 |
Description  |
|
|
FIELD OF THE INVENTION
This invention relates to a method and device useful for the
chromatographic and/or electrophoretic separation and detection of
materials. Such separation is particularly useful in the analysis of
biological molecules for research and diagnostic applications. In
particular, the device and accompanying methods may be used to separate
and detect microquantities of proteins and genetic material (RNA, DNA,
etc.) using such principles of electrophoresis and chromatography in
conduits of capillary and subcapillary dimensions.
1. BACKGROUND OF INVENTION
There exists a need for reliable, low-cost, automated analytical devices
that allow facile and rapid separation and detection of microquantities of
cellular tissue and genetic material for use in the research and diagnosis
of disease. DNA analysis is an effective approach for the detection and
identification of pathogenic microbes (i.e., viruses, bacteria, etc.) and
is essential to the identification of genetic disorders. The ability to
detect DNA with clinical specificity entails high resolution separation of
RNA or DNA fragments, appropriate labeling chemistry for such fragments,
and the adaption of high sensitivity sensors that are specific for the
labeling chemistry employed. DNA probe technology is now an established
tool of the molecular biologist for revealing the presence of
diagnostically significant cells, whether they be diseased cells from the
subject or infectious microorganisms.
Equally important to biomedical diagnosis is the ability to recognize
minute variations in protein structure. For example, it has been reported
that the detection of certain isoforms of the isoenzyme Creatine Kinase
(CK-MB) is a marker for early detection of myocardial infarct. Similarly,
proteins emanating from the core and envelope of viruses are used to
detect early stages of viral infection (i.e., Auto immune disease syndrome
AIDS).
On the biotechnology side, much of the success of modern molecular biology
can be attributed to the development of reliable methods for the chemical
structural analysis of nucleic acids. Determining the nucleotide sequence
of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) is essential to
recombinant DNA technology which aim is to alter the genes of
microorganisms so as to ultimately produce human proteins (drugs) such as
interferon, growth hormone, insulin, etc. In the plant world, DNA
sequencing information is useful in developing plant strains that are
resistant to adverse environmental conditions or disease. Without
exception, analytical information is required at both the DNA level of
information and also at the protein stage, to monitor gene expression
during cloning.
2. Description of the Prior Art
Most prior art separations have been confined to relatively large channel
dimensions (>50 micron diameter) dictated by the availability of fused
silica capillaries. Electro-osmosis has been used to pump solvents in both
thin layer and liquid chromatography [(D.L. Mould and R.L.M. Synge,
Analyst, London, 77, 1952, 964), (D.L. Mould and R.L.M. Synge, Biochem.
J., 58, 1954, 571), (V. Pretorius, B.J. Hopkins and J.D. Schieke, J.
Chromatography, 99, 1974, 23), (J.W. Jorgensen and K.D. LuKacs, Ana1.
Chem., 53, 1981, 1298), and (J.W. Jorgensen and K.D. LuKacs, J.
Chromatography, 218, 1981, 209). V. Pretorius, et al. used electroosmotic
pumping for packed columns and open tubes and Jorgensen, et al. (J.
Chromatooraohv Vol. 218, [1981] p. 209) for glass capillaries. Jorgensen
also used electrophoresis to separate charged molecules in solutions
pumped by electo-osmosis. A. S. Cohen, S. Terabe, J. A. Smith and B. L.
Karger (Analytical Chemistry [1987], Vol. 59, p. 1021) utilized the
interaction of solute and micelles to enhance the separation resolution of
nucleotides. A. S. Cohen and L. B. Karger (Journal of Chromatography, Vol.
397, [1987] p. 409) demonstrated high resolution electrophoresis
separation of proteins with polyacrylamide gel-filled fused silica
capillaries.
The success of silicon in microelectronics is due to its unique properties
as a structural material (K.E. Petersen, Preceedings of IEEE, 70, 1982,
420), as an electronic component, and as a chemical interface. A most
noteworthy implementation of silicon as an integrated analytical component
is the design of a gas chromatograph on a qilicon wafer [S.C. Terry, J.H.
Jerman, and J.B. Angell, IEEE Transactions on Electron Devices, ED-26,
(1979) 1880].
It is known in the art that fluids may be propelled through conduits by
electro-osmotic force. Electro-osmotic pressure is the consequence of
charge build-up on the conduit surface. The buffer solution supplies the
mobile counter ion to neutralize the surface charge and is the potential
energy equivalent of the electroosmotic pressure. The application of an
external voltage will cause a discharge via the mobile ions, resulting in
an electro-kinetic current.
The discharge of ions causes the fluid in tne conduit to flow. The fluid
flow is typically in the direction of the negative pole of the electric
field since the counter ions are usually cations. The fluid flow direction
is controlled by the magnitude of the applied voltage, its polarity, the
surface charge, the channel dimensions and the viscosity of the medium.
Unfortunately, the efforts in the prior art to utilize electro-osmotic
pumping have been limited by the relatively large channel dimensions
available in the glass capillaries used. Optimum flow velocity and control
is achieved when the channel diameter is twice the ion double-layer
thickness (i.e., .about.2.times.10.sup.-9 meter calculated for a 50
millimolar sodium phosphate solution). Furthermore, capillary
electrophoresis as practiced by Jorgensen et al. requires very high
voltages .about.25 KVolts) to achieve significant flow velocities. The
by-product of such high voltages is the formation of electrolysis products
in the vicinity of the electrodes, and thus an unwanted side effect. The
deployment of electrode implants within the silicon channels allows for
the application of smaller voltages to achieve equivalent electric fields
employed in capillary electrophoresis.
Capillary electrophoresis is practiced with fused silica capillaries with
nominal dimensions of 1 meter length and 80-100 .mu.m diameter. The
voltage used to electro-osmotically drive the fluids through such
capillaries at a rate of .about.0.2 .mu.l /minute is .about.25 KV. In the
practice of capillary electrophoresis, fluid flow is electro-osmotically
driven toward a down-stream detector and the separation of sample
components is accomplished electrophoretically (by charge), wall effects,
or chemical interactions in the mobile phase.
Electroosmosis is mediated by surface charge, buffer electrolyte
composition, viscosity of the fluid, channel or conduit diameter and the
applied voltage. Best fluid control and optimum velocity is achieved when
the capillary conduit diameter approaches twice the thickness of the Ion
Double Layer Thickness. We estimate an ion double layer thickness for a 50
mM Na.sub.2 H.sub.2 P0.sub.4 solution at approximately 10.sup.-9 meter vs
the 10.sup.4 meter diameter of a capillary.
The major factors that limit separation performance by capillary
electrophoresis are; diffusional zone spreading and dispersion of a zone
by thermal convection. Dispersion due to thermal gradients can be
controlled by the geometry of medium and the thermal conductivity and mass
of the structural material that comprises the separation device. These
same factors also limit resolution in gel-phase electrophoresis. Capillary
electrophoresis is effective in separating proteins on the basis of
charge/mass ratios. But, unlike gel-electrophoresis, capillary
electrophoresis cannot separate on the basis of molecular size.
It is also known in the art that analytical performance is improved in
capillary electrophoresis by reducing the capillary diameter. Similarly,
gel-phase separations improve in resolution as the gel-thickness is
reduced.
SUMMARY OF THE INVENTION
Many of the disadvantages of the prior art capillary type separation
devices and methods are overcome by the device and methods of this
invention. The device comprises a semiconductor wafer with micromachined
conduits which conduits and reservoirs contain an electrode and detection
electro-optics and/or electrochemical detectors. All analytical components
needed for the separation and detection of the separated components are
inclusive within the device and comprise sensing electrodes, drive
electrodes, light guides, photodiodes and compartments for sample and
reagent introduction.
According to the invention, an improved separation device, is constructed
comprising a capillary sized, closed conduit adapted to be filled with
liquid or solid materials for electrophoretic and/or chromatographic
separations, means to introduce a sample to be processed into the conduit.
The device is characterized by a semiconductor slab having a channel in
one face and a cover plate attached to the one slab face to form the
closed conduit, at least one interior dimension, transverse to the conduit
being less than 100 .mu.m, adapted to receive an ionizable liquid and
means for applying an electric potential along the length of the interior
of the conduit.
A method is described for electrophoretically separating a sample into
components using a capillary sized conduit defined by an elongated channel
in one face of a silicon slab, the channel being closed by a cover plate
attached to the slab face, and comprises the steps of: introducing the
sample into an ionic liquid; subjecting the liquid to a potential gradient
along the conduit length; the silicon providing good heat dissipation
characteristics which are enhanced by forming a channel with a high wall
surface to volume ratio.
In another embodiment of the invention, an improved gel electrophoresis
device with a first conduit having an inlet and an outlet is described,
the conduit being filled with an electrophoretic gel, and the device
includes means for applying an electric potential along the length of the
interior of the conduit inlet, and a detector positioned along the
conduit, the device characterized by all dimensions transverse to the
conduit being greater than 100 .mu.m, and the conduit being defined by a
silica slab having a channel in one face and a cover plate attached to the
one slab face to form the conduit. The benefits of the invention to such
fields as molecular biology research and biomedical diagnostic testing are
manifested as rapid, high resolution analysis of large, complex molecules
requiring only minute sample size. The field of application ranges from
DNA sequencing to diagnostic testing of genetic disease states and
microbial infection.
Using the devices or methods of this invention, the analysis of DNA and/or
proteins is accomplished with superior separation resolution and speed and
with concomitantly minute sample size. Furthermore, sensitivity is also
improved because of the integration of analytical components and
electronics within a silicon structure that includes chemistry, fluidics,
transducer and signal processing electronics. Silicon microelectronics
technology allows the precise structuring of channels in the micrometer
domain for the manipulation of minute sample volumes and the ability to
integrate electro-optics components within such channels for improved
signal/noise characteristics.
In accordance with another embodiment of the present invention, high
resolution separation of oligonucleotides, DNA fragments and proteins is
achieved in liquid-filled micro channels by applying the principles of
capillary electrophoresis. An electric field is applied to narrow bore
channels to effect both fluid flow and electrophoretic separation. The
narrow channels provide efficient field flux concentration with minimal
dispersion of migrating zones while also promoting nearly ideal plug flow
dynamics. When wall coating are deployed, chromatographic interactions
enhance the separation resolution of the migrating sample zone. The sample
composition may include but is not limited to DNA, RNA, proteins, lipids,
saccharides, and also intact cells such as viruses and bacteria.
It is the object of this invention to achieve superior DNA and proteins
separation resolution in liquid channels by electrophoresis and
chromatography. The focusing of electric fields within precisely machined
grooves and the elimination of thermal zone dispersion are primary
improvements.
Another object of this invention is to achieve high resolution separation
of biological molecules in gel filled channels.
Yet another object of this invention is to achieve separations at high
sample throughout rates and with minute sample size (<50 nanoliters).
A further object of this invention is to provide a means for actuating and
controlling fluid flow at the very low volumes necessary for biological
samples. Electro-osmotic pumping allows fluid flow control and EMF
actuated sample injection.
Especially it is the object of this invention to provide a fully integrated
diagnostic/analytical device that comprises: sample injection means,
separation means, marking means, detection, and onboard electronics to
effect signal processing and fluid movement.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other objects, advantages and features of this invention will
become apparent upon consideration of the following detailed description,
taken in conjunction with the following drawings:
FIG. 1 is a schematic representation of a separation device constructed in
accordance with a preferred embodiment of this invention;
FIG. 2 is a fragmentary plan view of a separation device constructed in
accordance with this invention utilizing a silicon slab;
FIG. 3 is a cross-sectional view of a conduit constructed using the silicon
slab of FIG. 2 together with a glass cover;
FIG. 4 is a schematic time representation of the manner in which the flow
of liquids through the conduit of this invention is maintained;
FIG. 5 is a diagrammatic representation of the manner in which a laser
detector is used in conjunction with the conduit and silicon slab of this
invention;
FIG. 6 is a pictorial representation of an alternative construction of the
conduit in a silicon slab of this invention utilizing a laser
photodetection system;
FIG. 7 is a plan view of plural channels formed in a single 100 mm diameter
silicon wafer;
FIGS. 8A, 8B, and 8C are plan side view and end elevation view of the
details of the reservoir construction used to provide an electrophoresis
separation device using a silicon slab according to another embodiment of
this invention;
FIG. 9 is a chromatogram in which emitted light is plotted as the ordinant
and time as the absissa depicting the separation of all of the
nucleotides; and
FIG. 10 is a chromatographic representation of ten polynucleotides in which
absorbed ultraviolet light is plotted as the ordinant and time as the
absissa.
DETAILED DESCRIPTION OF THE INVENTION
According to this invention, capillary sized conduits are constructed from
semiconductor materials using conduit dimensions in the 1 .mu.m domain by
fabrication methods generally applied by the
semiconductor-microelectronics industry Conduit dimensions and geometries
favorable for electro-osmotic fluid propulsion and electrophoretic and
chromatographic separations may be structured on semiconductor and
electrical insulator materials typically employed by the electronics
industry. Such materials include glass, silicon, germanium and metal
oxides. Especially appropriate are single crystal structural materials
such as silicon because very precise features may be micromachined on the
surface.
Silicon is a particularly suitable material because:
(1) Single crystal slab materials is obtainable in useful dimensions (i.g.
100 millimeter diameter, 500 .mu.m thickness).
(2) It has a high thermal conductivity.
(3) It is harder than steel.
(4) It may be modified to an insulator or conductor.
(5) It naturally develops electro-osmotic pressure with aqueous
electrolytes.
(6) Electronic and electro-optic components may be fabricated on it.
The preferred embodiment of this invention comprises: a channel <100
microns in diameter bounded by reservoirs (wells) etched onto a single
crystal Si slab, implanted in such channel and wells are electrodes and
the channel covered by a glass plate to allow the optical monitoring of
migrating molecules in the conduit thus formed. An alternate embodiment
comprises channel diameters >100 microns and filled with gels such as
polyacrylamide to achieve high resolution separation of large molecules.
The channel cross section is of trapezoidal geometry with the side walls
defined by the <111> crystal plane of silicon subtended by an angle of
54.7.degree. relative to the <100> crystal plane orientation. A laser beam
is directed across the conduit for photon capture considerations and with
optimal signal-to-noise characteristic. Other detectors may be adapted to
the structure including: chemFET's, electrodes, electrochemiluminescence,
mass spectrometry, optical fibers, wave guides, and piezoelectric sensors.
The structure of a preferred analytical device having a separation conduit
is seen with reference to FIGS. 1, 2, 3, and 5. FIG. 1 is a schematic
representation of the analytical device formed on a silicon slab
comprising the essential functional components, i.e., a separation conduit
10, electrodes 12 formed in the conduit, a reservoir 14, and a recipient
reservoir 16, a sample channel 18 and an injection conduit 20, and a
detector 22. Each electrode 12 is connected through a conductor lead 24 to
a bond pad 26, both being formed on the slab itself. A plan view of the
device is depicted in FIG. 2 and a three-dimensional cross-section of the
device is depicted in FIG. 3.
The analytical device is constructed by forming a channel or channels 32 in
a silicon wafer or slab 30 typically a silicon slab 500 .mu.m thick and
100 mm diameter yield channels that may be 80 ml long. These channels may
follow, of course, a tortuous path if longer channels are desired, as may
be seen by way of illustration in FIG. 7. Thus, a typical silicon slab 100
mm in diameter can provide several channels of varying lengths each with
its own buffer reservoir 14 and recipient reservoir 16. The channel 32 is
micromachined to have a trapezoidal cross-section. Preferably, the wall
angle from the groove bottom plane is 54.7.degree. and this angle (or wall
pitch) is uniformly maintained through the entire channel length. The
channel 32 as described is formed with a larger reservoir cavity 36 at
either end which provide the buffer and recipient reservoirs 14 and 16
respectively (FIG. 1). A silicon dioxide (SiO.sub.2) layer 32 is formed on
the top of the silicon slab and in the channel. Electrodes 12 are
implanted within the channel 32 and are connected to the bond pads 26 by
respective conductor leads 24. Electrical connections to external
instruments and power supplies may be attached to the bond pads. A glass
cover plate made of pyrex glass 38 is secured to the top surface 32 of the
slab 30 to provide a closed conduit to which the fluids may be directed
described. The cover plate 38 typically may be 2 mm thick while the
machine silicon slab 30 typically may be nominally taken from a circular
slab 100 mm in diameter and 500 .mu.m thick. The glass plate 38 is bonded
to the oxide layer 32 and sealed at the top edge of the trapezoid to
prevent liquid leaks that may shunt between several bond pads 26.
An auxiliary electrode 40 is formed on the underside of the glass plate 38
prior to securing it to the slab 30 such that each electrode 12 completely
surrounds the formed conduit for maximal contact with the fluids therein.
The auxiliary electrode 40 formed on the glass plate 38 may extend
slightly beyond the dimension of the channel 32 so as to afford good
contact with the conductor lead 32.
The bond pads 26, conductor leads 24, electrodes 12 and 40, are all formed
as will be described by vapor depositing a conductor directly on the
surface of either the silicon oxide or the glass as the case may be. Gold
is preferred for this purpose although other conductors used in the
semi-conductor industry such as tungsten, silver, copper, platinum, and
the like. Preferably a polyimide gasket is used to bond the silicon to the
glass plate and to prevent leakage beyond the confines of the channel,
which together with the glass plate form the conduit.
The polyimide gasket is formed of material typically 8 .mu.m thick and
during bonding undergoes a sixty percent compression particularly in the
region of the electrodes 12.
The electrodes 12 (and 40) are positioned at intervals along the length of
the conduit as will be later described and also in each reservoir 14 and
16.
The channel 32 is formed using conventional micro-machining techniques used
in the electronics industry in connection with semi-conductor devices. In
the preferred case at hand where the channel is formed in a silicon chip,
the device depicted in FIGS. 1, 2, and 3 is formed by the following steps:
(1) Develop the desired channel and reservoir pattern by photolithography
on a photo mask.
(2) Develop the etch protect mask pattern (by, SiO.sub.2 or Si.sub.3
N.sub.4) on a <100> oriented single crystal silicon wafer.
(3) Implant Boron (.about.10 .sup.20 atoms/cm.sup.3) at a prescribed depth
in the wafer as an etch stop.
(4) Anisotropically etch exposed silicon with ethylene diamine pyrocatechol
(in water). A timed etch alternatively may be used instead of the etch
stop.
(5) Thermally oxidize the top of the silicon to SiO.sub.2 especially within
the channel and reservoirs.
(6) Establish a desired mask pattern for the lead conductors, electrodes
and bond pads using suitable photo tools.
(7) Vapor deposit gold Au.degree. on the unmasked portions of the wafer to
form the lead conductors, electrodes and bond pads. Other conductors may
substitute for Au.degree. (e.g. W, Ag, Cu, Pt, etc.).
(8) Etch or ultrosonically drill access holes in the glass cover plate.
(9) Place a polyimide "gasket" pattern onto the silicon surface. Polyimide
is used to bond silicon to glass plate.
(10) Pressure bond glass cover plate to silicon slab. The polyimide gasket
is positioned along the groove top edges to effect a liquid tight seal. A
typical channel may be 10 .mu.m wide at the bottom, 38 .mu.m wide at the
top, and 20 .mu.m deep. Access holes (not shown) may be formed in the
glass plate 38 over each reservoirs 14, 16, to permit filling in the
buffer, as will be described, or sample 20.
Alternatively, steps 7 through 10 may be replaced by another fabrication
route. The Au.degree. lead conductor 24 may be formed by heavy doping of
the silicon by boron to yield a p.sup.30 (conductor) channel. The
electrophoresis drive electrode may be formed by either overlaying the
p.sup.+ termination within the channel by a layer of Au.degree. or the
p.sup.+ terminus itself can function as the electrode. Since the silicon
surface is relatively flat (i.e., encumbered by the Au.degree. lead
pattern that is elevated above the Si surface by the Au.degree.
thickness). A "Mallory" bond seals the glass plate directly to the silicon
slab. The Mallory bond is an electric field assisted glass - metal thermal
sealing process of the P.R. Mallory Co. The bond is formed by thermally
compressing at 400.degree. C. the Si/glass assembly under .about. 1200
volts.
Alternate channel-geometries cross-sections such as rectangular,
semicircular and V-shaped grooves may be created as follows:
(a) V-shaped channels are created by anisotropic etch of <100> silicon with
no etch stop.
(b) Rectangular channels are created by anisotropic etching of <110>
silicon.
(c) Semi-circular channels are created by isotropic etching with agitation
of etchant solution/substrate.
The trapezoidal channels are preferred because they accommodate light
absorbance detection measurements or more preferrably, a fluorescence
detection scheme as shown in FIG. (5). This diagram assumes nominal
channel dimensions of 38 .mu.m at the top of the trapezoid, 10 .mu.m at
the bottom and 20 .mu.m depth. An incident laser beam 40, 20 .mu.m wide,
can be comfortably positioned to penetrate the glass plate 38, reflect
from one wall 42 of the channel, cross the channel width and reflect from
the opposite wall 44 to pass back through the glass plate 38 and away from
the device surface. In so doing, any reporter labeled molecules within the
channel will fluoresce and the emitted radiation 46 is captured by a
photodetector 48 via a lens 50. A suitable reporter labeling, less
exciting and fluorescence detecting system is described in a published EPO
patent application No. 02 52 683 published Jan. 13, 1988. An improved
system is described in an article by Prober et al., Science, Vol. 238,
page 336, Oct. 16, 1987 and in EPO patent application No. 071,060,874,
issued May 23, 1989, as U.S. Pat. No. 4,833,332.
To increased channel length beyond the limitation of silicon wafer
diameter, the longitudinal geometry of the groove can assume "serpentine"
or coil patterns as seen in FIG. 7 these patterns can be built on devices
prepared with either Mallory and polyimide glass-to-silicon bonds.
In an alternative embodiment of the invention optical fibers or light
guides may be used to direct the laser beam through the channel. This
alternative embodiment shown in FIG. 6 preferrably uses a deep channel 60
having a rectangular cross section along its length and preferably a high
aspect ration of 10:1 (height:width). This enhances heat exchange with the
silicon which improves analytical performance. Optical fibers or light
guides 64 and 66 are positioned above the glass cover plate, which using
known techniques has a material density profile to focus light from the
fibers and compensate for the refractive index, to guide the light beam
directed through the fiber 64 will pass as depicted by the dotted lines 68
through the glass and the entire height of the conduit 60 be reflected
from the bottom surface 70 through the glass plate and return fiber 66 to
a suitable detector. Alternatively, radiation emitted from fluorescing
material passing through the channel is depicted by the line 72 and may be
detected by a suitable photo-detector 74 as hereinbefore described. The
deep channel pattern also improves the optical direction sensitivity by
increasing the optical path length while maintaining small channel
dimensions. Alternative detectors may be incorporated in the channel
geometry or adapted for use with the device. These detectors include Chem
FET's, electrochemiluminescence, mass spectrometers, waveguides and
pregoelectric sensors.
Returning now to FIGS. 1-3, the method of use of the analytical device is
now described. The reservoirs 14 and 16 are first filled with buffer
solution by injecting the fluid into buffer reservoir 14 via an access
hole (not shown) through the glass plate 38. Capillary action typically
fills the conduits 10 and 20 within seconds. Sample is injected via a
syringe through a hole (not shown) in the glass plate 38 into sample
chamber 26. Sample is then introduced into separation channel 20 by
electro-osmotic pumping, i.e., by applying a voltage between the
electrodes in the sample reservoir 18 and one of the downstream electrodes
12 (preferably electrode 70 which is closest to the injection conduits
intersection with the separation conduit. Excess sample is returned into
sample reservoir 26 by reverse polarity voltage applied to the sample
reservoir electrode and the electrode 72 upstream from the intersections.
The sample zone may be focused by establishing an EMF between the buffer
and recipient reservoirs electrodes to move through the separation conduit
past the detector 22. The detector signal is then recorded as a function
of time, reflecting the movement of molecules through the conduit 10 and
specifically the detector 22.
To achieve high resolution separation by molecular charge requires the
application of intense electric field gradients, of the order of 250
volts/cm. Rather than apply a large voltage along the entire length of the
conduit, much smaller voltages may be applied between more closely spaced
but staggered electrodes 122 as seen in FIG. 4 and yet maintain high
intensity fields. Such a voltage program scheme that applies small
voltages across staggered electrode pairs that straddle the sample zone as
it migrates down stream. The timing sequence for the application of
voltages follows the down-stream motion of the sample zone and the spacing
between electrode pairs is at least twice the zone width at the channel
end. In so doing the problem of electrolyte/solution break-down due to
electrolysis and gas generation is avoided. In the simplest case, a
voltage may be applied between the electrodes in the buffer and recipient
chambers to drive the electrophoresis.
Another embodiment of this invention is shown in FIGS. 7 and 8A, 8B, and
8C. In this embodiment a plurality of conduits are constructed as
previously described, but in this case are all constructed on a single
semi-conductor wafer. The conduits are all formed to accept gel
electrophoresis media and thus the conduit diameters are typically
provided to be >100 .mu.m. In this case reservoirs 14 and 16 are formed in
either end of the respective channels 32 which form the conduits. In this
case fifteen parallel channels, ranging typically in length from 56 mm to
135 mm, are illustrated. Two of the channels are in the form of
"serpentine" longitudinal geometry and thus are of greater length. In this
instance, however, the reservoirs are not formed in the semi-conductor
wafer, but rather are formed in the pyrex glass which covers the open
channels and the wafer to form the conduits. Thus, it is seen more clearly
in FIGS. 8A-8C. The silicon wafer 90 is formed as previously described to
have a channel 92 with an SiO.sub.2 upper surface. It also includes a
pyrex glass plate 94 which has an etched reservoir 96. The reservoir is
left open to the atmosphere. An electrode 98 is formed on the surface of
the reservoir and extends over to a bond pad 100. In this case the entire
bottom of the reservoir is formed with the electrode 98. A hole 102 is
formed in the bottom over the channel 92 so as to provide access thereto.
The hole passes through both the electrode and the glass 94. In a typical
case the reservoir 96, which is illustrated as circular, may be 3.2 mm in
diameter with a fill capacity of 3 .mu.m. In this case for electrophoretic
application the channel 92 may have a width of 380 .mu.m at the top, 100
.mu.m at the bottom, with a depth of 200 .mu.m. These dimensions allow a
wider diameter laser beam than that previously described and hence a
higher detection sensitivity, but with somewhat lower separation
resolution.
This device as noted above is specifically designed to be used with gel
media for electrophoresis separation. The method of use is as follows: The
groove is first filled with gel preparation fluid (e.g. monomer and
cross-linker of any suitable gel such as polyacrylamide gels). The liquid
is injected into reservoir 96 and the channel fills by capillary action up
to the reservoir 96. The gel typically sets within a few minutes. The
reservoirs are filled with an appropriate buffer. Sample is then injected
by a gas-chromatography type syringe (nominally 50 nanolites) into the
buffer filled reservoir 96 and the electrophoreses run, subsequently, by
applying a voltage to electrodes 98 between reservoirs at each end of each
conduit.
The invention employs a semiconductor microanalytical device and uses
principles of electrophoresis and chromatography to achieve separation
performance superior to that of the prior art on minute biological
samples. Electro-osmosis, electrophoresis and chromatography can be
practiced on silicon micromachined structures in a synergistic manner.
Electro-osmotic fluid flow actuation is best controlled when the channel
dimensions approach the electrical double layer thickness, which for all
practical purposes is accomplished with channel dimensions of <100 microns
diameter. Couple to the above the ability to integrate electronic
components in the conduit and the device of the invention is an analytical
instrument and method that can function in the microdimensional
environment to transport fluids and separate their content.
Several analytical functions become possible:
(a) The movement of nanoliter fluid volumes by proper application of
electromotive force (EMF).
(b) Electric field gates (or valves) to control passage of fluids and/or
particles.
(c) The routing of fluids through conduits by voltage actuation of
electrode implants within such conduits.
(d) Chromatographic separation of molecules by wall interactions.
(e) Integration of the detector within the device itself.
(f) All functions of an analytical instrument may be integrated within a
single Si wafer: Sample injection, separation, reagent introduction,
detection, signal conditioning circuitry, logic and on-board intelligence.
(g) The high resolution electrophoresis separation of molecules in liquid
and/or gel media.
EXAMPLE 1
(Demonstration of Principle-Capillary electrophoresis)
A capillary electrophoresis experiment run on a polynucleotide mixture
comprising polyadenosines of various polymer lengths from 2-mer to 20-mer
is shown on FIG. 9. This experiment was run on a fused silica tube, 50
microns in diameter and 60 cm length. 25 KV was applied and the separation
voltage and the sample injection was accomplished by a 5 sec. pulse at 5
KV. The oligonucleotides were detected by UV absorbance at 254 nm as they
migrate past the detector window as a function of time. This experiment is
an indication of the resolving power based solely on molecular charge
differentiation.
The test polynucleotide sample consists of polyadenosine fragments of
varying lengths, as opposed to oligonucleotides that comprise random
sequences of different nucleotides (e.g., adenosine, cytosine, guanosine,
and tyrosine). The fragment composition is as follows and corresponds to
the Peak Nos. as labeled on FIG. (5):
______________________________________
Peak # Component Theor. Plates
______________________________________
1 pb(A)2 131,777
2 pb(A)5 183,186
3 pb(A)6 141,859
4 pb(A)7 206,989
5 pb(A)8 181,468
6 pb(A)9 144,499
7 pb(A)10 133,510
8 pb(A)12 147,295
9 pb(A)16 183,322
10 pb(A)20 119,785
______________________________________
The sample was injected into the capillary by dipping the input capillary
orifice into a sample cup containing the polyadenosine fragments and
applying 5 KV for 5 seconds to a Pt electrode in the sample c | | |
