U.S. patent number 5,906,723 [Application Number 08/703,394] was granted by the patent office on 1999-05-25 for electrochemical detector integrated on microfabricated capillary electrophoresis chips.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Alexander N. Glazer, Kaiqin Lao, Richard A. Mathies, Adam T. Woolley.
United States Patent |
5,906,723 |
Mathies , et al. |
May 25, 1999 |
**Please see images for:
( Certificate of Correction ) ** |
Electrochemical detector integrated on microfabricated capillary
electrophoresis chips
Abstract
A microfabricated capillary electrophoresis chip which includes
an integral thin film electrochemical detector for detecting
molecules separated in the capillary.
Inventors: |
Mathies; Richard A. (Moraga,
CA), Glazer; Alexander N. (Orinda, CA), Lao; Kaiqin
(San Francisco, CA), Woolley; Adam T. (Albany, CA) |
Assignee: |
The Regents of the University of
California (Berkeley, CA)
|
Family
ID: |
24825205 |
Appl.
No.: |
08/703,394 |
Filed: |
August 26, 1996 |
Current U.S.
Class: |
204/603;
204/601 |
Current CPC
Class: |
G01N
27/4473 (20130101); G01N 27/44791 (20130101); G01N
30/6095 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); G01N 30/60 (20060101); G01N
30/00 (20060101); G01N 027/26 () |
Field of
Search: |
;204/451,452,601,603 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
4314755 |
|
Nov 1994 |
|
DE |
|
WO95/10040 |
|
Apr 1995 |
|
WO |
|
Other References
Jonathan M. Slater, et al. (On-chip Microband Array Electrochenical
Detector for use in Capillary Electrophoresis, Analyst, Nov. 1994,
vol. 119, pp. 2303-2307). .
Gavin, Peter F., et al.; "Continuous Separations with
Microfabricated Electrophoresis--Electrochemical Array Detection",
J. Am. Chem. Soc. (1996), vol. 118, pp. 8932-8936. .
Adam T. Woolley, et al., Ultra-high-speed DNA fragment separations
using microfabricated capillary array electrophoresis chips, Proc.
Natl. Acad. Sci., vol. 91, pp. 11348-11352, Nov. 1994, Biophysics.
.
Mark K. Shigenaga, et al., In Vivo Oxidative DNA Damage . . . ,
Methods in Enzymology, vol. 186, pp. 521-530. 1990 month
unavailable. .
Dan Wu, et al., Electrophoretically mediated mico-assay of alkaline
phosphatase using electrochemical and spectrophotometric detection
in capillary electrophoresis, Journal of Chromatography B, 656
(1994) month unavailable pp. 357-363. .
Dean H. Johnston, et al., Electrochemical Measurement of the
Solvent Accessibility of Nucleobases Using Electron Transfer
between DNA and Metal Complexes, J. Am. Chem. Soc. 1995 month
unavailable, 117, pp. 8933-8938. .
Teresa M. Olefirowicz, et al., Capillary Electrophoresis in 2 and 5
.mu.m Diameter Capillaries: Application to Cytoplasmic Analysis,
Anal. Chem. month unavailable 1990, 62, pp. 1872-1876. .
Karin Pihel, et al., Electrochemical Detection of Histamine and
5-Hydroxytryptamine at Isolated Mast Cells, Anal. Chem. month
unavailable 1995, 67, pp. 4514-4521. .
Fu-Ren F. Fan, et al. Electrochemical Detection of Single
Molecules, Science, vol. 267, Feb. 10, 1995, pp. 871-874. .
Shigeori Takenaka, et al., Electrochemically Active DNA Probes:
Detection of Target DNA Sequences at Femtomole Level . . . ,
Analytical Biochemistry 218, (1994) month available, pp. 436-443.
.
D. E. Smith, et al., Second Harmonic Alternating Current
Polarography with a Revrsible Electrode Process, Analytical
Chemistry, vol. 33, No. 4, Apr. 1961, pp. 482-485. .
Philip D. Voegel, et al., Electrochemical detection with copper
electrodes in liquid chromatography and capillary electrophoresis,
Ameican Laboratory, Jan. 1996, pp. 39-45. .
Jonathan M. Slater, et al., On-chip Microband Array Electrochemical
Detector for use in Capillary Electrophoresis, Analyst, Nov. 1994,
vol. 119, pp. 2303-2307. .
Andrew G. Ewing, et al., Electrochemical Detection in Microcolumn
Separations, Analytical Chemistry, vol. 66, No. 9, May 1, 1994, pp.
527A-536A. .
Xiaohua Huang, et al., On-Column Conductivity Detector for
Capillary Zone Electrophoresis, Analytical Chemistry, vol. 59, No.
23, Dec. 1, 1987, pp. 2747-2749. .
Xiaohua Huang, et al., End-Column Detection for Capillary Zone
Electropheresis, Analytical Chemistry, vol. 63, No. 2, Jan. 15,
1991, pp. 189-192. .
Mei-Cheng Chen, et al., An Electrochemical Cell for End-Column
Amperometric Detection in Capillary Electrophoresis, Analytical
Chemistry, vol. 67, No. 21, Nov. 1, 1995, pp. 4010-4014. .
Thomas J. O'Shea, et al., Capillary electrophoresis with
electrochemical detection employing an on-column Nafion joint,
Journal of Chromatography, 593, (1992) month unavailable, pp.
309-312. .
Adam T. Woolley, et al., Ultra-High-Speed DNA Sequencing Using
Capillary Electrophoresis Chips, Anal. Chem. 1995 month
unavailable, 67, pp. 3676-3680..
|
Primary Examiner: Warden; Robert
Assistant Examiner: Noguerola; Alex
Attorney, Agent or Firm: Flehr Hohbach Test Albritton &
Herbert LLP
Claims
What is claimed is:
1. In a microfabricated capillary electrophoresis chip including a
substrate with an elongated separation channel with conductive
means at each end of the channel for applying a separation voltage
along the channel, the improvement comprising an integrated
electrochemical detector having a thin film working electrode
extending into said separation channel at or near the very end of
said separation channel where the working electrode is close to
ground potential and has minimal influence from the high
electrophoresis potentials, in order to detect current generated by
molecules undergoing redox reaction as they migrate past the thin
film electrode after they have migrated the length of the channel,
the portion of said thin film electrode extending into said channel
being very narrow to minimize the electrophoresis voltage gradient
which it senses, and a reference electrode spaced from said working
electrode.
2. A microfabricated capillary electrophoresis chip as in claim 1
in which the reference electrode is a narrow thin film electrode
which extends into said channel towards and spaced from side
working electrode to form a detection region therebetween within
the electrophoresis channel or at or just beyond the end
thereof.
3. A microfabricated capillary electrophoresis chip as in claim 2
in which said channel is from 1-500 .mu.m wide, said narrow ends
are from 1-500 .mu.m wide and the spacing between the narrow ends
is from 1-500 .mu.m.
4. A microfabricated capillary electrophoresis chip as in claim 2
including thin film leads extending from said thin film working
electrode and said conductive means at each end of the separation
channel to the same edge of said chip.
5. A microfabricated capillary electrophoresis chip as in claim 1
in which said thin film working electrode is covered with an
insulating film which extends to a point near the end of the
electrode.
6. A microfabricated capillary electrophoresis chip as in claim 1
in which the channel at one end transitions to a larger volume
portion and said thin film working electrode is placed in or near
the transition to a larger volume.
7. A microfabricated capillary electrophoresis chip as in claim 1
including a plurality of pairs of spaced working electrodes
disposed along said end of said channel to perform multiple
electrochemical detection.
8. A microfabricated capillary electrophoresis chip including a
substrate with an elongated separation channel and a cover plate
mounted on said substrate to form, with said channel, a separation
capillary comprising:
thin film conductive means at each end of said capillary for
applying a separation voltage along said capillary, and
an electrochemical detector having a thin film working electrode
near the very end of said channel where the working electrode is
close to ground potential and has minimal influence from the high
electrophoresis potentials, wherein the thin film working electrode
is adapted to detect molecules undergoing redox reaction as they
migrate past the thin film electrode, the portion of said thin film
working electrode extending into said channel being narrow and near
the end of said capillary to minimize the separation voltage
gradient which is senses, and a reference electrode spaced from
said working electrode.
9. A microfabricated capillary electrophoresis chip as in claim 8
in which the thin film reference electrode extends into said
channel towards and spaced from said working electrode to form a
detection region therebetween within the capillary.
10. A microfabricated capillary electrophoresis chip as in claim 8
in which said thin film working electrode is covered with an
insulating film which extends to a point near the end of the
electrode.
11. A microfabricated capillary electrophoresis chip as in claim 8
in which said channel is from 1-500 .mu.m wide, said narrow ends
are 1-500 .mu.m wide and the spacing between the ends is from 1-500
.mu.m.
12. A microfabricated capillary electrophoresis chip as in claim 8
in which the channel transitions to a larger volume channel portion
and said thin film working electrode is placed less than 500 .mu.m
from the transition to a larger volume.
13. A microfabricated capillary electrophoresis chip as in claim 8
including a plurality of pairs of spaced working electrodes
disposed along said end of said capillary to perform multiple
electrochemical detection.
14. A microfabricated capillary electrophoresis chip as in claim 8
including thin film leads extending from said thin film working
electrode and said conductive means to one edge of said chip.
15. A microfabricated capillary electrophoresis chip including an
etched glass substrate with an elongated separation channel and an
injection channel and a cover plate mounted on said substrate to
form, with said channels a separation and injection capillary
comprising:
thin film leads extending from the same edge of said chip to
connect to the ends of the separation and injection channels,
an integrated electrochemical detector having a thin film working
electrode extending from said one edge into said separation channel
at or near the very end of said channel where the working electrode
is close to ground potential and has minimal influence from the
high electrophoresis potentials, in order to detect current
generated by molecules undergoing redox reaction as they migrate
past the thin film electrode after they have migrated the length of
the channels, the portion of said thin film electrode extending
into said channel being narrow to minimize the electrophoresis
voltage gradient which it senses, and
a reference electrode spaced from said working electrode.
16. A microfabricated capillary electrophoresis chip as in claim 15
in which the reference electrode is a thin film electrode extending
from said one edge with a narrow end extending into said channel
towards and spaced from said working electrode to form a detection
region therebetween.
17. A microfabricated capillary electrophoresis chip as in claim 16
in which said channel is from 1-500 .mu.m wide, said narrow ends
are from 1-500 .mu.m wide and the spacing between the ends is from
1-500 .mu.m.
18. A microfabricated capillary electrophoresis chip as in claim 15
in which the channel transitions to a wider channel portion and
said thin film electrodes are closely spaced from said channel
transition.
Description
BRIEF DESCRIPTION OF THE INVENTION
This invention relates generally to an electrochemical detector as
a component of an integrated separation and detection module on a
microfabricated capillary electrophoresis chip and to a method of
fabricating the electrochemical detector and more particularly to
the design of a thin film electrochemical detector which can be
precisely positioned in a microfabricated capillary.
BACKGROUND OF THE INVENTION
Electrochemical detection has been employed in liquid
chromatography and in capillary electrophoresis (CE). It has been
demonstrated that electrochemical detection is very sensitive and
can measure 10.sup.-16 to 10.sup.-19 moles of sample with typical
detection volumes from nL to pL.sup.1,2. Electrochemical methods
have also been used to detect DNA,.sup.3-5 single cells,.sup.6,7
and even single molecules..sup.8 The operation of these
electrochemical detectors is typically based on the use of three
electrodes called the working, counter, and reference electrodes.
There are three configurations which have been used to detect CE
separations: on-column,.sup.9 where the electrodes of the detector
are placed within the capillary; end-column,.sup.10,11 where the
electrodes are placed directly at the end of the separation
capillary; and off-column,.sup.6,12,13 where the electrodes are
electrically isolated from the electrophoresis voltage by a
grounded porous glass tube. On-column electrochemical detection of
CE separations has been performed by fixing two platinum wires
through diametrically opposed holes drilled by a laser in a
capillary tube. This structure is very difficult to manufacture and
align, and the placement of the detection electrodes within the
high voltage region of the separation column is problematic. In
this format, one is trying to detect small currents or voltages
while applying many kV to the separation column. The mechanical
instability and poor definition of the electrode alignment can lead
to significant electrical pickup or fluctuation in the background,
making the desired signal very difficult to detect. The presence of
high voltage gradients and significant electrophoretic currents in
the column near the electrodes can induce stray signals. The
end-column and off-column detection formats are important because
they minimize the influence of the electrophoresis voltage. In the
end-column format, one wants to place the detection electrodes as
close to the end of the electrophoresis channel as possible so the
detection is performed as close to ground potential as possible.
This is very difficult to do with conventional manufacturing
techniques. The electrodes must be placed with micron precision at
the end of the capillary. Any error in the placement will cause
loss of analyte signal if the electrodes are too far from the
opening or high voltage pick up if the electrodes are placed within
the separation column. Furthermore, fluctuations in electrode
placement or electrode--electrode gap can cause severe fluctuations
in the background signal producing noise. Typically, one must use
micromanipulators and a microscope to assemble the detector.
Furthermore, the engineering of the electrical isolation by
connection of the separation and detection capillary tubes with a
grounded porous glass tube in the off-column format is rather
difficult to assemble and operate, and the junction can be
mechanically unstable and poorly defined. In one case, although
Slater and Watt (17) photolithographically fabricated electrodes on
a substrate, because they did not make a fully integrated
separation and detection device, they were forced to use said
undesirable junctions to couple their detector to a conventional
cylindrical capillary.
There is a need for a microfabricated capillary electrophoresis
chip with integral thin film electrochemical detector and
electrophoresis leads which can be easily connected to associated
electrical electrophoresis and detector apparatus.
OBJECTS AND SUMMARY OF THE INVENTION
It is a general object of the present invention to provide an
electrochemical detector for capillary electrophoresis on a
microfabricated planar glass chip that overcomes the aforementioned
short comings of the prior art.
It is another object of the present invention to provide a
microfabricated capillary electrophoresis chip with a
microelectrochemical detector that minimizes the effect of
interference from applied electrophoresis fields.
It is another object of the present invention to provide detector
electrodes which are reproducibly, accurately and conveniently
placed, robust and sensitive.
It is a further object of the present invention to provide detector
electrodes which are precisely and stably positioned at the very
end of the capillary where they are close to ground potential and
thereby immune to pick up from the high electrophoresis
potentials.
It is a further object of the present invention to provide a
microfabricated capillary electrophoresis chip with integrated thin
film electrochemical detector electrodes and electrophoresis
electrodes which can be produced accurately and at low cost.
The foregoing and other objects of the invention are achieved by
integrating an electrochemical detector on a microfabricated
capillary electrophoresis chip of the type including a substrate
having at least an elongated separation channel and a cover plate
bonded to said substrate to form with said channel a separation
capillary. A thin film electrochemical detector is fabricated on
the surface of said substrate or cover plate with thin narrow
electrodes extending into said channel near one end of said
channel.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects of the present invention will be
more clearly understood from the following description when read in
conjunction with the accompanying drawings, of which:
FIG. 1 shows a microfabricated capillary electrophoresis chip in
accordance with the prior art;
FIG. 2 is a sectional view taken along the line 2--2 of FIG. 1;
FIG. 3 is a perspective view of a microfabricated capillary
electrophoresis chip incorporating the present invention;
FIG. 4 is an enlarged view of the indicated detector region 4--4 of
FIG. 3;
FIG. 5 is a sectional view taken along the line 5--5 of FIG. 4;
FIG. 6 is a sectional view taken along the line 6--6 of FIG. 5;
FIG. 7 is a sectional view showing another embodiment of the
electrochemical electrodes shown in FIGS. 3 and 4;
FIG. 8 is a sectional view taken along the line 8--8 of FIG. 7;
FIG. 9 is a sectional view taken along the line 9--9 of FIG. 7;
FIG. 10 is an enlarged view of another detector embodiment;
FIG. 11 is an electropherogram of norepinephrine and epinephrine
separated on a capillary electrophoresis chip with integrated
electrochemical detection;
FIGS. 12A-12C are electropherograms of norepinephrine separations
obtained with a capillary electrophoresis chip with integrated
electrochemical detection for three consecutive experiments;
FIGS. 13A-13B are perspective views of a microfabricated capillary
electrophoresis chip with integrated electrochemical detection
including thin film connections to the separation and injection
channels;
FIG. 14 is an enlarged view of the section 14--14 of FIG. 13B;
FIG. 15 is a perspective view of a substrate including an
integrated electrochemical detector and leads connected to the
injection and separation channels;
FIG. 16 is a sectional view taken along the lines 16--16 of FIG.
15;
FIG. 17 is an enlarged view taken along the direction of arrow 17
of FIG. 16;
FIG. 18 is a partial enlarged view showing a plurality of
electrochemical detection electrodes formed along the separation
channel;
FIG. 19 is a block diagram of an apparatus for joining a capillary
electrophoresis chip into an overall electrochemical separation and
analysis system in accordance with the present invention.
DESCRIPTION OF PREFERRED EMBODIMENTS
FIGS. 1 and 2 show a microfabricated capillary electrophoresis (CE)
chip formed in accordance with the prior art. The capillary
channels are formed on an etched glass substrate 11 by
photolithography and chemical etching. The process is described by
Woolley et al., Ultra-High-Speed DNA Fragment Separations Using
Microfabricated Capillary Array Electrophoresis Chips, Proc. Nat'l.
Acad. Sci., USA, 91, 11348-11352 (1994).sup.14. The separation
channel 12 and the injection channel 13 for injecting sample into
the channel by stack or plug injection are described in the above
reference. In one example, all channels were etched to a depth of 8
.mu.m; the separation channels were 100 .mu.m wide, and the
injection channels were 50 .mu.m wide. The separation channels were
46 mm long, with a distance of 39 mm from the point of injection to
the electrochemical detector. The injection channels were 22 mm
long with a distance of 12 mm from the point of sample introduction
to the injection region. A top plate 14 was bonded to the etched
glass substrate to form the capillaries which are filled with a
separation matrix. The top plate includes drilled holes 1-4 which
provide reagent reservoirs to the ends of the separation channel
and the ends of the injection channel.
In the prior art, the electrophoretic DNA separations in the
microfabricated capillary channels were detected by bulky,
inconvenient and costly systems employing external lasers, optical
systems, photomultiplier tubes, etc. It has thus far not been
possible to integrate the optical detection system onto a
microfabricated CE chip. Similarly, although electrochemical
detection of conventional capillary electrophoresis separations
performed in hollow silica capillaries has been performed with a
variety of external electrode and detector formats, such a detector
has never been integrated within a CE electrophoresis chip system
with a single microfabrication technology.
In accordance with one embodiment of the present invention,
platinum electrodes for electrochemical detectors are fabricated on
the substrate or top plate by RF by sputtering and photolithography
before the top or cover plate is bonded to the etched substrate.
The electrodes can be accurately positioned at the ends of the
separation column where they are close to ground potential thereby
providing a stable, easy to manufacture, inexpensive
electrochemical detector. Other suitable electrode materials are
gold, chromium, carbon and other relatively inert easily deposited
conductive materials.
Referring to FIGS. 3-5, a CE chip is shown with thin film platinum
electrodes. The electrodes comprise a reference electrode 21, a
working electrode 22, and a counter electrode 23 (not shown)
connected to an external circuit by thin film conductors 21a, 22a
and 23a. The substrate is preferably etched so that the electrodes
and thin film conductors are inset as shown in FIG. 6 whereby the
top plate 14 can be effectively sealed to the substrate. The
reference and working electrodes include a narrow portion extending
into the channel with the ends separated and adapted to detect
current or voltage as molecules undergo redox reactions or conduct
current as they migrate past spaced electrodes. The electrodes are
connected to wider thin film leads 21a, 22a and 23a which extend to
the edge of the chip for insertion into a connector (not shown) to
provide electrical connection to the electrical measuring circuits.
In order to limit the exposed area of the narrow portions of the
working and reference electrodes which extend into the channel, the
electrodes can be covered with an insulating dielectric film such
as SiO.sub.2. This is illustrated in FIGS. 7-9 where the electrodes
21 and 22 are covered by an insulating film 24. In one example, the
Pt electrodes were deposited using RF sputtering; the thickness of
the electrodes was 3000 .ANG.. The working and reference electrodes
were 20 .mu.m wide Pt electrodes that were precisely aligned on
opposite sides of the channel (to minimize the potential difference
between electrodes) and extended 40 .mu.m into the channel, with a
spacing of 20 .mu.m (see FIG. 4). The 100 .mu.m channel widens to
1000 .mu.m at the end to increase the volume of separation channel.
The working and reference electrodes were placed 20 .mu.m from the
point of widening. The counter electrode was 2 mm wide and extended
into the widened portion at the end of channel. The advantage of
this design is that it minimizes the influence of the
electrophoresis voltage by working very close (20 .mu.m) to the
ground end of the channel where the analyte is still highly
concentrated, while still performing on-column detection. After
careful alignment, the etched bottom plate or substrate 11 with the
Pt electrodes was thermally bonded to a top glass plate 14 with 0.8
mm holes 1-4. The detector electrodes can also be formed adjacent
the end of the channel as shown in FIG. 10. The detector electrodes
21b and 22b are covered by an insulating film 25 with the ends
exposed. Although specific dimensions have been given for the
described embodiment, the channel width and depth can be between
1-500 .mu.m, the electrode width 1-500 .mu.m and the electrode
spacing 1-500 .mu.m.
The advantages of such fabrication and design are that (i) the
working and reference electrodes can be easily and precisely
positioned near, at, or just beyond the opening of the separation
channel where pickup and interference from the electrophoresis
voltage is minimal and where the analyte concentration in the
separated zone is still high. This precise (micron) alignment is
only possible with an integrated microfabricated device. (ii) The
electrodes in the channel are very small in the electrophoresis
dimension. This is advantageous because it facilitates the
placement of multiple electrodes, FIG. 18, at essentially (compared
to the zone size) the same point in the channel. It is also
advantageous because we have observed that wider electrodes tend to
nucleate electrolysis bubbles presumably because they sample more
of the electrophoretic voltage gradient. This effect can be reduced
by covering the body of the electrode (not the tip) with an
insulating layer. Such thin electrodes can only be produced via
photolithography on an integrated device. Finally, one wants to
have a precise and small electrode gap so that each detector
functions the same and has a similar sensitivity and probed volume.
The ability to fabricate a small gap will produce low backgrounds
because the effective volume of conductive and capacitive solution
between the electrode is small. The ability to make detectors with
small gaps is also advantageous because it permits the fabrication
and detection of narrow separation channels which require only
small amounts of sample and which have very high electrophoretic
resolution.
It is noted that the channel widens at the end just past or at the
point of detection. This is important because it keeps the first
zones in the separation from raising the background as a result of
diffusion of analyte back into the detector zone. By having a
larger channel beyond the detector to provide a greater volume, the
early zones are effectively diluted by the large solution volume
around the counter electrode thereby keeping them from raising the
background for the detection of subsequent bands. The wide section
also has a low resistance because of its large cross section. This
means that the voltage drop from the detector to the counter
electrode will be much smaller thereby further reducing stray
voltages at the detector and pickup and background. It will be
appreciated that in addition to widening the channel to provide a
greater volume, the depth may be increased.
Capillary zone electrophoresis separation of two neurotransmitters,
epinephrine and norepinephrine was performed using a CE microchip
having the dimensions given in the above examples following in
general the methods outlined in Woolley et al.sup.14-15. A 30 mM
solution of 2-(N-morpholino) ethanesulfonic acid (MES) adjusted to
pH 5.6 with NaOH and modified with 20% (v/v) 2-propanol was used as
the buffer. Stock solutions (10 .mu.m) of epinephrine and
norepinephrine (Sigma, St. Louis) were prepared in 0.01 M
perchloric acid. Samples were serially diluted to the desired
concentration in MES buffer. After placing the sample in reservoir
3, the samples were injected by applying 90 V/cm between reservoirs
1 and 3 (FIG. 3) for 20 seconds and the approximate injection
volume was calculated as 40 pL. Separations were performed by
applying 45 V/cm between reservoirs 2 and 4. The electrophoresis
currents were typically 0.3 .mu.A.
A Macintosh computer equipped with a National Instruments
NB-MIO-16XL-18 I/O board was used to set voltages, store data and
control the home-built three electrode potentiostat. The working
electrode 22 was biased at +0.5 V relative to the reference
electrode 21; the counter electrode 23 was used to complete the
circuit. The potentiostat measured the current generated by
molecules undergoing redox reactions as they migrated past the gap
between the reference and working electrodes. Small currents (<1
pA could be detected even in the presence of the larger DC
electrophoresis current (0.3 .mu.A) in the channels. Alternatively,
the small currents could be detected by biasing the working
electrode with an AC potential..sup.16 A lock-in amplifier could
then be used to distinguish the signal from the DC electrophoresis
current. Prior to experiments, the electrodes were cleaned using 1M
H.sub.2 SO.sub.4 with a sine wave potential (V.sub.p--p =0.5V)
applied to the electrodes for 20 minutes.
FIG. 11 shows the separation of two neurotransmitters, epinephrine
and norepinephrine, performed on the microfabricated CE chip with
integrated electrochemical detection. Norepinephrine and
epinephrine were detected at 2.6 min and 3.4 min, respectively, and
the peaks were baseline resolved. The separation time was short,
approximately 3 minutes.
FIGS. 12A-12C present the injection and detection of 0.48 nM
epinephrine in three consecutive times. The reproducibility of
migration times for these runs is excellent. The reproducibility of
the signal strength is within a factor of 1.5, and most of the
variability can be attributed to tailing from the later
injections.
In addition to the use of thin film detection electrodes, thin film
connections can be made from the edge of the chip to the ends of
the separation and injection channels, 12 and 13. This would then
permit insertion of the chip 30 into the socket 31, (FIG. 19,)
which provides electrical connection to electrophoresis and
detection electronics 32, for example, a processor of the type
described above. The processor can be used to control stack or plug
injection of sample into the separation channel and to apply
electrophoresis voltages to the separation channel. Furthermore,
the processor can apply voltages to the detector and analyze redox
currents to provide a display or printout 33.
Referring to FIGS. 13 and 14, thin film leads 36, 37, and 38 are
shown connected to the ends of the injection channel and to one end
of the separation channel or column. A thin film connection 40 to
the other end of the channel is also shown. The thin film leads
terminate at the edge 39 of the substrate. The thin film leads are
carefully placed in all the reservoirs so that they are far from
the end of the channels so that hydrolysis bubbles due to current
flow at the lead do not enter the adjacent channel. This is
illustrated in FIG. 14 for one end of the injection channel. The
chip can then be inserted into the socket for carrying out sample
analysis. After the thin film leads are formed by photolithography
and sputtering, the cover 14 is bonded to the substrate spaced from
the end so that the leads can be contacted.
In another example, thin film leads 36a, 37a, 38a and 40a can be
formed at the bottom of the substrate, FIGS. 15-17 with lead
through connections 39 to the bottom of the etched channels and
spaced from the ends of the channel.
Discrimination between species with different half-cell potentials
can be achieved by sweeping over different bias voltages at the
working electrode or by using multiple pairs of working and
reference electrodes 21-1, 21-2 and 21-3, and 22-1, 22-2 and 22-3
as shown in FIG. 18.
It should be apparent that the various thin film detector
electrodes and thin film connections to the injection and
separation channel can alternatively be made on the top cover plate
which is then accurately positioned with respect to the
channels.
Thus, there has been provided an improved integrated
electrochemical detector on a microfabricated CE chip. This opens
the way to a variety of interesting and useful analytes. For
example, electrochemical detection on CE chips could be used for
numerous analytes which are redox active. A microfabricated chip
and electrochemical detector can be used for remote analysis of
hazardous substances without the need for operator intervention.
This invention is an important step towards complete integration of
DNA and other analyses on microfabricated chips.
REFERENCES
1. Ewing, A. G.; Mesaros, J. M.; Gavin, P. F., Electrochemical
Detection in Microcolumn Separations, Anal. Chem., 66, 527A-536A,
(1994).
2. Voegel, P. D.; Baldwin, R. P., Electrochemical Detection with
Copper Electrodes in Liquid Chromatography and Capillary
Electrophoresis, American Laboratory, 28(2), 39-45, (1996).
3. Shigenaga, M. K.; Park, J.-W.; Cundy, K. C.; Gimeno, C. J.;
Ames, B. N., In Vivo Oxidative DNA Damage: Measurement of
8-hydroxy-2'-deoxyguanosine in DNA and Urine by High-Performance
Liquid Chromatography with Electrochemical Detection, Methods in
Enzymol., 186, 521-530, (1990).
4. Takenaka, S.; Uto, H.; Knodo, H.; Ihara, T.; Takagi, M.,
Electrochemically Active DNA Probes-Detection of Target DNA
Sequences at Femtomole Level by High-Performance Liquid
Chromatography with Electrochemical Detection, Anal. Biochem., 218,
436-443, (1994).
5. Johnston, D. H.; Glasglow, D. C.; Thorp, H. H., Electrochemical
Measurement of the Solvent Accessibility of Nucleobases Using
Electron Transfer Between DNA and Metal Complexes, J. Am. Chem.
Soc., 117 8933-8938, (1995).
6. Olefirowicz, T. M.; Ewing, A. G., Capillary Electrophoresis in 2
and 5 .mu.M Diameter Capillaries: Application to Cytoplasmic
Analysis, Anal. Chem., 62, 1872-1876, (1990).
7. Pihel, K.; Hsieh, S.; Jorgenson, J. W.; Wightman, R. M.,
Electrochemical Detection of Histamine and 5-Hydroxytryptamine at
Isolated Mast Cells, Anal. Chem., 67, 4514-4521, (1995).
8. Fan, F.-R. F.; Bard, A. J., Electrochemical Detection of Single
Molecules, Science, 267, 871-874, (1995).
9. Huang, X.; Pang, T.-K. J.; Gordon, M. J.; Zare, R. N., On-Column
Conductivity Detector for Capillary Zone Electrophoresis, Anal.
Chem., 59, 2747-2749, (1987).
10. Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G., End-Column
Detection for Capillary Zone Electrophoresis, Anal. Chem., 63,
189-192, (1991).
11. Chen, M.-C.; Huang, H.-J., An Electrochemical Cell for
End-Column Amperometric Detection in Capillary Electrophoresis,
Anal. Chem., 67, 4010-4014, (1995).
12. O'Shea, T. J.; Greenhagen, R. D.; Lunte, S. M.; Lunte, C. E.;
Smyth, M. R.; Radzik, D. M.; Watanabe, N., Capillary
Electrophoresis with Electrochemical Detection Employing an
On-Column Nafion Joint, J. Chromatogr., 593, 305-312, (1992).
13. Wu, D.; Regnier, F. E.; Linhares, M. C., Electrophoretically
Mediated Micro-Assay of Alkaline Phosphatase using Electrochemical
and Spectrophotometric Detection in Capillary Electrophoresis, J.
Chromatogr. B, 657, 357-363, (1994).
14. Woolley, A. T.; Mathies, R. A., Ultra-High-Speed DNA Fragment
Separations Using Microfabricated Capillary Array Electrophoresis
Chips, Proc. Nat'l. Acad. Sci., USA, 91, 11348-11352, (1994).
15. Woolley, A. T.; Mathies, R. A., Ultra-High-Speed DNA Sequencing
Using Capillary Electrophoresis Chips, Anal. Chem., 67, 3676-3680,
(1995).
16. Smith, D. E.; Reinmuth, W. H., Second Harmonic Alternating
Current Polarography with a Reversible Electrode Process, Anal.
Chem., 33, 482-485, (1961).
17. Slater, J. M.; Watt, E. J., On-chip Microbond Array
Electrochemical Detector for use in Capillary Electrophoresis
Analyst, 1994, 119, 2303-2307.
* * * * *