Note: Descriptions are shown in the official language in which they were submitted.
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CONTROL OF FLOW AND MATERIALS FOR
MICRO DEVICES
INTRODUCTIQN
The present invention generally relates to methods and devices for the
control of the movement of fluids and electrically charged sample components
within
those fluids. More particularly, the present invention permits exclusion or
concentration of specifically chosen sample components within a fluid.
The present invention provides an analytical device, either microchip-
or capillary-based, having the means to exclude specific sample components of
interest from a capillary or channel for the purpose of preconcentration or
control of
movement of sample components. Such a control system includes a means for
controlling the flow of the fluid in the channel and the placement of an
electrode at
the immediate entrance of each channel on such devices so that material may be
directly manipulated by either or both of the effects of both bulk flow and
electrically
driven migration.
BACKGROUND OF THE INVENTION
Capillary zone electrophoresis (CZE) is an efficient analytical
separation technique which utilizes differences in mobility of sample
components in
an electric field based on the electrical charge and molecular site and shape
of the
sample component. Conventional CZE systems typically comprise a buffer-filled
capillary with outlet and inlet ends disposed in two reservoirs into which one
sample
is injected, a means for applying voltage to the capillary resulting in
migration of the
sample through the capillary, and a means for detecting the sample zone.
Sample injection systems and capillary zone electrophoresis channel
systems have been integrated together on planar glass substrates for
separation of
sample components as described by Harrison et al. (1992, Anal. Chem.
64:19261932)
and Seller et al. (1993, Anal. Cem. 65:1481-1488). Additionally, capillary
electrophoresis on microchips has been described by Manz et ai., (1992, 3. of
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Chromatography 593:253-258). Total chemical analysis systems {TAS) in which
sample transport, chromatography or electrophoretic separations and detection
are all
performed have also been developed.
One of the limitations of conventional CZE is the extremely small
amount of sample which must be used in order to obtain separation or
resolution of
sample components. The use of small volume samples results in low amount of
sample components of interest representing a major limitation in the
detectability of
sample components. On the other hand, the larger the sample volume introduced
into
the capillary, the broader the sample component peaks will be. Attempts to
increase
injection sample volume typically leads to a breakdown in resolution due to
broadening of the peaks attributable to individual sample components which one
is
actually trying to resolve or separate and possibly leads to generation of
laminar flow
inside the capillary.
A number of techniques have been developed for increasing the
i5 concentration of specific sample components of interest and narrowing the
width of
the injected sample. One such technique involves the use of a solid-phase
adsorption
medium followed by a sequential combination of pressure- and electrically-
driven
flows as described in United States Patent No. 5,453,382. Using such a
technique, the
solution containing the sample component of interest is applied to the solid
phase
adsorption medium under conditions which permit sorption of the sample
component
of interest to the adsozption medium. The environment of the medium is then
altered
to promote desorption of the concentrated sample component and a voltage
gradient is
induced across the medium to induce electroosmosis. United States Patent No.
5,340,452 also describes a similar method for increasing the concentration of
sample
components prior to electrophoresis by using an active material which
selectively
retains the sample components of interest at the inlet end of the capillary
tube.
For some specialized samples, another obstacle to successful
separation of components of a solution results from the low strength of the
electric
field in the buffer bordering the sample solution and the column buffer. To
circumvent this problem, water or diluted buffer may be removed from the
capillary
or column using electro-osmotic flow while the sample components are stacked
in a
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support buffer thereby concentrating the sample components in a sample with a
minimum amount of laminar flow. Such a method is described in United States
Patent No. 5,116,471.
For large volume samples in constrained containers, pressurized flow
and countermigration can be used to increase the overall concentration as
described by
Hori et al. (1993, Anal. Chem. 65:2882-2886). The sample is introduced into a
first
vessel containing buffer which is connected to another vessel by a glass tube.
An
electrode extending into the first vessel applies a voltage to the sample
while suction
pressure is applied. The sample concentration increases throughout the first
vessel
rather than concentrating the sample in a discrete portion of that vessel
because the
applied potential field is unconstrained throughout the buffer volume. Because
the
concentration increase and electric fields are dispersed throughout the entire
first
vessel volume this technique is not applicable as a small volume
injection/preconcentration technique. Moreover, this arrangement does not
allow for
micromanipulations such as electrophoretic separation within the vessel
containing
the concentrated sample.
Hence, none of the aforedescribed methods provide for concentration
of sample components upon immediate introduction into a constrained small
volume
flow path which receives a fluid sample without the use of complicated systems
such
as discontinuous buffer systems and, in some instances, microengineered
absorption
devices. Accordingly, there exists a need in the art for more precise and
efficient
methods and devices for increasing the concentration of sample components of
interest within a fluid sample while maintaining a consistent buffer and
without
microengineering absorption systems.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a novel,
more efficient method far controlling the movement of fluids and electrically
charged
species, referred to as sample components, within those fluids which permits
exclusion or concentration of specifically chosen species within a constrained
fluid-
flow path.
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It is another object of the invention to provide an analytical
electrophoretic arrangement including microchips or capillaries which excludes
specific sample components of interest from a capillary or channel for the
purposes of
preconcentration or control of movement of materials.
It is a further object of the present invention to provide an arrangement
in which preconcentration and manipulation is achieved within a single
constrained
flow pathway system. More particularly, the sample is preconcentrated in a
portion of
the constrained flow pathway and is manipulated as it travels through the
pathway.
These and other objects of the invention are obtained by a method for
controlling the movement of a specific sample component in a fluid sample
comprising:
(a) providing a constrained fluid pathway having an inlet;
(b) introducing the fluid sample into the inlet of the constrained
fluid pathway;
(c) providing an electrode mounted at the inlet of the fluid
pathway, the electrode being entirely external to the constrained
fluid pathway;
(d) applying voltage to the electrode to create a voltage gradient
within the constrained fluid pathway to promote electrophoretic
migration of the sample component; and
(e) adjusting the flow rate of the fluid approximately equal to and
opposite to the electrophoretic migration o.f the sample.
(f) adjusting the electrophoretic migration rate to be approximately
equal and opposite to the flow rate of the fluid.
wherein movement of the specific sample component ceases.
The invention further provides an electrophoretic apparatus for
controlling the movement of an sample component in a fluid sample comprising:
(a) at least one constrained fluid pathway having an inlet and an
electrode mounted at the inlet of the constrained fluid pathway
and entirely external to the constrained fluid pathway; and -
(b) a power supply for supplying a voltage to the electrode.
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It is another object of the invention to provide an electrophoretic
apparatus for controlling the movement of an sample component in a fluid
sample
comprising:
{a) at least one injection fluid pathway having an electrode
S mounted at the inlet of said the pathway;
(b) at least one separation fluid pathway having an electrode
mounted at the inlet of said pathway;
(c) at least one power supply for providing voltage between the
electrodes; and
I O (d) means for regulating the bulk flow within the channels.
The present invention can be utilized in methods and devices for
manipulating, testing, probing, or analyzing sample fluids of any kind where
fluid
manipulations are utilized for preconcentration, chemical reaction, injection,
detection, or movement, or cessation of movement, of components of interest in
a
1 S sample fluid.
In one embodiment, the present invention is directed to an analytical
device having a plurality of channels with electrodes placed at the immediate
entrance
of all or selected channels and a method for regulating the bulk flow within
the
channels. When the bulk flow is set approximately equal to and opposite the
2U electrophoretic migration of specific sample components of interest, the
movement of
those specific sample components ceases. The introduction ofan electric field
between the electrodes within the channel, coupled with control of bulk flow,
allows
selected sample components of interest to be excluded or preconcentrated
immediately upon introduction of the fluid sample into the channel.
BRIEF DESCRIPTIOl~] OF THE DRAWINGS
Further objects and advantages of the invention will be apparant from a
reading of the following descriptian in conjunction with the accompanying
drawings,
in which:
Figure 1 is a schematic drawing of a fused silica capillary arrangement
with electrodes placed immediately at the inlet to provide the voltage control
within
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the capillary in accordance with the invention;
Figures 2(a), 2(b) and 2(c) are schematic drawings of a micro-device
apparatus having an injection channel and a separation channel in accordance
with the
invention;
Figure 3 is a schematic drawing of a micro-device apparatus indicating
the preconcentration of materials at the immediate entrance to a channel where
the
voltage with in the buffer reservoir is held constant in accordance with the
invention;
Figure 4 is a schematic drawing of the theoretical profile of the
pi-econcentration of material at the immediate entrance to a capillary showing
the
concentration of desired materials;
Figure 5 is a graph showing the normalized fluorescence intensity
versus distance outside the capillary entrance for two control experiments;
Figure 6 is a graph showing the normalized fluorescence intensity
versus number of pixels (1 pixel = 0.24 um) outside a capillary entrance ;and
Figures 7(a) and {b) are fluorescence micrographs of a capillary
entrance before and after, respectively, preconcentration of 200 nm
fluorescently
labeled latex micro spheres far 270 seconds.
DETAILED DESCRT'PTION OF THE PREFERRED EMBODIMENTS
The present invention provides novel methods and devices for
exclusion or concentration of specifically chosen sample components within
fluids
through the control of fluid movement and electrophoretic migration of charged
sample components within those fluids. Typically the fluid sample is delivered
or
injected into a restricted flow path such as a channel or capillary. For
purposes of the
present invention, the flow path is preferably less than 200 microns in
diameter.
Precise control of fluid manipulation, sample component movement and solution
injection systems are accomplished by carefully controlling the voltage field
gradients
and the bulk flow within each channel on a micro-device.
The principle of electrophoretic focusing as a means of sample
component exclusion from a capillary or channel can be applied to the
microscale
analytical device described herein. The apparatus and processes disclosed
herein may
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be used on microchip instrumentation in conjunction with control fluid
dynamics in
channels formed into or onto semiconductor devices. As used herein, the term
"microchip" includes a semiconductor device comprising silica or any other
substrate
which may be used in microfluidic devices, which may be used in or in
conjunction
with a computer.
The present invention also provides for the placement of an electrode
at the immediate entrance of each channel on a micro-device so that material
movement may be directly manipulated by electrically-driven migration, i.e.,
electraphoretic migration. The present invention also provides control of bulk
flow
of the fluid within the channel. Bulk flow may be positive or negative
depending
upon the magnitude and direction of electrically-driven flow, i.e.,
electroosmosis, or
various other sources of flow such as pressure, convection, capillarity, etc.
Voltage
gradients may likewise be manipulated to provide electrophoretic migration in
either
direction.
The introduction of an electric field resulting in electrophoretic
migration of a specific sample component, coupled with manipulation of bulk
flow
equal and opposite to electrophoretic migration, results in cessation of
movement of
those specific sample components. Thus, the independent control of these
parameters
provides for absolute control of movement of sample components within the
fluid
about a micro-device.
The method of the invention comprises as a first step, the introduction
of a sample containing the sample component of interest into a channel or
capillary
that has been filled with buffer. Sample introduction may be accomplished
using a
syringe by which the sample solution is injected into the channel.
Alternatively, the
introduction of the sample can be performed according to standard procedures,
including but not limited to the use of electroosmotic flow, electro-kinetic
pumping,
or pneumatic pumping.
An electrophoretic arrangement in which a capillary is utilized to
create the restricted flow path is shown in Figure 1. In this arrangement
electrodes 20
are located external to and mounted onto a fused silica capillary. A counter
electrode
24 is placed at a location remote from electrodes 20 and forms a circuit
therewith. A
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high voltage is applied to the electrodes 2U and 24 by power supply 26. A
reservoir
28 including buffer bulk flow materials is in fluid contact with the
capillary. A
sample 5 including charged components is introduced into the reservoir and
moves
towards the entrance 9 of the capillary in the presence of the applied voltage
which
induces electrophoretic migration. Thus, the charged components in analyte 5
axe
concentrated at the entrance 9 of the capillary 22.
The present invention also provides a micro-analytical separation
device comprised of etched or molded channels whereby various channels are
used for
separation and analysis purposes and others are distinctly used for the
purpose of
1U injection or material movement illustrated in Figs. 2(a-c). As shaven in
Figure 2a the
system includes an injection channel 2 and separation channel 4 . Sample
material is
injected to fill the injection channel 2 in between the separation channels 4
as depicted
in Figure 2b. To prevent unintentional introduction of material movement,
commonly
referred to as trailing or leaking, into the main separation channel after
injection
15 ceases, a small voltage is applied to the two injection channel electrodes
5. As
illustrated in Figure Zc, after the initial injection the electrodes are used
to create an
appropriate voltage gradient to prevent unwanted introduction of materials
into the
separation channel thereby concentrating desired components in separation
channel 4.
By manipulating flow and the voltage fields independently, positive, negative
and
20 neutral molecules may be manipulated as a group or individually.
A high voltage is applied by power supply means between the inlet and
outlet end of the channel or capillary through electrode means. The voltage
used is
not critical to the invention and may vary widely depending on the sample
components) to be excluded or concentrated. Conditions for selecting
appropriate
25 voltage conditions will depend on the physical properties of the sample
components)
and can be determined by those of skill in the art.
To preconcentrate sample components of either positive or negative
charge, the method of the invention further comprises setting the bulk flow in
the
channel or capillary approximately equal to and opposite to the
eiectrophoretic
3U migration rate of the material. The bulk flow in the capillary may be
generated and
controlled by either electroosmosis, pressure or various other mechanisms.
Bulk flow
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may be created and controlled by electroosmotic pumping devices, pneumatic
devices, or directly by electroosmasis with dynamic control and monitoring.
Thus the
sample component of interest is drawn toward the channel by bulk flow, but is
excluded from the channel by the voltage field effects on a narrow range of
materials
with similar eiectrophoretic migration rates thereby excluding or
concentrating the
sample component of interest at the immediate entrance of the capillary or
channel.
Alternatively, any constrained fluid pathway, for example a fused silica
or teflon capillary, where separation or injection of materials of interest
are performed
may be included in the device. Each channel or continuous fluid pathway where
control of material movement is desired is constructed with an electrode
adjoining the
entrance and.exit of the channel or pathway. Electrodes are placed at the
entrance of
the side channels to control the voltage field allowing electrophoretic
migration to
occur, and electroosmosis if the source of flow in the particular channel. In
this
manner the invention provides for integration of preconcentration and analysis
within
the constrained fluid pathway.
In the preferred embodiment of the invention as shown in Figure 2a-c,
the injection channel 2 is perpendicular to the separation channel 4, although
the
geometry of this intersection is not of direct importance to the concepts
presented
here. Electrodes 5, 6 are located at the immediate entrances of channels S, G
and are
electrically connected to the junction where the two channels 2, 4 intersect.
Placement of an electrode at the immediate entrance of a capillary or channel
and at
the junction with another channel or buffer reservoir, creates a chemical
voltage gate,
in that movement of materials may be independently controlled by simply
varying the
voltage field gradient and the flow rate within the particular channel. At
this chemical
voltage gate, materials of interest may be totally excluded from entering the
adjoining
channel or selectively permitted to enter the channel by using electrophoretic
focusing
techniques.
In another embodiment of the invention shown in Figure 3 a reservoir
containing a buffer solution 5 is placed in fluid contact with a channel 12
and an
electrode 9 is placed at the immediate entrance to that channel 11. The buffer
reservoir is maintained at the same voltage as the entrance electrode, thus
the material
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will not undergo electrophoretic migration within the reservoir. However, the
charged
materials will move toward the channel entrance at the same rate as the bulk
flow. At
the immediate entrance of the channel the effects of the applied voltage field
influences the charged materials, thus inducing electrophoretic migration.
Since the
5 bulk flow within the channel is approximately equal to and opposite the
electroplaoretic migration, the charged material of interest stops.
The flow rate of fluids may be controlled by, for example, the
following techniques: pressure induced flow, capillary, and electroosmosis as
taught
by Giddings (1991, Unified Separations Science, Wiley-Interscience, New York,
10 Chapt. 3). More specifically, pressure can be controlled by any physical or
chemical
means which will generate controllable flow or pressure. Capillarity can be
controlled
via chemical, electrochemical or photo-induced surface or solution changes as
taught
by Gallardo et al. (1999, Science 283:57-60). Electroosmosis can be controlled
by
external radial electrostatic fields as taught by Tsuda ( I 998, Handbook of
Capillary
Electrophoresis, Ed. J.P. Landers, 2"'' ed., CRC Press, Boca Raton , Chap.
22).
The methods and devices of the present invention may be used for
purposes of manipulating, testing; probing, or analyzing fluids of any kind
where fluid
manipulations may be used for preconcentration, chemical reaction, injection,
detection, or movement or restriction of movement, of the materials of
interest. The
manipulations provided for by the methods and devices described herein will
allow
for precise liquid injection and handling within a micro-chemical analysis
device in
addition to the ability to increase local concentration of materials by
several orders of
magnitude.
Preparation of specific embodiments in accordance with the present
invention will now be described in further detail. These examples are intended
to be
illustrative and the invention is not limited to the specific materials and
methods set
forth in these embodiments.
The examples discussed hereinafter were conducted using the
following standard chemicals and instrumentation, unless otherwise stated:
Chetnicc~ls and Materials. Sodium dihydrogen phosphate and
anhydrous ethyl alcohol (Aldrich Chemical Company, Milwaukee, WT); and
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phosphoric acid (EMGINCV Science, Gibbstown, NEW JERSEY) were used as
received. Capillaries were 45 cm in length (150 um o.d. - 20 ~tm i.d.) fused
silica and
were purchased from Polymicro Technologies (Phoenix, AZ). 0.2 pm carboxylate
modified yellow-green fluorescent (SOSI515) latex micro spheres were purchased
from Molecular Probes {Eugene, OR). The capillary electrophoresis buffer used
for
the latex micro sphere experiments was 100 mM phosphate buffer, adjusted with
phosphoric acid to pH 5.1.
Instrmnentation. The capillary electrophoresis system was built and
used a CZE1fl00R high voltage power supply from Spellman High Voltage
Electronics Corporation (Hauppauge, New York). The vacuum pump system was
purchased from Cenco Hyvac {Fort Wayne, IN). The laser source was a 4421325 nm
100 MPA: (Omnichrome Laser, Chino, Cat Scan). Image viewing was accomplished
with a case closed-5E CCD camera {HutchNet; East Hartford, Construction)
integrated to an Olympus Vanex stereo microscope (Tokyo, Japan). Data
collection
and analysis were accomplished using Labview software and an Imaq Pci- i 408
image acquisition board by in-house program development (National Instruments,
Austin, TX}. Data analysis was also performed on Microsoft Excel spreadsheet
program using an Optiplex GXI Pentium 233 (Dell Computer Corporation, Round
Rock, TX). The fluorescent signal was monitored from the carboxylate modified
latex
micro spheres as vacuum and voltage fields were adjusted.
Example 1
Experiments were performed to effectively demonstrate the increased
local concentration of specific materials using a capillary 30 and reservoir
32
arrangement shown in Fig. 4. The tip of the capillary was coated with metal 34
thereby providing a metal electrode. These experiments were performed with
fluorescence microscopy, fluorescently labeled latex microspheres, vacuum flaw
and
a metal-coated capillary tip.
The presence or location of carboxylate-modified latex spheres were
directly observed with the microscope under the effects of vacuum induced
flow.
The voltage was then empirically adjusted until the micro spheres were
excluded from
entering the capillary due to the electrophoretic migration rate of the micro
spheres.
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The intensity of the fluorescent signal which is directly related to
concentration was
monitored. Only a selected probe area, of approximately 2.5 pm x 120 pm
parallel,
and centered with the bore of the capillary immediately outside the entrance
was
quantitated for the fluorescence.intensity changes.
First, control experiments were performed to determine if adsorption or
other unknown processes were responsible for the increased fluorescence. These
control experiments consisted of using either the voltage field only (- 14 kV)
or the
vacuum-induced flow only 1.2 in Hg across a 45 cm Long; 20 ~m i.d. capillary.
As
illustrated in Figure. S the fluorescent signal was monitored and quantitated
for 4
minutes. The fluorescent signal was normalized with the fluorescent signal
obtained at
t = 0 minutes to eliminate any existing background fluorescence from the
temporal
data. The normalized fluorescent signal of the control experiments remained at
a value
of 1.7519.32 (n = 11 ) throughout the 4 minutes of the experiment (Figure G).
No
increase in fluorescent intensity was observed over the experimental period
indicating
that no unknown mechanisms nor adsorption to the capillary tip and walls
contributed
to the increased fluorescent intensity in the following experiments.
Experiments were performed to demonstrate preconcentration once the
electrophoretic migration rate within the channel in the capillary was
adjusted to be
equal to and opposite the bulk buffer low rate. As with the control
experiments, the
fluorescence intensity was normalized and then monitored for 4 minutes (n =
4), The
voltage empirically determined to generate an electrophoretic migration rate
which
counterbalanced the bulk flow rate was l4kV. Figures 7(a) and (b) are
fluorescence
micrographs of a capillary entrance before and after, respectively,
preconcentration of
200 nm fluorescently labeled latex micro spheres for 270 seconds. As
illustrated in
Figure 7b and Figure 4 the largest fluorescence intensity changes occurred
within 33
p.m of the capillary entrance. Due to dynamic range limitations, the
fluorescent
intensity at the entrance to the capillary saturated the CCD and therefore
quantitation
of this effect must be performed some 19.2 um outside the entrance to the
capillary.
The normalized fluorescent signal at 19.2 pm resulted in an increase in
fluorescence
intensity approximated by a linear equation (y = mx + h) where m is 0.042 arb.
-
units/min and h is 0.99 arb. units (Rz= 0. 938, P <_ 0.01). The initial
concentration of
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the micro-spheres was 1. 473 x 10'° micro spheres/mL.
The preconcentration build-up over time can be modeled as the
formation as an exponential zone superimposed on a background of constant
solute
concentration for materials accumulating up behind a partially rejecting
barrier such a
filter. The filter in this case is the exclusion of the micro spheres from the
capillary by
the applied voltage field and the resulting electrophoretic migration rate.
Assuming
the system will reach steady state conditions after a given time, the
background
concentration of the micro spheres is equal to Jq/v , initial flux over
velocity. The
concentration build-up of the micro spheres is given by the following
equation:
c = Jav + (cue Jal v)exp(-Ivly/DT)
where the concentration of the micro spheres is given by c, the flux of the
micro
spheres is given by J«, the velocity of the flow towards the barrier is v, the
original
concentration of micro spheres is given by c~" the distance from the barrier
is given by
y, and the total diffusion of the micro spheres is given by D1-. A plot of
concentration
of desired components versus location in the arrangement is shown in Figure 4.
Although the present invention has been described with reference to
latex micro spheres and fused silica capillaries providing the constrained
fluid
pathway, it should be understood that various modifications and variations can
be
easily made by those skilled in the art without departing from the spirit of
the
invention. Such modifications are intended to fall within the scope of the
claims.
Accordingly, the foregoing disclosure should be interpreted as illustrative
only and
not in a limiting sense. Various publications are cited herein, the contents
of which
are incorporated, by reference, in their entireties.
