. This will allow
injection and separation to be performed with a single polarity
power supply.
[0075] An enlarged view of the intersection 40A is shown in FIG. 4.
The directional arrows indicate the time sequence of the flow
profiles at the intersection 40A. The solid arrows show the initial
flow pattern. Voltages at the various reservoirs are adjusted to
obtain the described flow patterns. The initial flow pattern brings
a second material from the second reservoir 16A at a sufficient
rate such that all of the first material transported from reservoir
12A to the intersection 40A is pushed toward the third reservoir
18A. In general, the potential distribution will be such that the
highest potential is in the second reservoir 16A, a slightly lower
potential in the first reservoir 12A, and yet a lower potential in
the third reservoir 18A, with the fourth reservoir 20A being
grounded. Under these conditions, the flow towards the fourth
reservoir 20A is solely the second material from the second
reservoir 16A.
[0076] To dispense material from the first reservoir 12A through
the intersection 40A, the potential at the second reservoir 16A can
be switched to a value less than the potential of the first
reservoir 12A or the potentials at reservoirs 16A and/or 18A, can
be floated momentarily to provide the flow shown by the short
dashed arrows in FIG. 4. Under these conditions, the primary flow
will be from the first reservoir 12A down towards the separation
channel waste reservoir 20A. The flow from the second and third
reservoirs 16A, 18A will be small and could be in either direction.
This condition is held long enough to transport a desired amount of
material from the first reservoir 12A through the intersection 40A
and into the separation channel 34A. After sufficient time for the
desired material to pass through the intersection 40A, the voltage
distribution is switched back to the original values to prevent
additional material from the first reservoir 12A from flowing
through the intersection 40A toward the separation channel 34A.
[0077] One application of such a "gated dispenser" is to inject a
controlled, variable-sized plug of analyte from the first reservoir
12A for electrophoretic or chromatographic separation in the
separation channel 34A. In such a system, the first reservoir 12A
stores analyte, the second reservoir 16A stores an ionic buffer,
the third reservoir 18A is a first waste reservoir and the fourth
reservoir 20A is a second waste reservoir. To inject a small
variable plug of analyte from the first reservoir 12A, the
potentials at the buffer and first waste reservoirs 16A, 18A are
simply floated for a short period of time (.apprxeq.100 ms) to
allow the analyte to migrate down the separation column 34A. To
break off the injection plug, the potentials at the buffer
reservoir 16A and the first waste reservoir 18A are reapplied.
Alternatively, the valving sequence could be effected by bringing
reservoirs 16A and 18A to the potential of the intersection 40A and
then returning them to their original potentials. A shortfall of
this method-is that the composition of the injected plug has an
electrophoretic mobility bias whereby the faster migrating
compounds are introduced preferentially into the separation column
34A over slower migrating compounds.
[0078] In FIG. 5, a sequential view of a plug of analyte moving
through the intersection of the FIG. 3 embodiment can be seen by
CCD) images The analyte being pumped through the laboratory system
10A was rhodamine B (shaded area), and the orientation of the CCD
images of the injection cross or intersection is the same as in
FIG. 3. The first image, (A), shows the analyte being pumped
through the injection cross or intersection toward the first waste
reservoir 18A prior to the injection. The second image, (B), shows
the analyte plug being injected into the separation column 34A. The
third image, (C), depicts the analyte plug moving away from the
injection intersection after an injection plug has been completely
introduced into the separation column 34A. The potentials at the
buffer and fist waste reservoirs 16A, 18A were floated for 100 ms
while the sample moved into the separation column 34A. By the time
of the (C) image, the closed gate mode has resumed to stop further
analyte from moving through the intersection 40A into the
separation column 34A, and a clean injection plug with a length of
142 .mu.m has been introduced into the separation column. As
discussed below, the gated injector contributes to only a minor
fraction of the total plate height. The injection plug length
(volume) is a function of the time of the injection and the
electric field strength in the column. The shape of the injected
plug is skewed slightly because of the directionality of the
cleaving buffer flow. However, fear a given injection period, the
reproducibility of the amount injected, determined by integrating
the peak area, is 1% RSD for a series of 10 replicate
injections.
[0079] Electrophoresis experiments were conducted using the
microchip laboratory system 10A of FIG. 3, and employed methodology
according to the present invention. Chip dynamics were analyzed
using analyte fluorescence. A charge coupled device (CCD) camera
was used to monitor designated areas of the chip and a
photomultiplier tube (PMT) tracked single point events. The CCD
(Princeton Instruments, Inc. TE/CCD-512TKM) camera was mounted on a
stereo microscope (Nikon SMZ-U), and the laboratory system 10A was
illuminated using an argon ion laser (514.5 nm, Coherent Innova 90)
operating at 3 W with the beam expanded to a circular spot
.apprxeq.2 cm in diameter. The PMT, with collection optics, was
situated below the microchip with the optical axis perpendicular to
the microchip surface. The laser was operated at approximately 20
mW, and the beam impinged upon the microchip at a 45.degree. angle
from the microchip surface and parallel to the separation channel.
The laser beam and PMT observation axis were separated by a
135.degree. angle. The point detection scheme employed a
helium-neon laser (543 nm, PMS Electro-optics LITGP-0051) with an
electrometer (Keithley 617) to monitor response of the PMT (Oriel
77340). The voltage controller 46 (Spellman CZE 1000R) for
electrophoresis was operated between 0 and +4.4 kV relative to
ground.
[0080] The type of gated injector described with respect to FIGS. 3
and 4 show electrophoretic mobility based bias as do conventional
electroosmotic injections. Nonetheless, this approach has
simplicity in voltage switching requirements and fabrication and
provides continuous unidirectional flow through the separation
channel. In addition, the gated injector provides a method for
valving a variable volume of fluid into the separation channel 34A
in a manner that is precisely controlled by the electrical
potentials applied.
[0081] Another application of the gated dispenser 10A is to dilute
or mix desired quantities of materials in a controlled manner. To
implement such a mixing scheme in order to mix the materials from
the first and second reservoirs 12A, 16A, the potentials in the
first and second channels 26A, 30A need to be maintained higher
than the potential of the intersection 40A during mixing. Such
potentials will cause the materials from the first and second
reservoirs 12A and 16A to simultaneously move through the
intersection 40A and thereby mix the two materials. The potentials
applied at the first and second reservoirs 12A, 16A can be adjusted
as desired to achieve the selected concentration of each material.
After dispensing the desired amounts of each material, the
potential at the second reservoir 16A may be increased in a manner
sufficient to prevent further material from the first reservoir 12A
from being transported through the intersection 40A toward the
third reservoir 30A.
[0082] Analyte Injector
[0083] Shown in FIG. 6 is a microchip analyte injector 10B
according to the present invention. The channel pattern 24B has
four distinct channels 26B, 30B, 32B, and 34B micromachined into a
substrate 49 as discussed above. Each channel has an accompanying
reservoir mounted above the terminus of each charnel portion, and
all four channels intersect at one end in a four way intersection
40B. The opposite ends of each section provide termini that extend
just beyond the peripheral edge of a cover plate 49' mounted on the
substrate 49. The analyte injector 10B shown in FIG. 6 is
substantially identical to the gated dispenser 10A except that the
electrical potentials are applied in a manner that injects a volume
of material from reservoir 16% through the intersection 40B rather
than from the reservoir 12B and the volume of material injected is
controlled by the size of the intersection.
[0084] The embodiment shown in FIG. 6 can be used for various
material manipulations. In one application, the laboratory system
is used to insect an analyte from an analyte reservoir 16B through
the intersection 40B for separation in the separation channel 34B.
The analyte injector 10B can be operated in either "load" mode or a
"run" mode. Reservoir 16B is supplied with an analyte and reservoir
12B with buffer. Reservoir 18B acts as an analyte waste reservoir,
and reservoir 20B acts as a waste reservoir.
[0085] In the "load" mode, at least two types of analyte
introduction are possible. In the first, known as a "floating"
loading, a potential is applied to the analyte reservoir 16B with
reservoir 18B grounded. At the same time, reservoirs 12B and 20B
are floating, meaning that they are neither coupled to the power
source, nor grounded.
[0086] The second load mode is "pinched" loading mode, wherein
potentials are simultaneously applied at reservoirs 12B, 16B, and
20B, with reservoir 18B grounded in order to control the injection
plug shape as discussed in more detail below. As used herein,
simultaneously controlling electrical potentials at plural
reservoirs means that the electrodes are connected to a operating
power source at the same chemically significant time period.
Flouting a reservoir means disconnecting the electrode in the
reservoir from the power source and thus the electrical potential
at the reservoir is rot controlled.
[0087] In the "run" mode, a potential is applied to the buffer
reservoir 12B with reservoir 20B grounded and with reservoirs 16B
and 18B at approximately half of the potential of reservoir 12B.
During the run mode, the relatively high potential applied to the
buffer reservoir 12B causes the analyte in the intersection 40B to
move toward the waste reservoir 20B in the separation column
34B.
[0088] Diagnostic experiments were performed using rhodamine B and
sulforhodamine 101 (Exciton Chemical Co., Inc.) as the analyte at
60 .mu.M for the CCD) images and 6 .mu.M for the point detection. A
sodium tetraborate buffer (50 mM, pH 9.2) was the mobile phase in
the experiments. An injection of spatially well defined small
volume (.apprxeq.100 pL) and of small longitudinal extent
(.apprxeq.100 .mu.m), injection is beneficial when performing these
types of analyses.
[0089] The analyte is loaded into the injection crass as a frontal
electropherogram, and once the front of the slowest analyte
component passes through the injection cross or intersection 40B,
the analyte is ready to be analyzed. In FIG. 7, a CCD image (the
area of which is denoted by the broken line square) displays the
flow pattern of the analyte 54 (shaded area) and the buffer (white
area) through the region of the injection intersection 40B.
[0090] By pinching the flow of the analyte, the volume of the
analyte plug is stable over time. The slight asymmetry of tie plug
shape is due to the different electric field strengths in the
buffer channel 26B (470 V/cm) and the separation channel 34B (100
V/cm) when 1.0 kV is applied to the buffer, the analyte and the
waste reservoirs, and the analyte waste reservoir is grounded.
However, the different field strengths do not influence the
stability of the analyte plug injected. Ideally, when the analyte
plug is injected into the separation channel 34B, only the analyte
in the injection cross or intersection 40B would migrate into the
separation channel.
[0091] The volume of the injection plug in the injection cross is
approximately 120 pL with a plug length of 130 .mu.m. A portion of
the analyte 54 in the analyte channel 30B and the analyte waste
channel 32B is drawn into the separation channel 34B. Following the
switch to the separation (run) mode, the volume of the injection
plug is approximately 250 pL with a plug length of 208 .mu.m. These
dimensions are estimated from a series of CCD images taken
immediately after the switch is made to the separation mode.
[0092] The two modes of loading were tested for the analyte
introduction into the separation channel 34B. The analyte was
placed in the analyte reservoir 16B, and in both injection schemes
was `transported` in the direction of reservoir 18B, a waste
reservoir. CCD images of the two types of injections are depicted
in FIGS. 8(a)-8(c). FIG. 8(a) schematically shows the intersection
40B, as well as the end portions of channels.
[0093] The CCD) image of FIG. 8(b) is of loading in the pinched
mode, just prior to being switched to the run mode. In the pinched
mode, analyte (shown as white against the dark background) is
pumped electrophoretically and electroosmotically from reservoir
16B to reservoir 19B (left to right) with buffer from the buffer
reservoir 12B (top) and the waste reservoir 20B (bottom) traveling
toward reservoir 18B (right). The voltages applied to reservoirs
12B, 16B, 18B, and 20B were 90%, 90%, 0, and 100% respectively, of
the power supply output which correspond to electric field
strengths in the corresponding channels of 400, 270, 690 and 20
V/cm, respectively. Although the voltage applied to the waste
reservoir 20B is higher than voltage applied to the analyte
reservoir 18B, the additional length of the separation channel 34B
compared to the analyte channel 30B provides additional electrical
resistance, and thus the flow from the analyte buffer 16B into the
intersection predominates. Consequently, the analyte in the
injection cross or intersection 40B has a trapezoidal shape and is
spatially constricted in the channel 32B by this material transport
pattern.
[0094] FIG. 8(c) shows a floating mode loading. The analyte is
pumped from reservoir 16B to 18B as in the pinched injection except
no potential is applied to reservoirs 12B and 20B. By not
controlling the flow of mobile phase (buffer) in channel portions
26B and 34B, the analyte is free to expand into these channels
through convective and diffusive flow, thereby resulting in an
extended injection plug.
[0095] When comparing the pinched and floating injections, the
pinched injection is superior in three areas: temporal stability of
the injected volume, the precision of the injected volume, and plug
length. When two or more analytes with vastly different mobilities
are to be analyzed, an injection with temporal stability insures
that equal volumes of the faster and slower moving analytes are
introduced into the separation column or channel 34B. The high
reproducibility of the injection volume facilitates the ability to
perform quantitative analysis. A smaller plug length leads to a
higher separation efficiency and, consequently, to a greater
component capacity for a given instrument and to higher speed
separations.
[0096] To determine the temporal stability of each mode, a series
of CCD fluorescence images were collected at 1.5 second intervals
starting just prior to the analyte reaching the injection
intersection 40B. An estimate of the amount of analyte that is
injected was determined by integrating the fluorescence in the
intersection 40B and channels 26B and 34B. This fluorescence is
plotted versus time in FIG. 9.
[0097] For the pinched injection, the injected volume stabilizes in
a few seconds and has a stability of 1% relative standard deviation
(RSD), which is comparable to the stability of the illuminating
laser. For the floating injection, the amount of analyte to be
injected into the separation channel 34B increases with time
because of the dispersive flow of analyte into channels 26B and
34B. For a 30 second injection, the volume of the injection plug is
ca. 90 pL and stable for the pinched injection versus ca. 300 pL
and continuously increasing with time for a floating injection.
[0098] By monitoring the separation channel at a point 0.9 cm from
the intersection 40B, the reproducibility for the pinched injection
mode was tested by integrating the area of the band profile
following introduction into the separation channel 34B. For six
injections with a duration of 40 seconds, the reproducibility for
the pinched injection is 0.7% RSD. Most of this measured
instability is from the optical measurement system. The pinched
injection has a higher reproducibility because of the temporal
stability of the volume injected. With electronically controlled
voltage switching, the RSD is expected to improve for both
schemes.
[0099] The injection plug width and, ultimately, the resolution
between analytes depends largely on both the flow pattern of the
analyte and the dimensions of the injection cross or intersection
40B. For this column, the width of the channel at the top is 90
.mu.m, but a channel width of 10 .mu.m is feasible which would lead
to a decrease in the volume of the injection plug from 90 pL down
to 1 pL with a pinched injection.
[0100] There are situations where it may not be desirable to
reverse the flow in the separation channel as described above for
the "pinched" and "floating" injection schemes. Examples of such
cases might be the injection of a new sample plug before the
preceding plug has been completely eluted or the use of a
post-column rector where reagent is continuously being injected
into the end of the separation column. In the latter case, it would
in general not be desirable to have the reagent flowing back up
into the separation channel.
[0101] Alternate Analyte Injector
[0102] FIG. 10 illustrates an alternate analyte injector system 10C
having six different ports or channels 26C, 30C, 32C, 34C. 56, and
58 respectively connected to six different reservoirs 12C, 16C,
18C, 20C, 60, and 62. The letter C after each element number
indicates that the indicated clement is analogous to a
correspondingly numbered elements of FIG. 1. The microchip
laboratory system 10C is similar to laboratory systems 10, 10A and
10B described previously, in that an injection cross or
intersection 40C is provided. In the FIG. 10 embodiment, a second
intersection 64 and two additional reservoirs 60 and 62 are also
provided to overcome the problems with reversing the flow in the
separation channel.
[0103] Like the previous embodiments, the analyte injector system
10C can be used to implement an analyte separation by
electrophoresis or chromatography or dispense material into some
other processing element. In the laboratory system 10C, thc
reservoir 12C contains separating buffer, reservoir 16C contains
the analyte, and reservoirs 18C and 20C are waste reservoirs.
Intersection 40C preferably is operated in the pinched mode as in
the embodiment shown in FIG. 6. The lower intersection 64, in fluid
communication with reservoirs 60 and 62, are used to provide
additional flow so that a continuous buffer stream can be directed
down towards the waste reservoir 20C and, when needed, upwards
toward the injection intersection 40C. Reservoir 60 and attached
channel 56 are not necessary, although they improve performance by
reducing band broadening as a plug passes the lower intersection
64. In many cases, the flow from reservoir 60 will be symmetric
with that from reservoir 62.
[0104] FIG. 11 is an enlarged view of the two intersections 40C and
64. The different types of arrows show the flow directions at given
instances in time for injection of a plug of analyte into the
separation channel. The solid arrows show the initial flow pattern
where the analyte is electrokinetically pumped into the upped
intersection 40C and "pinched" by material flow from reservoirs
12C, 60, and 62 toward this same intersection. Flow away from the
injection intersection 40C is carried to the analyte waste
reservoir 18C. The analyte is also flowing from the reservoir 16C
to the analyte waste reservoir 18C. Under these conditions, flow
from reservoir 60 (and reservoir 62) is also going down the
separation channel 34C to the waste reservoir 20C. Such a flow
pattern is created by simultaneously controlling the electrical
potentials at all six reservoirs.
[0105] A plug of the analyte is injected through the injection
intersection 40C into the separation channel 34C by switching to
the flow profile shown by the short dashed arrows Buffer flows down
from reservoir 12C to the injection intersection 40C and towards
reservoirs 16C, 18C, and 20C. This flow profile also pushes the
analyte plug toward waste reservoir 20C into the separation channel
34C as described before. This flow profile is held for a sufficient
length of time so as to move the analyte plug past the lower
intersection 64. The flow of buffer from reservoirs 60 and 62
should be low as indicated by the short arrow and into the
separation channel 34C to minimize distortion.
[0106] The distance between the upper and lower intersections 40C
and 64, respectively, should be as small as possible to minimize
plug distortion and criticality of timing in the switching between
the two flow conditions. Electrodes for sensing the electrical
potential may also be placed at the lower intersection and in the
channels 56 and 58 to assist in adjusting the electrical potentials
for proper flow control. Accurate flow control at the lower
intersection 64 may be necessary to prevent undesired band
broadening.
[0107] After the sample plug passes the lower intersection, the
potentials are switched back to the initial conditions to give the
original flow profile as shown with the long dashed arrows. This
flow pattern will allow buffer flow into the separation channel 34C
while the next analyte plug is being transported to the plug
forming region in the upper intersection 40C. This injection scheme
will allow a rapid succession of injections to be made and may be
very important for samples that are slow to m grate or if it takes
a long time to achieve a homogeneous sample at the upper
intersection 40C such as with entangled polymer solutions. This
implementation of the pinched injection also maintains
unidirectional flow through the separation channel as might be
required for a post-column reaction as discussed below with respect
to FIG. 22.
[0108] Serpentine Channel
[0109] Another embodiment of the invention is the modified analyte
injector system 10D shown in FIG. 12. The laboratory system 10D
shown in FIG. 12 is substantially identical to the laboratory
system 00B shown in FIG. 6, except that the separation channel 34D
follows a serpentine path. The serpentine pith of the separation
channel 34D allows the length of the separation channel to be
greatly increased without substantially increasing the area of the
substrate 49D needed to implement the serpentine path. Increasing
the length of the separation channel 34D increases the ability of
the laboratory system 10D to distinguish elements of an analyte in
one particularly preferred embodiment, the enclosed length (that
which is covered by the cover plate 49D) of the channels extending
from reservoir 16D to reservoir 18D is 19 mm, while the length of
channel portion 26D is 6.4 mm and channel 34D is 171 mm. The turn
radius of each turn of the channel 34D, which serves as a
separation column, is 0.16 mm.
[0110] To perform a separation using the modified analyte injector
system 10D, an analyte is first loaded into the injection
intersection 40D using one of the loading methods described above.
After the analyte has been loaded into the intersection 40D of the
microchip laboratory system 10, the voltages are manually switched
from the loading mode to the run (separation) mode of operation.
FIGS. 13(a)-13(e) illustrate a separation of rhodamine B (less
retained) and sulforhodamine (more retained) using the following
conditions: E.sub.irij=400 V/cm, E=150 V/cm, buffer=50 mM sodium
tetraborate at pH 9.2. The CCD images demonstrate the separation
process at 1 second intervals, with FIG. 13(a) showing a schematic
of the section of the chip imaged, and with FIGS. 13(b)-13(e)
showing the separation unfold.
[0111] FIG. 13(b) again shows the pinched injection with the
applied voltages at reservoirs 12D, 16D, and 20D equal and
reservoir 18D grounded. FIGS. 13(c)-13(e) shows the plug moving
away from the intersection at 1, 2, and 3 seconds, respectively,
after switching to the run mode. In FIG. 13(c), tile injection plug
is migrating around a 90.degree. turn and band distortion is
visible due to the inner portion of the plug traveling less
distance than the outer portion. By FIG. 13(d), the analytes have
separated into distinct bands, which are distorted in the shape of
a parallelogram. In FIG. 13(c), the bands are well separated and
have attained a more rectangular shape, i.e., collapsing of the
parallelogram, due to radial diffusion, an additional contribution
to efficiency loss.
[0112] When the switch is made from the load mode to the run mode,
a clean break of the injection plug from the analyte stream is
desired to avoid tailing. This is achieved by pumping the mobile
phase or buffer from channel 26D into channels 30D, 32D, and 34D
simultaneously by maintaining the potential at the intersection 40D
below the potential of reservoir 12D and above the potentials of
reservoirs 16D, 18D, and 20D.
[0113] In the representative experiments described herein, the
intersection 40D was maintained at 66% of the potential of
reservoir 12D during the run mode. This provided sufficient flow of
the analyte back away from the injection Intersection 40D down
channels 30D and 32D without decreasing the field strength in the
separation channel 34D significantly. Alternate channel designs
would allow a greater fraction of the potential applied at
reservoir 12D to be dropped across the separation channel 34D,
thereby improving efficiency This three way flow is demonstrated in
FIGS. 13(c)-13(e) as the analytes in channels 30D and 32D) (left
and right, respectively) move further away from the intersection
with time. Three way flow permits well-defined reproducible
injections with minimal bleed of the analyte into the separation
channel 34D.
[0114] Detectors
[0115] In most applications envisaged for these integrated
microsystems for chemical analysis or synthesis it will be
necessary to quantify the material present in a channel at one or
more positions similar to conventional laboratory measurement
processes. Techniques typically utilized for quantification
include, but are not limited to, optical absorbance, refractive
index changes, fluorescence emission, chemiluminescence, various
forms of Raman spectroscopy, electrical conductometric
measurements, electrochemical amperiometric measurements, acoustic
wave propagation measurements.
[0116] Optical absorbence measurements are commonly employed with
conventional laboratory analysis systems because of the generality
of the phenomenon in the UV portion of the electromagnetic
spectrum. Optical absorbence is commonly determined by measuring
the attenuation of impinging optical power as it passes through a
known length of material to be quantified. Alternative approaches
are possible with laser technology including photo acoustic and
photo thermal techniques. Such measurements can be utilized with
the microchip technology discussed here with the additional
advantage of potentially integrating optical wave guides on
microfabricated devices. The use of solid-state optical sources
such as LEDs and diode lasers with and without frequency conversion
elements would be attractive for reduction of system size.
Integration of solid state optical source and detector technology
onto a chip does not presently appear viable but may one day be of
interest.
[0117] Refractive index detectors have also been commonly used for
quantification of flowing stream chemical analysis systems because
of generality of the phenomenon but have typically been less
sensitive than optical absorption. Laser based implementations of
refractive index detection could provide adequate sensitivity in
some situations and have advantages of simplicity. Fluorescence
emission (or fluorescence detection) is an extremely sensitive
detection technique and is commonly employed for the analysis of
biological materials. This approach to detection has much relevance
to miniature chemical analysis and synthesis devices because of the
sensitivity of the technique and the small volumes that can be
manipulated and analyzed (volumes in the picoliter range are
feasible). For example, a 100 pL sample Volume with 1 nM
concentration of analyte would have only 60,000 analyte molecules
to) be processed and detected. There are several demonstrations in
the literature of detecting a single molecule in solution by
fluorescence detection. A laser source is often used as the
excitation source for ultrasensitive measurements but conventional
light sources such as rare gas discharge lamps and light emitting
diodes (LEDs) are also used. The fluorescence emission can be
detected by a photomultiplier tube, photodiode or other light
sensor. An array detector such as a charge coupled device (CCD)
detector can be used to image an analyte spatial distribution.
[0118] Raman spectroscopy can be used as a detection method for
microchip devices with the advantage of gaining molecular
vibrational information, but with the disadvantage of relatively
poor sensitivity. Sensitivity has been increased through surface
enhanced Raman spectroscopy (SERS) effects but only at the research
level. Electrical or electrochemical detection approaches are also
of particular interest for implementation on microchip devices due
to the ease of integration onto a microfabricated structure and the
potentially high sensitivity that car be attained. The most general
approach to electrical quantification is a conductometric
measurement, i.e., a measurement of the conductivity of an ionic
sample. The presence of an ionized analyte can correspondingly
increase the conductivity of a fluid and thus allow quantification.
Amperiometric measurements imply the measurement of the current
through an electrode at a given electrical potential due to the
reduction or oxidation of a molecule at the electrode. Some
selectivity can be obtained by controlling the potential of the
electrode but it is minimal. Amperiometric detection is a less
general technique than conductivity because not all molecules can
be reduced or oxidized within the limited potentials that can be
used with common solvents. Sensitivities in the 1 nM range have
been demonstrated in small volumes (10 nL). The other advantage of
this technique is that the number of electrons measured (through
the current) is equal to the number of molecules present. The
electrodes required for either of these detection methods can be
included on a microfabricated device through a photolithograpitic
patterning and metal deposition process. Electrodes could also be
used to initiate a chemiluminescence detection process, i.e., an
excited state molecule is generated via an oxidation-reduction
process which then transfers its energy to an analyte molecule,
subsequently emitting a photon that is detected.
[0119] Acoustic measurements can also be used for quantification of
materials but have not been widely used to date. One method that
has been used primarily for gas phase detection is the attenuation
or phase shift of a surface acoustic wave (SAW). Adsorption of
material to the surface of a substrate where a SAW is propagating
affects the propagation characteristics and allows a concentration
determination. Selective sorbents on the surface of the SAW device
are often used. Similar techniques may be useful in the devices
described herein.
[0120] The mixing capabilities of the microchip laboratory systems
described herein lend themselves to detection processes that
include the addition of one or more reagents. Derivatization
reactions are commonly used in biochemical assays. For example,
amino acids, peptides and proteins are commonly labeled with
dansylating reagents or o-phthaldialdehyde to produce fluorescent
molecules that are easily detectable. Alternatively, an enzyme
could be used as a labeling molecule and reagents, including
substrate, could be added to provide an enzyme amplified detection
scheme, i.e., the enzyme produces a detectable product. The are
many examples where such an approach has been used in conventional
laboratory procedures to enhance detection, either by absorbence or
fluorescence. A third example of a detection method that could
benefit from integrated mixing methods is chemiluminescence
detection. In these types of detection scenarios, a reagent and a
catalyst are mixed with an appropriate target molecule to produce
an excited state molecule that emits a detectable photon.
[0121] Analyte Stacking
[0122] To enhance the sensitivity of the microchip laboratory
system 10D, an analyte pre-concentration can be performed prior to
the separation. Concentration enhancement is a valuable toot
especially when analyzing environmental samples and biological
materials, two areas targeted by microchip technology. Analyte
stacking is a convenient technique to incorporate with
electrophoretic analyses. To employ analyte stacking, the analyte
is prepared in a buffer with a lower conductivity than the
separation buffer. The difference in conductivity causes the ions
in the analyte to stack at the beginning or end of the analyte
plug, thereby resulting in a concentrated analyte plug portion that
is detected more easily. More elaborate preconcentration techniques
include two and three buffer systems, i.e., transient
isotachophoretic preconcentration. It will be evident that the
greater the number of solutions involved, the more difficult the
injection technique is to implement. Pre concentration steps are
well suited for implementation on a microchip. Electroosmotically
driven flow enables separation and sample buffers to be controlled
without the use of valves or pumps. Low dead volume connections
between channels can be easily fabricated enabling fluid
manipulation with high precision, speed and reproducibility.
[0123] Referring again to FIG. 12, the pre-concentration of the
analyte is performed at the top of the separation channel 34D using
a modified gated injection to stack the analyte. First, an analyte
plug is introduced onto the separation channel 34D using
electroosmotic flow. The analyte plug is then followed by more
separation buffer from the buffer reservoir 16D. At this point, the
analyte stacks at the boundaries of the analyte and separation
buffers. Dansylated amino acids were used as the analyte, which are
anions that stack at the rear boundary of the analyte buffer plug.
Implementation of the analyte stacking is described along with the
effects of the stacking on both the separation efficiency and
detection limits.
[0124] To employ a gated injection using the microchip laboratory
system 10D, the analyte is stored in the top reservoir 12D and the
buffer is stored in the left reservoir 16D. The gated injection
used for the analyte stacking is performed on an analyte having an
ionic strength that is less than that of the running buffer. Buffer
is transported by electroosmosis from the buffer reservoir 16D
towards both the analyte waste and waste reservoirs 18D, 20D. This
buffer stream prevents the analyte from bleeding into the
separation channel 34D. Within a representative embodiment, the
relative potentials at the buffer, analyte, analyte waste and waste
reservoirs are 1. 0.9, 0.7 and 0, respectively. For 1 kV applied to
the microchip, the field strengths in the buffer, analyte, analyte
waste, and separation channels during the separation are 170, 130,
180, and 120 V/cm, respectively.
[0125] To inject the analyte onto the separation channel 34D, the
potential at the buffer reservoir 16D is floated (opening of the
high voltage switch) for a brief period of time (0.1 to 10 s), and
analyte migrates into the separation channel. For 1 kV applied to
the microchip, the field strengths in the buffer, sample, sample
waste, and separation channels during the injection are 0, 240,
120, and 110 V/cm, respectively. To break off the analyte plug, the
potential at the buffer reservoir 16D is reapplied (closing of a
high voltage switch). The volume of the analyte plug is a function
of the injection time, electric field strength, and electrophoretic
mobility.
[0126] The separation buffer and analyte compositions can be quite
different, yet with the gated injections the integrity of both the
analyte and buffer streams can be alternately maintained in the
separation channel 34D to perform the stacking operation. The
analyte stacking depends an the relative conductivity of the
separation buffer to analyte, .gamma.. For example, with a 5 mM
separation buffer and a 0.516 mM sample (0.016 mM dansyl-lysine and
0.5 mM sample buffer), .gamma. is equal to 9.7. FIG. 14 shows two
injection profiles for didansyl-lysine injected for 2 s with
.gamma. equal to 0.97 and 9.7. The injection profile with
.gamma.=0.97 (the separation and sample buffers are both 5 mM shows
no stacking. The second profile with .gamma.=9.7 shows a modest
enhancement of 3.5 for relative peak heights over the injection
with .gamma.=0.97. Didansyl-lysine is an anion, and thus stacks at
the rear boundary of the sample buffer plug. In addition to
increasing the analyte concentration, the spatial extent of the
plug is confined. The injection profile with .gamma.=9.7 has a
width at half-height of 0.41 s, while the injection profile with
.gamma.=0.97 has a width at half-height of 1.88 s. The electric
field strength in then separation channel 34D during the injection
(injection field strength) is 95% of the electric field strength in
the separation channel during the separation (separation field
strength). These profiles are measured while the separation field
strength is applied. For an injection time of 2 s, an injection
plug width of 1.9 s is expected for .gamma.=0.97.
[0127] The concentration enhancement due to stacking was evaluated
for several sample plug lengths and relative conductivities of the
separation buffer and analyte. The enhancement due to stacking
increases with increasing relative conductivities, .gamma.. In
Table 1 the enhancement is listed for g from 0.97 to 970. Although
the enhancement is largest when .gamma.=970, the separation
efficiency suffers due to an electroosmotic pressure originating at
the concentration boundary when the relative conductivity is too
large. A compromise between the stacking enhancement and separation
efficiency must be reached and .gamma.=10 has been found to be
optimal. For separations performed using stacked injections with
.gamma.=97 and 970, didansyl-lysine and dansyl-isoleucine could not
be resolved due to a loss in efficiency. Also, because the
injection process on the microchip is computer controlled, and the
column is not physically transported from vial to vial, the
reproducibility of the stacked injections is 2.1% rsd (percent
relative standard deviation) for peak area for 6 replicate
analyses. For comparisor, the non-stacked, gated injection has a
1.4% rsd for peak area for 6 replicate analyses, and the pinched
injection has a 0.75% rsd for peak area for 6 replicate analyses.
These correspond well to reported values for large-scale,
commercial, automated capillary electrophoresis instruments.
However, injections made on the microchip are .apprxeq.100 times
smaller in volume, e.g. 100 pL on the microchip versus 10 nL on a
commercial instrument.
1TABLE 1 Variation or stacking enhancement with relative
conductivity, .gamma.. .gamma. Concentration Enhancement 0.97 1 9.7
6.5 97 11.5 970 13.8
[0128] Buffer streams of different conductivities can be accurately
combined on microchips. Described herein is a simple stacking
method, although more elaborate stacking schemes can be employed by
fabricating a microchip with additional buffet reservoirs. In
addition, the leading and trailing electrolyte buffer can be
selected to enhance the sample stacking, and ultimately, to lower
the detection limits beyond that demonstrated here. It is also
noted that much larger enhancements are expected for inorganic
(elemental) cations due to the combination of field amplified
analyte injection and better matching of analyte and buffer ion
mobilities.
[0129] Regardless of whether sample stacking is used, the microchip
laboratory system 10D of FIG. 12 can be employed to achieve
electrophoretic separation of an analyte composed of rhodamine B
and sulforhodamine. FIG. 15aare electropherograms at (a) 3.3 cm,
(b) 9.9 cm, and (c) 16.5 cm from the point of injection for
rhodamine B (less retained) and sulforhodamine (more retained).
These were taken using the following conditions: injection type was
pinched, E.sub.=500V/cm, E.sub.run=170 V/cm, buffer=50 mM sodium
tetraborate at pH 9.2. To obtain electropherograms in the
conventional manner, single point detection with the helium-neon
laser (green line) was used at different locations down the axis of
the separation channel 34D.
[0130] An important measure of the utility of a separation system
is the number of plates generated per unit time, as given by the
formula
N/t=L/(Ht)
[0131] where N is the number of theoretical plates, t is the
separation time, L is the length of the separation column, and H is
the height equivalent to a theoretical plate. The plate height, H,
can be written as
H=A+B/u
[0132] where A is the sum of the contributions from the injection
plug length and the detector path length, B is equal to 2D.sub.,
where D.sub.is the diffusion coefficient for the analyte in the
buffer, and u is the linear velocity of the analyte.
[0133] Combining the two equations above and substituting u=.mu.E
where .mu. is the effective electrophoretic mobility of the analyte
and E is the electric field strength, the plates per unit time can
be expressed as a function of the electric field strength:
N/t=(.mu.E).sup.2/(A.mu.E+B)
[0134] At low electric field strengths when axial diffusion is the
dominant form of band dispersion, the A.mu.ME is small relative to
B and consequently, the number of plates per second increases with
the square of the electric field strength.
[0135] As the electric field strength increases, the plate height
approaches a constant value, and the plates per unit time increases
linearly with the electric field strength because B is small
relative to A.mu.E. It is thus advantageous to have A as small as
possible, a benefit of the pinched injection scheme.
[0136] The efficiency of the electrophorectic separation of
rhodamine B and sulforhodamine at ten evenly spaced positions was
monitored, each constituting a separate experiment. At 16.5 cm from
the point of injection the efficiencies of rhodamine B and
sulforhodamine are 38,100 and 29,000 plates, respectively.
Efficiencies of this magnitude are sufficient for many separation
applications. The linearity of the data provides information about
the uniformity and quality of the channel along its length. If a
defect in the channel, e.g., a large-pit, was present a sharp
decrease in the efficiency would result; however, none was
detected. The efficiency data are plotted in FIG. 16 (conditions
for FIG. 16 were the same as for FIG. 15).
[0137] A similar separation experiment was performed using the
microchip analyte injector 10B of FIG. 6. Because of the straight
separation channel 34B, the analyte injector 10B enables faster
separations than are possible using the serpentine separation
channel 34D of the alternate analyte injector 10D should in FIG.
12. In addition, the electric field strengths used were higher (470
V/cm and 100 V/cm for the buffer and separation channels 26B, 34B,
respectively), which further increased the speed of the
separations.
[0138] One particular advantage to the planar microchip laboratory
system 10B of the present invention is that with laser induced
fluorescence the poi it of detection can be placed anywhere along
the separation column. The electropherograms are detected at
separation lengths of 0.9 mm, 1.6 mm and 11.1 mm from the injection
intersection 40B. The 1.6 mm and 11.1 mm separation lengths were
used over a range of electric field strengths from 0.06 to 1.5
kV/cm, and the separations had baseline resolution over this range.
At an electric field strength of 1.5 kV/cm, the analytes, rhodamine
B and fluorescein, are resolved in less than 150 ms for the 0.9 mm
separation length, as shown in FIG. 17(a), in less than 260 ms for
the 1.6 mm separation length as shown in FIG. 17(b), and in less
than 1.6 seconds for the 11.1 mm separation length, as shown in
FIG. 17(c).
[0139] Due to the trapezoidal geometry of the channels, the upper
corners make it difficult to cut the sample plug away precisely
when the potentials are switched from the sample loading mode to
the separation mode. Thus, the injection plug has a slight tail
associated with it, and this effect probably accounts for the
tailing observed in the separated peaks.
[0140] In FIG. 18, the number of plates per second for the 1.6 mm
and 11.1 mm separation lengths are plotted versus the electric
field strength. The number of plates per second quickly becomes a
linear function of the electric field strength, because the plate
height approaches a constant value. The symbols in FIG. 18
represent the experimental data collected for the two analytes at
the 1.6 mm and 11.1 mm separation lengths. The lines are calculated
using the previously-stated equation and the coefficients arc
experimentally determined. A slight deviation is seen between the
experimental data and the calculated numbers for rhodamine B at the
11.1 mm separation length. This is primarily due to experimental
error.
[0141] Electrochromatography
[0142] A problem with electrophoresis for general analysis is its
inability to separate uncharged species. All neutral species in a
particular sample will have zero electrophoretic mobility, and
thus, the same migration time. The microchip analyte injector 10D
shown in FIG. 12 can also be used to perform electrochromatography
to separate non-ionic analytes. To perform such
electrochromaography, the surface of the separation channel 34D was
prepared by chemically bonding a reverse phase coating to the walls
of the separation channel after bonding the cover plate t.) the
substrate to enclose the channels. The separation channel was
treated with 1 M sodium hydroxide and then rinsed with water. The
separation channel was dried at 125.degree. C. for 24 hours while
purging with helium at a gauge pressure of approximately 50 kPa. A
25% (w/w) is solution of chlorodimethyloctaldecylsilane (ODS,
Aldrich) in toluene was loaded into the separation channel with an
over pressure of helium at approximately 90 kPa. The ODS/toluene
mixture was pumped continuously into the column throughout the 18
hour reaction period at 125.degree. C. The channels are rinsed with
toluene and then with acetonitrile to remove the unreacted ODS. The
laboratory system 10D was used to perform electrochromatography on
an analytes composed of coumarin 440 (C440), coumarin 450 (C450)
and coumarin 460 (C460; Exciton Chemical Co., Inc.) at 10 .mu.M for
the direct fluorescent measurements of the separations and 1 .mu.M
for the indirect fluorescent measurements of the void time. A
sodium tetraborate buffer (10 mM, pH 9.2) with 25% (v/v)
acetonitrile was the buffer.
[0143] The analyte injector 10D was operated under a pinched
analyte loading mode and a separation (run) mode as described above
with respect to FIG. 6. The analyte is loaded into the injection
cross via a frontal chromatogram traveling from the analyte
reservoir 16D to the analyte waste reservoir 18D, and once the
Front of the slowest analyte passes through the injection
intersection 40D, the sample is ready to be analyzed. To switch to
the separation mode, the applied potentials sire reconfigured, for
instance by manually throwing a switch. After switching the applied
potentials, the primary flow path for the separation is from the
buffer reservoir 12D to the waste reservoir 20D. In order to inject
a small analyte plug into the separation channel 34D and to prevent
bleeding of the excess analyte into the separation channel, the
analyte and the analyte waste reservoirs 16D, 18D are maintained at
57% of the potential applied to the buffer reservoir 12D. This
method of loading and injecting the sample is time-independent,
non-biased and reproducible.
[0144] In FIG. 19, a Chromatogram of the coumarins is shown for a
linear velocity of 0.65 mm/s. For C440, 11700 plates was observed
which corresponds to 120 plates/s. The most retained component,
C460, has an efficiency nearly an order of magnitude lower than for
C440, which was 1290 plates. The undulating background in the
chromatograms is due to background fluorescence from the glass
substrate and shows the power instability of the laser. This,
however, did not hamper the quality of the separations or
detection. These results compare quite well with conventional
laboratory High Performance LC (HPLC) techniques in terms of plate
numbers and exceed HPLC in speed by a factor of ten. Efficiency is
decreasing with retention faster than would be predicted by theory.
This effect may be due to overloading of the monolayer stationary
or kinetic effects due to the high speed of the separation.
[0145] Micellar Electrokinetic Capillary Chromatography
[0146] In the elcctrochromatography experiments discussed above
with respect to FIG. 19, sample components were separated by their
partitioning interaction with a stationary phase coated on the
channel walls. Another method of separating neutral analytes is
micellar electrokinetic capillary chromatography (MECC). MECC is an
operational mode of electrophoresis in which a surfactant such as
sodium dodecylsulfate (SDS) is added to the buffer in sufficient
concentration to form micelles in the buffer. In a typical
experimental arrangement, the micelles move much more slowly toward
the cathode than does the surrounding buffer solution. The
partitioning of solutes between the micelles and the surrounding
buffer solution provides a separation mechanism similar to that of
liquid chromatography.
[0147] The microchip laboratory 10D of FIG. 12 was used to perform
on an analyte composed of neutral dyes coumarin 440 (C440),
coumarin 450 (C450), and coumarin 460 (C460, Exciton Chemical Co.,
Inc.). Individual stock solutions of each dye were prepared in
methanol, then diluted into the analysis buffer before use. The
concentration of each dye was approximately 50 .mu.M unless
indicated otherwise. The MECC buffer was composed of 10 mM sodium
borate (pH 9.1), 50 mM SDS, and 10% (v/v) methanol. The methanol
aids in solubilizing the coumarin dyes in the aqueous buffer system
and also affects the partitioning of some of the dyes into the
micelles. Due care must be used in working with coumarin dyes as
the chemical, physical, and toxicological properties of these dyes
have not been fully investigated.
[0148] The microchip laboratory system 10D was operated in the
"pinched injection" mode described previously. The voltages applied
to the reservoirs are set to either loading mode or a "run"
(separation) mode. In the loading mode, a frontal chromatogram of
the solution in the analyte reservoir 16D is pumped
electroosmotically through the intersection and into the analyte
waste reservoir 18D. Voltages applied to the buffer and waste
reservoirs also cause weak flows into the intersection from the
sides, and then into the analyte waste reservoir 18D. The chip
remains in this mode until the slowest moving component of the
analyte has passed through the intersection 40D. At this point, the
analyte plug in the intersection is representative of tile analyte
solution, with no electrokinetic bias.
[0149] An injection is made by switching the chip to the "run" mode
which changes the voltages applied to the reservoirs such that
buffer now flows from the buffer reservoir 12D through the
intersection 40D into the separation channel 34D toward the waste
reservoir 20D. The plug of analyte that was in the intersection 40D
is swept into the separation channel 34D. Proportionately lower
voltages are applied to the analyte and analyte waste reservoirs
16D, 18D to cause a weak flow of buffer from the buffer reservoir
12D into these channels. These flows ensure that the sample plug is
cleanly "broken off" from the analyte stream, and that no excess
analyte leaks into the separation channel during the analysis.
[0150] The results of the MECC analysis of a mixture of C440, C450,
and C460 are shown in FIG. 20. The peaks were identified by
individual analyses of each dye. The migration time stability of
the first peak, C440, with changing methanol concentration was a
strong indicator that this dye did not partition into the micelles
to a significant extent. Therefore it was considered an
electroosmotic flow marker with migration time t0. The last peak,
C460, was assumed to be a marker for the micellar migration time,
tm. Using these values of t0 and trn from the data in FIG. 20, the
calculated elution range, t0/tm, is 0.43. This ads well with a
literature value of t0/tm=0.4 for a similar buffer system and
supports our assumption. These results compare well with
conventional MECC performed in capillaries and also shows some
advantages over the electrochromatography experiment described
above in that efficiency is retained with retention ratio. Further
advantages of this approach to separating neutral species is that
no surface modification of the walls is necessary and that the
stationary phase is continuously refreshed during experiments.
[0151] Inorganic Ion Analysis
[0152] Another laboratory analysis that can be performed on either
the laboratory system 10B of FIG. 6 or the laboratory system 10D of
FIG. 12 is inorganic ion analysis. Using the laboratory system 10B
of FIG. 6, inorganic ion analysis was performed on metal ions
complexed with 8-hydroxyquinoline-5-sulfonic acid (HQS) which are
separated by electrophoresis and detected with UV laser induced
fluorescence. HQS has been widely used as a ligand for optical
determinations of metal ions. The optical properties and the
solubility of HQS in aqueous media have recently been used for
detection of metal ions separated by ion chromatography and
capillary electrophoresis. Because uncomplexed HQS does not
fluoresce, excess ligand is added to the buffer to maintain the
complexation equilibria during the separation without contributing
a large background signal. This benefits both the efficiency of the
separation and detectability of the sample. The compounds used for
the experiments arc zinc sulfate, cadmium nitrate, and aluminum
nitrate. The buffer is sodium phosphate (60 mM, pH 6.9) with
8-hydroxyquinoline-5-sulf- onic acid (20 mM for all experiments
except FIG. 5; Sigma Chemical Co.). At least 50 mM sodium phosphate
buffer is needed to dissolve up to 20 mM HQS. The substrate 49B
used was fused quartz, which provides greater visibility than glass
substrates.
[0153] The floating or pinched analyte loading, as described
previously with respect to FIG. 6, is used to transport the analyte
to the injection intersection 40B. With the floating sample
loading, the injected plug has no electrophoretic bias but the
volume of sample is a function of the sample loading time Because
the sample loading time is inversely proportional to the field
strength used, for high injection field strengths a shorter
injection time is used than for low injection field strengths. For
example, for an injection field strength of 630 V/cm (FIG. 3a), the
injection time is 12 s, and for an injection field strength of 520
V/cm (FIG. 3b), the injection time is 14.5 s. Both the pinched and
floating sample loading can be used with and without suppression of
the electroosmotic flow.
[0154] FIGS. 21(a) and 21(b) show the separation of three metal
ions complexed with 8-hydroxyquinoline-5-sulfonic acid. All three
complexes have a net negative charge. With the electroosmotic flow
minimized by the covalent bonding of polyacrylamide to the channel
walls, negative potentials relative to ground are used to
manipulate the complexes during sample loading and separation. In
FIGS. 21(a) and 21(b), the separation channel field strength is 870
and 720 V/cm, respectively, and the separation length is 16.5 mm.
The volume of the injection plug is 120 pL which corresponds to 16,
7, and 19 fmol injected for Zn, Cd, and Al, respectively, for FIG.
4a. In FIG. 4b, 0.48, 0.23, and 0.59 fmol of Zn, Cd, and Al,
respectively, are injected onto the separation column. The average
reproducibility of the amounts injected is 1.6% rsd (percent
relative standard deviation) as measured by peak areas (6 replicate
analyses). The stability of the laser used to excite the complexes
is 1% rsd. The detection limits are in a range where useful
analyses can be performed.
[0155] Post-Separation Channel Reactor
[0156] An alternate microchip laboratory system 10E is shown in
FIG. 22. The five-port pattern of channels is disposed on a
substrate 49E and with a cover slip 49E as in the
previously-described embodiments. The microchip laboratory system
10E embodiment was fabricated using standard photolithographic, wet
chemical etching, and bonding techniques. A photomask was
fabricated by sputtering chrome (50 nm) onto a glass slide and
ablating the channel design into the chrome film via a CAD/CAM
laser ablation system (Resoneties, Inc.). The channel design was
then transferred onto the substrates using a positive photoresist.
The channels were etched into the substrate in a dilute
Hf/Nh.sub.4F bath. To form the separation channel 34E a cover late
was bonded to the substrate over the etched channels using a direct
bonding technique. The surfaces were hydrolyzed in dilute
NH.sub.4OH/H.sub.2O.sub.2 solution, rinsed in deionized, filtered
H.sub.2, joined and then annealed at 500.degree. C. Cylindrical
glass reservoirs were affixed on the substrate using RTV silicone
(made by General Electric). Platinum electrode; provided electrical
contact from the voltage controller 46E (Spellman CZE1000R) to the
solutions in the reservoirs.
[0157] The channel 26E is in one embodiment 2.7 mm in length from
the first reservoir 12E to the intersection 40E, while the channel
30E is 7.0 mm, and the third channel 32E is 6.7 mm. The separation
channel 34E is modified to, be only 7.0 mm in length, due to the
addition of a reagent reservoir 22E which has a reagent channel 36E
that connects to the separation channel 34E at a mixing tee 44E.
Thus, the length of the separation channel 34E is measured from the
intersection 40E to the mixing tee 44E. The channel 56 extending
from the mixing tee 44E to the waste reservoir 20E is the reaction
column or channel, and in the illustrated embodiment this channel
is 10.8 mm in length. The length of the reagent channel 36E is 11.6
mm.
[0158] In a representative example, the FIG. 22 embodiment was used
to separate an analyte and the separation was monitored
on-microchip via fluorescence using an argon ion laser (351.1 nm,
50 mW, Coherent Innova 90) for excitation. The fluorescence signal
was collected with a photomultiplier tube (Pry, Oriel 77340) for
point detection and a charge coupled device (CCD, Princeton
Instruments, Inc. TE/CCD-512TKM) for imaging a region of the
microchip 90. The compounds used for testing the apparatus were
rhodamine B (Exciton Chemical Co., Inc.) arginine, glycine
threonine and o-phthaldialdehyde (Sigma Chemical Co.). A sodium
tetraborate buffer (20 A, pH 9.2) with 2% (v/v) methanol and 0.5%
(v/v) .beta.-mercaptoethanol was the buffer in all tests. The
concentrations of the amino acid, OPA and rhodamine ES solutions
were 2 mM, 3.7 mM, and 50 .mu.M, respectively. Several run
conditions were utilized.
[0159] The schematic view in FIG. 23 demonstrates one an example
when 1 kV is applied to the entire system. With this voltage
configuration, the electric field strengths in the separation
channel 34E (E.sub.scp) and the reaction channel 36E (E.sub.rxa)
are 200 and 425 V/cm, respectively. This allows the combining of 1
part separation effluent with 1.125 parts reagent at the mixing tee
44E. An analyte introduction system such as this, with or without
post-column reaction, allows a very rapid cycle time for multiple
analyses.
[0160] The electropherograms; (A) and (B) in FIG. 2c demonstrate
the separation of two pairs of amino acids. The voltage
configuratior is the same as in FIG. 23, except the total applied
voltage is 4 kV which corresponds; to an electric field strength of
800 V/cm in the separation column (E.sub.scp) and 1,700 V/cm in the
reaction column (E.sub.rxa). The injection times were 100 ms for
the tests which correspond to estimated injection plug lengths of
384, 245, and 225 .mu.m for arginine, glycine and threonine,
respectively. The injection volumes of 102, 65, and 60 pL
correspond to 200, 130, and 120 fmol injected for arginine, glycine
and threonine, respectively. The point of detection is 6.5 mm
downstream from the mixing tee which gives a total column length of
13.5 mm for the separation and reaction.
[0161] The reaction rates of the amino acids with the OPA are
moderately fast, but not fast enough on the time scale of these
experiments. An increase in the band distortion is observed because
the mobilities of the derivatized compounds are different from the
pure amino acids. Until the reaction is complete, the zones of
unreacted and reacted amino acid will move at different velocities
causing a broadening of the analyte zone. As evidenced in FIG. 24,
glycine has the greatest discrepant in electrophoretic mobilities
between the derivatized and un-derivatized amino acid. To ensure
that the excessive band broadening was not a function of the
retention time, threonine was also tested. Threonine has a slightly
longer retention time than the glycine; however the broadening is
not as extensive as for glycine.
[0162] To test the efficiency of the microchip in both the
separation column and the reaction column, a fluorescent laser dye,
rhodamine B, was used as a probe Efficiency measurements calculated
from peak widths at half height were made using the point detection
scheme at distances of 6 mm and 8 mm from the injection criss, or 1
mm upstream and 1 mm downstream from the mixing tee. This provided
information on the effects of the mixing of the two streams.
[0163] The electric field strengths in the reagent column and the
separation column were approximately equal, and the field strength
in the reaction column was twice that of the separation column.
This configuration of the applied voltages allowed an approximately
1:1 volume ratio of derivatizing reagent and effluent from the
separation column. As the field strengths increased, the degree of
turbulence at the mixing tee increased. At the separation distance
of 6 mm (1 mm upstream from the mixing tee), the plate height as
expected as the inverse of the linear velocity of the analyte. At
the separation distance of 8 mm (1 mm upstream from the mixing
tee), the plate height data decreased as expected as the inverse of
the velocity of the analyze. At the separation distance of 8 mm (1
mm downstream from the mixing tee), the plate height data decreases
from 140 V/cm to 280 V/cm to 1400 V/cm- This behavior is abnormal
and demonstrates a band broadening phenomena when two streams of
equal volumes converge. The geometry of the mixing tee was not
optimized to minimize this band distortion. Above separation field
strength of 840 V/cm, the system stabilize and again the plate
height decreases with increasing linear velocity. For E.sub.scp
=1400 V/cm, the ratio of the plate heights at the 8 mm and 6 mm
separation lengths is 1.22 which is not an unacceptable loss in
efficiency for the separation.
[0164] The intensity of the fluorescence signal generated from the
reaction of OPA with an amino acid was tested by continuously
pumping glycine down the separation channel to mix with the OPA at
the mixing tee. The fluorescence signal from the OPA/amino acid
reaction was collected using a CCD as the product moved downstream
from the mixing tee. Again, the relative volume ratio of the OPA
and glycine streams was 1.125. OPA has a typical half-time of
reaction with amino acids of 4 s. The average residence times of an
analyte molecule in the window of observation are 4.68, 2.34, 1.17,
and 0.58 s for the electric field strengths in the reaction column
(E) of 240, 480, 960, and 1920 V/cm, respectively. The relative
intensities of the fluorescence correspond qualitatively to this 4
s half-time of reaction As the field strength increases in the
reaction channel, the slope and maximum of the intensity of the
fluorescence shifts further downstream because the glycine and OPA
arm swept away from the mixing tee faster with higher field
strengths. Ideally, the (observed fluorescence from the product
would have a step function of a response following the mixing of
the separation effluent and derivatizing reagent. However, the
kinetics of the reaction and a finite rate of mixing dominated by
diffusion prevent this from occurring.
[0165] The separation using the post-separation channel reactor
employed a gated injection scheme in order to keep the analyte,
buffer and reagent streams isolated as discussed above with respect
to FIG. 3. For the post-separation channel reactions, the microchip
was operated in a continuous analyte loading/separation mode
whereby the analyte was continuously pumped from the analyte
reservoir 12E through the injection intersection 40E toward the
analyte waste reservoir 18E. Buffer was simultaneously pumped from
the buffer reservoir 16E toward the analyte waste and waste
reservoirs 18E, 20E to deflect the analyte stream and prevent the
analyte from migrating down the separation channel. To inject a
small aliquot of analyte, the potentials at the buffer and analyte
waste reservoirs 16E, 18E are simply floated for a short period of
time (.apprxeq.100 ms) to allow the analyte to migrate down the
separation channel as an analyte injection plug. To break off the
injection plug, the potentials at the buffer and analyte waste
reservoirs 16E, 18E are reapplied.
[0166] The use of micromachined post-column reactors can improve
the power of post-separation channel reactions as an analytical
tool by minimizing the volume of the extra-channel plumbing,
especially between the separation and reagent channels 34E, 36E.
This microchip design (FIG. 22) was fabricated with modest lengths
for the separation channel 34E (7 mm) and reagent channel 36E (10.8
mm) which were more than sufficient for this demonstration Longer
separation channels can be manufactured on a similar size microchip
using a serpentine path to perform more difficult separations as
discussed above with respect to FIG. 12. To decrease post-mixing
tee band distortions, the ratio of the channel dimensions between
the separation channel 34E and reaction channel 56 should be
minimized so that the electric field strength in the separation
channel 34E is large, i.e. narrow channel, and in the reaction
channel 56 is small i.e., wide channel.
[0167] For capillary separation systems, the small detection
volumes can limit the number of detection schemes that can be used
to extract information. Fluorescence detection remains one of the
most sensitive detection techniques for capillary electrophoresis.
When incorporating fluorescence detection into a system that does
not have naturally fluorescing analytes, derivatization of the
analyte must occur either pre- or post-separation. When the
fluorescent "tag" is short lived or the separation is hindered by
pre-separation derivatization, post-column addition of derivatizing
reagent becomes the method of choice. A variety of post-separation
reactors have been demonstrated for capillary electrophoresis.
However, the ability to construct a post-separation reactor with
extremely low-volume connections to minimize band distortion has
been difficult. The present invention takes the approach of
fabricating a microchip device for electrophoretic separations with
an integrated post-separation reaction channel 56 in a single
monolithic device enabling extremely low volume exchanges between
individual channel functions.
[0168] Pre-Separation Channel Reaction System
[0169] Instead of the post-separation channel reactor design shown
in FIG. 22, the microchip laboratory system 10F shown in FIG. 25
includes a pre-separation channel reactor. The pre-separation
channel reactor design shown in FIG. 25 is similar to that shown in
FIG. 1, except that the first and second channels 26F, 28F form a
"goal-post" design with the reaction chamber 42F rather than the
"Y" design of FIG. 1. The reaction chamber 42F was designed to be
wider than the separation channel 34F to give lower electric field
strengths in the reaction chamber and thus longer residence times
for the reagents. The reaction chamber is 96 .mu.m width at
half-depth and 6.2 .mu.m deep, and the separation channel 34F is 31
.mu.m wide at half-depth and 6.2 .mu.m deep.
[0170] The microchip laboratory system 10F was used to perform
on-line pre-separation channel reactions coupled with
electrophoretic analysis of the reaction products. Here, the
reactor is operated continuously with small aliquots introduced
periodically into the separation channel 34F using the gated
dispenser discussed above with respect to FIG. 3. The operation of
the microchip consists of three elements: the derivatization of
amino acids with o-phthaldialdehyde (OPA), injection of the sample
onto the separation column, and the separation/detection of the
components of the reactor effluent. The compounds used for the
experiments were arginine (0.48 mM), glycine (0.58 mM), and OPA
(5.1 mM; Sigma Chemical Co.) The buffer in all of the reservoirs
was 20 mM sodium tetraborate with 2% (v/v) methanol and 0.5% (v/v)
2-mercaptoethanol. 2-mercaptoethanol is added to the buffer as a
reducing agent for the derivatization reaction.
[0171] To implement the reaction the reservoirs 12F, 14F, 16F, 18F,
and 20F were simultaneously given controlled voltages of 0.5 HV,
0.5 HV, HV. 0.2 HV, and ground, respectively. This configuration
allowed the lowest potential drop across the reaction chamber 42F
(25 V/cm for 1.0 kV applied to the microchip) and highest across
the separation channel 34F (300 V/cm for 1.0 kV applied to the
microchip) without significant bleeding of the product into the
separation channel when using the gated injection scheme. The
voltage divider used to establish the potentia's applied to each of
the reservoirs had a total resistance of 100 M.OMEGA. with 10
M.OMEGA. divisiors. The analyte from the first reservoir 12F and
the reagent from the second reservoir 14F are electroosmotically
pumped into the reaction chamber 42F with a volumetric ratio of
1:1.06. Therefore, the solutions from the analyte and reagent
reseivoirs 12F, 14F are diluted by a factor of .apprxeq.2. Buffer
was simultaneously pumped by electroosmosis from the buffer
reservoir 16F toward the analyte waste and waste reservoirs 18F,
20F. This buffer strum prevents the newly formed product from
bleeding into the separation channel 34F.
[0172] Preferably, a gated injection scheme, described above with
respect to FIG. 3, is used to inject effluent from the reaction
chamber 42F into the separation channel 34F. The potential at the
buffer reservoir 16F is simply floated for a brief period of time
(0.1 to 1.0 s), and sample migrates into the separation channel
34F. To break off the injection plug, the potential at the buffer
reservoir 16F is reapplied. The length of the injection plug is a
function of both the time of the injection and the electric field
strength. With this configuration of applied potentials, the
reaction of the amino acids with the OPA continuously generate
fresh product to be analyzed.
[0173] A significant shortcoming of many capillary electrophoresis
experiments has been the poor reproducibility of the injections.
Here, because thc microchip injection process is computer
controlled, and the injection process involves the opening of a
single high voltage switch, the injections can be accurately timed
events. FIG. 26 shows the reproducibility of the amount injected
(percent relative standard deviation % 'rsd, for the integrated
areas of the peaks) for both arginine and glycine at injection
field strengths of 0.6 and 1.2 kV/cm and injection times ranging
from 0.1 to 1.0 s. For injection times greater than 0.3 s, the
percent relative standard deviation is below 1.8%. This is
comparable to reported values for commercial, automated capillary
electrophoresis instruments. However, injections made on the
microchip arc .apprxeq.100 times smaller in volume, e.g. 100 pL on
the microchip versus 10 nL on a commercial instrument. Part of this
fluctuation is due to the stability of the laser which is
.apprxeq.0.6%. For injection times >0.3 s, the error appears to
be independent of the compound injected and the injection field
strength.
[0174] FIG. 27 shows the overlay of three electrophoretic
separations of arginine and glycine after on-microchip pre-column
derivatization with OPA with a separation field strength of 1.8
kV/cm and a separation length of 10.mm The separation field
strength is the electric field strength in the separation channel
34F during the separation. The field strength in the reaction
chamber 42F is 150 V/cm. The reaction times for the analytes is
inversely related to their mobilities e.g., for arginine the
reaction time is 4.1 s and for glycine the reaction time is 8.9 s.
The volumes of the injected plugs were 150 and 71 pL for arginine
and glycine, respectively, which correspond to 35 and 20 fmol of
the amino acids injected onto the separation channel 34F. The gated
injector allows rapid sequential injections to be made. In this
particular case, an analysis could be performed every 4 s. The
observed electrophoretic mobilities for the compounds are
determined by a linear fit to the variation of the linear velocity
with the separation field strength. The slopes were 29.1 and 13.3
mm.sup.2/(kV-s) for arginine and glycine, respectively. No evidence
of Joule heating was observed as indicated by the linearity of the
velocity versus field strength data, A linear fit produced
correlation coefficients of 0.999 for arginine and 0.996 for
glycine for separation field strengths from 0.2 to 2.0 kV/cm.
[0175] With increasing potentials applied to the microchip
laboratory system 10F, the field strengths in the reaction chamber
42F and separation channel 34F increase. This leads to shorter
residence times of the reactants in the reaction chamber and faster
analysis times for the products. By varying the potentials applied
to the microchip, the reaction kinetics can be studied. The
variation in amount of product generated with reaction time is
plotted in FIG. 28. The response is the integrated area of the peak
corrected for the residence time in the detector observation window
and photobleaching of the product. The offset between the data for
the arginine and the glycine in FIG. 28 is due primarily to the
difference in the amounts injected, i.e. different electrophoretic
mobilities, for the amino acids. A ten-fold excess of OPA was used
to obtain pseudo-first order reaction conditions. The slopes of the
lines fitted to the data correspond to the rates of the
derivatization reaction. The slopes are 0.13 s.sup.-1 for arginine
and 0.11 s.sup.-1 for glycine corresponding to half-times of
reaction of 5.1 and 6.2 s, respectively. These half-times of
reaction are comparable to the 4 s previously reported for alanine.
We have found no previously reported data for arginine or
glycine.
[0176] These results show the potential power of integrated
microfabricated systems for performing chemical procedures. The
data presented in FIG. 28 can be produced under computer control
within five approximately five minutes consuming on the order of
100 nL of reagents. These results are unprecedented in terms of
automation, speed and volume for chemical reactions
[0177] DNA Analysis
[0178] To demonstrate a useful biological analysis procedure, a
restriction digestion and electrophoretic sizing experiment arc
performed sequentially on the integrated biochemical
reactor/electrophoresis microchip system 10G shown in FIG. 29. The
microchip laboratory system 10G is identical to tie laboratory
system shown in FIG. 25 except that the separation channel 34G of
the laboratory system 10G follows a serpentine path. The sequence
for plasmid pBP322 and the recognition sequence of the enzyme Hinf
I are known. After digestion, determination of the fragment
distribution is performed by separating the digestion products
using electrophoresis in a sieving medium in the separation channel
34G. For these experiments, hydroxyethyl cellulose is used as the
sieving medium. At a fixed point downstream in the separation
channel 34G, migrating fragments are interrogated using on-chip
laser induced fluorescence with an intercalating dye, thiazole
orange dimer (TOTO-1), as the fluorophore.
[0179] The reaction chamber 42G and separation channel 34G shown in
FIG. 29 are 1 and 67 mm long, respectively, having a width at
half-depth of 60 .mu.m and a depth of 12 .mu.m. In addition, the
channel walls are coated with polyacrylamide to minimize
electroosmotic flow and adsorption. Electropherograms are generated
using single point detection laser induced fluorescence detection.
An argon ion laser (10 mW) is focused to a spot onto the chip using
a lens (100 mm focal length) The fluorescence signal is collected
using a 21.times. objective lens (N.A.=0.42), followed by spatial
filtering (0.6 mm diameter pinhole) and spectral filtering (560 nm
bandpass, 40 nm bandwidth), and measured using a photomultiplier
tube (PMT). The data acquisition and voltage switching apparatus
are computer controlled. The reaction buffer is 10 mM Tris-acetate,
10 mM magnesium acetate, and 50 mM potassium acetate. The reaction
buffer is placed in the DNA, enzyme and waste 1 reservoirs 12G,
14G, 18G shown in FIG. 29. The separation buffer is 9 mM
Tris-borate with 0.2 mM EDTA and 1% (w/v) hydroxyethyl cellulose.
The separation buffer is placed in the buffer and waste 2
reservoirs 16F, 20F. The concentrations of the plasmid pBR322 and
enzyme Hinf I are 125 ng/.mu.t and 4 units/.mu.l, respectively. The
digestions and separations are performed at room temperature
(20.degree. C.).
[0180] The DNA and enzyme are electrophoretically loaded into the
reaction chamber 42G from their respective reservoirs 12G, 14G by
application of proper electrical potentials. The relative
potentials at the DNA (12G), ceze (14G), buffer (16G), waste 1
(18G), and waste 2 (20G) reservoirs are 10%, 10%, 0, 30%, and 100%,
respectively. Due to the electrophoretic mobility differences
between the DNA and enzyme, the loading period is made sufficiently
long to reach equilibrium. Also, due to the small volume of the
reaction chamber 42G, 0.7 nL, rapid diffusional mixing occurs. The
electroosmotic flow is minimized by the covalent immobilization of
linear polyacrylamide, thus only anions migrate from the DNA and
enzyme reservoirs 12G, 14G into the reaction chamber 42G with the
potential distributions used. The reaction buffer which contains
cations, required for the enzymatic digestions, e.g. Mg.sup.2+, is
also placed in the waste 1 reservoir 18G. This enables the cations
to propagate into the reaction chamber countercurrent to the DNA
and enzyme during the loading of the reaction chamber. The
digestion is performed statically by removing all electrical
potentials after loading the reaction chamber 42G due to the
relatively short transit time of the DNA through the reaction
chamber.
[0181] Following the digestion period, the products are migrated
into the separation channel 34F for analysis by floating the
voltages to the buffer and waste 1 reservoirs 16F, 18F. The
injection has a mobility bias where the smaller fragments are
injected in favor of the larger fragments. In these experiments the
injection plug length for the 75-base pair (bp) fragment is
estimated to be 0.34 mm whereas for the 1632-bp fragment only 0.22
mm. These plug lengths correspond to 34% and 22% of the reaction
chamber volume, respectively. The entire contents of the reaction
chamber 42F cannot be analyzed under current separation conditions
because the contribution of the injection plug length to the plate
height would be overwhelming.
[0182] Following digestion and injection onto the separation
channel 34F, the fragments are resolved using 1.0% (w/v)
hydroxyethyl cellulose as the sieving medium. FIG. 30 shows an
electropherogram of the restriction fragments of the plasmid pBR322
following a 2 min digestion by the enzyme Hinf I. To enable
efficient on-column staining of the double-stranded DNA after
digestion but pr or to interrogation, the intercalating dye, TOTO-1
(1 .mu.M), is placed in the waste 2 reservoir 20G only and migrates
countercurrent to the DNA. As expected, the relative intensity of
the bands increases with increasing fragment size because more
intercalation sites exist in the larger fragments. The unresolved
220/221 and 507/511-bp fragments having higher intensities than
adjacent single fragment peaks due to the band overlap. The
reproducibility of the migration times and injection volumes are
0.55 and 3.1% relative standard deviation (%rsd), respectively, for
5 replicate analyses.
[0183] This demonstration of a microchip laboratory system 10G that
performs plasmid DNA restriction fragment analysis indicates the
possibility of automating and miniaturizing more sophisticated
biochemical procedures This experiment represents the most
sophisticated integrated microchip chemical analysis device
demonstrated to date. The device mixes a reagent with an analyte,
incubates the analyte/reagent mixture, labels the products, and
analyzes the products entirely under computer control while
consuming 10,000 times less material than the typical small volume
laboratory procedure.
[0184] In general, the present invention can be used to mix
different fluids contained in different ports or reservoirs. This
could be used for a liquid chromatography separation experiment
followed by post-column labeling reactions in which different
chemical solutions of a given volume are pumped into the primary
separation channel and other reagents or solutions can be injected
or pumped into the stream at different times to be mixed in precise
and known concentrations To execute this process, it is necessary
to accurately control and manipulate solutions in the various
channels.
[0185] Pre-/Post-Separation Reactor System
[0186] FIG. 31 shows the same six port microchip laboratory system
10 shown in FIG. 1, which could take advantage of this novel mixing
scheme. Particular features attached to the different ports
represent solvent reservoirs. This laboratory system could
potentially be used for a liquid chromatography separation
experiment followed by post-column labeling reactions In such an
experiment, reservoirs 12 and 14 would contain solvents to be used
in a liquid chromatography solvent programming type of separation,
e.g., water and acetonitrile.
[0187] The channel 34 connected to the waste reservoir 20 and to
the two channels 26 and 28 connecting the analyte and solvent
reservoirs 12 and 14 is the primary separation channel, i.e., where
the liquid chromatography experiment would take place. The
intersecting channels 30, 32 connecting the buffer and analyte
waste reservoirs 16 and 18 are used to make an injection into the
liquid chromatography or separation channel 34 as discussed above.
Finally, reservoir 22 and its channel 36 attaching to the
separation channel 34 are used to add a reagent, which is added in
proportions to render the species separated in the separation
channel detectable.
[0188] To execute this process, it is necessary to accurately
control and manipulate solutions in the various channels. The
embodiments described above took very small volumes of solution
(.apprxeq.100 pl) from reservoirs 12 and 40 and accurately injected
them into the separation channel 34. For these various scenarios, a
given volume of solution needs to be transferred from one channel
to another. For examples solvent programming for liquid
chromatography or reagent addition for post-column labeling
reactions requires that streams of solutions be mixed in precise
and known concentrations.
[0189] The mixing of various solvents in known proportions can be
done according to the present invention by controlling potentials
which ultimately control electroosmotic flows as indicated in
equation l. According to equation 1 the electric field strength
needs to be known to determine the linear velocity of the solvent.
In general, in these types of fluidic manipulations a known
potential or voltage is applied to a given reservoir. The field
strength can be calculated from the applied voltage and the
characteristics of the channel. In addition, the resistance or
conductance of the fluid in the channels must also be known.
[0190] The resistance of a channel is given by equation 2 where R
is the resistance, .kappa. is the resistivity, L is the length of
the channel, and A is the cross-sectional area. 2 R i = i L i A i (
2 )
[0191] Fluids are usually characterized by conductance which is
just the reciprocal of the resistance as shown in equation 3. In
equation 3, K is the electrical conductance, .rho. is the
conductivity, A is the cross-sectional area, aid L is the length as
above. 3 K i = i A i L i ( 3 )
[0192] Using ohms law and equations 2 and 3 we can write the field
strength in a given channel, i, in terms of the voltage drop across
that channel divided by its length which is equal to the current,
I.sub.i through channel i times the resistivity of that channel
divided by the cross-sectional area as shown in equation 4. 4 E i =
V i L i = I i P i A i = I i i A i ( 4 )
[0193] Thus, if the channel is both dimensionally and electrically
characterized, the voltage drop across the channel or the current
through the channel can be used to determine the solvent velocity
or flow rate through that channel as: expressed in equation 5. It
is also noted that fluid flow depends on the zeta potential of the
surface and thus on the chemical make-ups of the fluid and
surface.
V.sub.i'I.sub.i.varies.Flow
[0194] Obviously the conductivity, .kappa., or the resistivity,
.rho., will depend upon the characteristics of the solution which
could vary from channel to channel. In many CE applications the
characteristics of the buffer will dominate the electrical
characteristics of the fluid, and thus the conductance will be
constant. In the case of liquid chromatography where solvent
programming is performed, the electrical characteristics of the two
mobile phases could differ considerably if a buffer is not used.
During a solvent programming run where the mole fraction of the
mixture is changing, the conductivity of the mixture may change in
a nonlinear fashion but it will change monotonically from the
conductivity of the one neat solvent to the other. The actual
variation of the conductance with mote fraction depends on the
dissociation constant of the solvent in addition to the
conductivity of the individual ions.
[0195] As described above, the device shown schematically in FIG.
31 could be used for performing gradient elution liquid
chromatography with post-column labeling for detection purposes,
for example. FIG. 31(a), 31(b), and 31(c) show the fluid flow
requirements for carrying out the tasks involved in a liquid
chromatography experiment as mentioned above. The arrows in the
figures show the direction and relative magnitude of the flow in
the channels. In FIG. 31(a), a volume of analyte from the analyte
reservoir 16 is loaded into the separation intersection 40. To
execute a pinched injection it is necessary to transport the sample
from the analyte reservoir 16 across the intersection to the
analyte waste reservoir 18. In addition, to confine the analyte
volume, material from the separation channel 34 and the solvent
reservoirs 12,14 must flow towards the intersection 40 as shown.
The flow from the first reservoir 12 is much larger than that from
the second reservoir 14 because these are the initial conditions
for a gradient elution experiment. At the being of the gradient
elution experiment, it is desirable to prevent the reagent in the
reagent reservoir 22 from entering the separation channel 34. To
prevent such reagent flow, a small flow of buffer from the waste
reservoir 20 directed toward the reagent channel 36 is desirable
and this flow should be as near to zero as possible. After a
representative analyte volume is presented at the injection
intersection 40, the separation can proceed.
[0196] In FIG. 31(b), the run (separation) mode is shown, solvents
firm reservoirs 12 and 14 flow through the intersection 40 and down
the separation channel 34. In addition, the solvents flow towards
reservoirs 4 and 5 to make a clean injection of the analyte into
the separation channel 34. Appropriate flow of reagent from the
reagent reservoir 22 is also directed towards the separation
channel. The initial condition a shown in FIG. 31(b) is with a
large mole fraction of solvent 1 and a small mole fraction of
solvent 2. The voltages applied to the solvent reservoirs 12, 14
are changed as a function of time so that the proportions of
solvents 1 and 2 are changed from a dominance of solvent 1 to
mostly solvent 2, this is shown in FIG. 31(c). The latter monotonic
change in applied voltage effects the gradient elution liquid
chromatography experiment. As the isolated components pass the
reagent addition channel 36, appropriate reaction can take place
between this reagent and the isolated material to form a detectable
species.
[0197] FIG. 32 shows how the voltages to the various reservoirs are
changed for a hypothetical gradient elution experiment. The
voltages shown in this diagram only indicate relative magnitudes
and not absolute voltages. In the loading mode of operation, static
voltages are applied to the various reservoirs. Solvent flow from
au reservoirs except the reagent reservoir 22 is towards the
analyte chaste reservoir 18. Thus, the analyte reservoir 18 is at
the lowest potential and all the other reservoirs are at higher
potential. The potential at the reagent reservoir should be
sufficiently below that of the waste reservoir 20 to provide only a
slight flow towards the reagent reservoir. The voltage at the
second solvent reservoir 14 should be sufficiently great in
magnitude to provide a net flow towards the injection intersection
40, but the flow should be a low magnitude.
[0198] In moving to the run (start) mode depicted in FIG. 31(b),
the potentials are readjusted as indicated in FIG. 32 The flow now
is such that the solvent from the solvents reservoirs 12 and 14 is
moving down the separation channel 34 towards the waste reservoir
20. There is also a slight flow of solvent away from the injection
intersection 40 towards the analyte and analyte waste reservoirs 16
and 18 and an appropriate flow of reagent from the reagent
reservoir 22 into the separation channel 34. The waste reservoir 20
now needs to be at the minimum potential and the first solvent
reservoir 12 at the maximum potential. All other potentials are
adjusted to provide the fluid flow directions and magnitudes as
indicated in FIG. 31(b). Also, as shown in FIG. 37, the voltages
applied to the solvent reservoirs 12 and 14 are monotonically
changed to move from the conditions of a large mole faction of
solvent 1 to a large mole fraction of solvent 2.
[0199] At the end of tie solvent programming run, the device is now
ready to switch back to the inject condition to load another
sample. The voltage variations shown in FIG. 32 are only to be
illustrative of what might be done to provide the various fluid
flows in FIGS. 31(a)-(c). In an actual experiment some to the
various voltages may well differ in relative magnitude.
[0200] While advantageous embodiments have been chosen to
illustrate the invention, it will be understood by those skilled in
the art that various changes and modifications can be made therein
without departing from the scope of the invention as defined in the
appended claims.
* * * * *