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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.
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