U.S. patent application number 09/745891 was filed with the patent office on 2001-08-16 for integrated monolithic microfabricated electrospray and liquid chromatography system and method.
Invention is credited to Davis, Timothy J., Galvin, Gregory J., Moon, James E..
Application Number | 20010013490 09/745891 |
Document ID | / |
Family ID | 22557808 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010013490 |
Kind Code |
A1 |
Moon, James E. ; et
al. |
August 16, 2001 |
Integrated monolithic microfabricated electrospray and liquid
chromatography system and method
Abstract
An electrospray device, a liquid chromatography device and an
electrospray liquid chromatography system are disclosed. The
electrospray device comprises a substrate defining a channel
between an entrance orifice on an injection surface and an exit
orifice on an ejection surface, a nozzle defined by a portion
recessed from the ejection surface surrounding the exit orifice,
and an electrode for application of an electric potential to the
substrate to optimize and generate an electrospray; and,
optionally, additional electrode(s) to further modify the
electrospray. The liquid chromatography device comprises a
separation substrate defining an introduction channel between an
entrance orifice and a reservoir and a separation channel between
the reservoir and an exit orifice, the separation channel being
populated with separation posts perpendicular to the fluid flow; a
cover substrate bonded to the separation substrate to enclose the
reservoir and the separation channel adjacent the cover substrate;
and, optionally, electrode(s) for application of a electric
potential to the fluid. The exit orifice of the liquid
chromatography device may be homogeneously interfaced with the
entrance orifice of the electrospray device to form an integrated
single system. An array of multiple systems may be fabricated in a
single monolithic chip for rapid sequential fluid processing and
generation of electrospray for subsequent analysis, such as by
positioning the exit orifices of the electrospray devices near the
sampling orifice of a mass spectrometer.
Inventors: |
Moon, James E.; (Ithaca,
NY) ; Davis, Timothy J.; (Trumansburg, NY) ;
Galvin, Gregory J.; (Ithaca, ~) |
Correspondence
Address: |
BROWN PINNISI & MICHAELS
400 M & T BANK BUILDING
118 NORTH TIOGA ST
ITHACA
NY
14850
|
Family ID: |
22557808 |
Appl. No.: |
09/745891 |
Filed: |
December 21, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09745891 |
Dec 21, 2000 |
|
|
|
09156037 |
Sep 17, 1998 |
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Current U.S.
Class: |
210/198.3 |
Current CPC
Class: |
G01N 30/6095 20130101;
Y10T 29/49002 20150115; Y10T 436/255 20150115; Y10T 436/25375
20150115; G01N 30/7266 20130101; H01J 49/167 20130101; H01J 49/0018
20130101 |
Class at
Publication: |
210/198.3 |
International
Class: |
B01D 015/08 |
Claims
What is claimed is:
1. A chemical separation device, comprising: a substrate defining a
channel, said channel extending between a channel exit on one end
and a channel entrance on another end; a plurality of posts
fabricated from a portion of said substrate and extending from and
perpendicular to a bottom of said channel, wherein at least one of
said channel and said plurality of posts are formed at least in
part by reactive-ion etching; and said substrate is of silicon.
2. The device of claim 1, wherein said substrate further comprises
an isolating layer provided over a surface of said channel and a
surface of said plurality of posts.
3. The device of claim 2, wherein said isolating layer chemically
and/or electrically isolates an exterior surface of said device
from said substrate.
4. The device of claim 3, wherein said isolating layer is silicon
oxide.
5. The device of claim 4, wherein said silicon oxide is grown by
thermal oxidation of silicon.
6. The device of claim 4, wherein said silicon oxide layer is
deposited using a deposition technique.
7. The device of claim 1, wherein said plurality of posts are
spaced apart from each other by no more than 5 microns measured
edge to edge.
8. The device of claim 1, wherein said plurality of posts in said
separation channel are arranged at least one of periodically,
semi-periodically, and randomly.
9. The device of claim 1, wherein an arrangement of said plurality
of posts in said separation channel is such that only nonlinear
lines of fluid flow exist between a fluid reservoir connected to
said channel entrance and said channel exit.
10. The device of claim 1, further comprising a stationary phase
bound to said plurality of posts, said plurality of posts providing
interaction with an analyte introduced into said channel, thereby
producing separation of said analyte.
11. The device of claim 1, further comprising means for applying
electrical potential to a fluid at one or more locations in said
channel.
12. The device of claim 1, wherein said substrate defines at least
one additional channel, said at least one additional channel
containing a plurality of posts fabricated from said substrate and
extending from and perpendicular to a bottom of said at least one
additional channel.
13. The device of claim 12, further comprising means for applying
electrical potential to fluids at one or more locations in said at
least one additional channel.
14. The device of claim 1, further comprising controlling circuitry
for said chemical separation device integrated on said
substrate.
15. A chemical separation system, comprising: a first substrate
having a first surface and a second surface, said first substrate
defining (a) an entrance opening on said first surface, (b) a fluid
reservoir recessed from said second surface, (c) a first channel
extending between said entrance opening and said reservoir, (d) a
second channel recessed from said second surface, and (e) a
plurality of posts extending from and perpendicular to a bottom of
said second channel; and a cover substrate attached to said first
substrate to enclose said reservoir and said second channel
adjacent said cover substrate; wherein at least one of said first
substrate and said cover substrate defines an exit; wherein said
second channel extends between said exit and said reservoir, and
wherein at least one of said entrance opening, said reservoir, said
first channel, said second channel, and said plurality of posts are
formed at least in part by reactive-ion etching.
16. The system of claim 15, wherein at least one of said first
substrate and said cover substrate is of silicon.
17. The system of claim 15, further comprising an insulating layer
over said fluid reservoir, said first channel, said second channel,
and said plurality of posts.
18. The system of claim 17, wherein said insulating layer is
silicon oxide.
19. The system of claim 18, wherein said silicon oxide is grown by
thermal oxidation of silicon.
20. The system of claim 18, wherein said silicon oxide is deposited
by a deposition technique.
21. The system of claim 15, further comprising a stationary phase
bound to said plurality of posts, said plurality of posts providing
interaction with an analyte introduced into said second channel for
producing separation in said analyte.
22. The system of claim 15, further comprising means for applying
electrical potential to a fluid at least one location, said
location selected from the group consisting of said fluid
reservoir, said second channel, and said exit.
23. The system of claim 15, wherein each of said plurality of posts
are spaced apart from each other by no more than 5 microns,
measured edge to edge.
24. The system of claim 15, wherein said plurality of posts in said
second channel are arranged at least one of periodically,
semi-periodically, and randomly.
25. The device of claim 15, wherein an arrangement of said
plurality of posts in said second channel is such that only
nonlinear lines of fluid flow exist between said fluid reservoir
and said exit.
26. The system of claim 15, further comprising: a multiplicity of
entrance openings on said first surface; an equal multiplicity of
fluid reservoirs recessed from said second surface; a multiplicity
of first channels extending between each of said multiplicity of
entrance openings and a corresponding one of said multiplicity of
fluid reservoirs; wherein said second channel recessed from said
second surface extends between said exit and every one of said
multiplicity of fluid reservoirs.
27. The system of claim 15, further comprising: a plurality of
additional entrance openings on said first surface; a plurality of
additional reservoirs recessed from said second surface, each
additional reservoir corresponding to one of said plurality of
additional entrance openings; a plurality of additional first
channels, each corresponding to and extending between one of said
plurality of additional entrance openings and its corresponding
additional reservoir; and a plurality of additional second channels
recessed from said second surface, wherein one of said first
substrate and said cover substrate defines a plurality of
additional exits, each additional exit corresponding to one of said
plurality of additional reservoirs; wherein each additional second
channel corresponds to and extends between one of said plurality of
additional reservoirs and said corresponding additional exit.
28. The system of claim 27, further comprising at least one means
for applying electrical potential to fluids at one or more
locations in said plurality of additional fluid reservoirs,
additional second channels and additional exits.
29. The system of claim 15, further comprising controlling
circuitry for said chemical separation system, said circuitry being
integrated on at least one of said first and
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional patent application of copending
application Ser. No. 09/156,037, filed Sep. 17, 1998, entitled
"Integrated Monolithic Microfabricated Electrospray And Liquid
Chromatography System and Method", which received notice of
allowance on Sep. 12, 2000. The aforementioned application is
hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to an integrated
miniaturized chemical analysis system fabricated using
microelectromechanical systems (MEMS) technology. In particular,
the present invention relates to an integrated monolithic
microfabricated electrospray and liquid chromatography device. This
achieves a significant advantage in terms of high-throughput
analysis by mass spectrometry, as used, for example, in drug
discovery, in comparison to a conventional system.
BACKGROUND OF THE INVENTION
[0003] New developments in drug discovery and development are
creating new demands on analytical techniques. For example,
combinatorial chemistry is often employed to discover new lead
compounds, or to create variations of a lead compound.
Combinatorial chemistry techniques can generate thousands or
millions of compounds (combinatorial libraries) in a relatively
short time (on the order of days to weeks). Testing such a large
number of compounds for biological activity in a timely and
efficient manner requires high-throughput screening methods which
allow rapid evaluation of the characteristics of each candidate
compound.
[0004] The compounds in combinatorial libraries are often tested
simultaneously against a molecular target. For example, an enzyme
assay employing a colorimetric measurement may be run in a 96-well
plate. An aliquot of enzyme in each well is combined with tens or
hundreds of compounds. An effective enzyme inhibitor will prevent
development of color due to the normal enzyme reaction, allowing
for rapid spectroscopic (or visual) evaluation of assay results. If
ten compounds are present in each well, 960 compounds can be
screened in the entire plate, and one hundred thousand compounds
can be screened in 105 plates, allowing for rapid and automated
testing of the compounds.
[0005] Often, however, determination of which compounds are present
in certain portions of a combinatorial library is difficult, due to
the manner of synthesis of the library. For example, the
"split-and-pool" method of random peptide synthesis in U.S. Pat.
No. 5,182,366, describes a way of creating a peptide library where
each resin bead carries a unique peptide sequence. Placing ten
beads in each well of a 96-well plate, followed by cleavage of the
peptides from the beads and removal of the cleavage solution, would
result in ten (or fewer) peptides in each well of the plate. Enzyme
assays could then be carried out in the plate wells, allowing
100,000 peptides to be screened in 105 plates. However, the
identity of the peptides would not be known, requiring analysis of
the contents of each well.
[0006] The peptides could be analyzed by removing a portion of
solution from each well and injecting the contents into a
separation device such as liquid chromatography or capillary
electrophoresis instrument coupled to a mass spectrometer. Assuming
that such a method would take approximately 5 minutes per analysis,
it would require over a month to analyze the contents of 105
96-well plates, assuming the method was fully automated and
operating 24 hours a day.
[0007] This example illustrates the critical need for a method for
rapid analysis of large numbers of compounds or complex mixtures of
compounds, particularly in the context of high-throughput
screening. Techniques for generating large numbers of compounds,
for example through combinatorial chemistry, have been established.
High-throughput screening methods are under development for a wide
variety of targets, and some types of screens, such as the
calorimetric enzyme assay described above and ELISA (enzyme linked
immunosorbent assay) technology, are well established. As indicated
in the example above, a bottleneck often occurs at the stage where
multiple mixtures of compounds, or even multiple individual
compounds, must be characterized.
[0008] This need is further underscored when current developments
in molecular biotechnology are considered. Enormous amounts of
genetic sequence data are being generated through new DNA
sequencing methods. This wealth of new information is generating
new insights into the mechanism of disease processes. In
particular, the burgeoning field of genomics has allowed rapid
identification of new targets for drug development efforts.
Determination of genetic variations between individuals has opened
up the possibility of targeting drugs to individuals based on the
individual's particular genetic profile. Testing for cytotoxicity,
specificity, and other pharmaceutical characteristics could be
carried out in high-throughput assays instead of expensive animal
testing and clinical trials. Detailed characterization of a
potential drug or lead compound early in the drug development
process thus has the potential for significant savings both in time
and expense.
[0009] Development of viable screening methods for these new
targets will often depend on the availability of rapid separation
and analysis techniques for analyzing the results of assays. For
example, an assay for potential toxic metabolites of a candidate
drug would need to identify both the candidate drug and the
metabolites of that candidate. An assay for specificity would need
to identify compounds which bind differentially to two molecular
targets such as a viral protease and a mammalian protease.
[0010] It would therefore be advantageous to provide a method for
efficient proteomic screening in order to obtain the
pharmacokinetic profile of a drug early in the evaluation process.
An understanding of how a new compound is absorbed in the body and
how it is metabolized can enable prediction of the likelihood for
an increased therapeutic effect or lack thereof.
[0011] Given the enormous number of new compounds that are being
generated daily, an improved system for identifying molecules of
potential therapeutic value for drug discovery is also critically
needed.
[0012] It also would be desirable to provide rapid sequential
analysis and identification of compounds which interact with a gene
or gene product that plays a role in a disease of interest. Rapid
sequential analysis can overcome the bottleneck of inefficient and
time-consuming serial (one-by-one) analysis of compounds.
[0013] Accordingly, there is a critical need for high-throughput
screening and identification of compound-target reactions in order
to identify potential drug to. candidates.
[0014] Microchip-based separation devices have been developed for
rapid analysis of large numbers of samples. Compared to other
conventional separation devices, these microchip-based separation
devices have higher sample throughput, reduced sample and reagent
consumption and reduced chemical waste. The liquid flow rates for
microchip-based separation devices range from approximately 1-300
nanoliters (nL) per minute for most applications.
[0015] Examples of microchip-based separation devices include those
for capillary electrophoresis (CE), capillary electrochromatography
(CEC) and high-performance liquid chromatography (HPLC). See
Harrison et al, Science 1993, 261, 859-897; Jacobson et al. Anal.
Chem. 1994, 66, 1114-1118; and Jacobson et al. Anal. Chem. 1994,
66, 2369-2373. Such separation devices are capable of fast analyses
and provide improved precision and reliability compared to other
conventional analytical instruments.
[0016] Liquid chromatography (LC) is a well-established analytical
method for separating components of a fluid for subsequent analysis
and/or identification. Traditionally, liquid chromatography
utilizes a separation column, such as a cylindrical tube, filled
with tightly packed beads, gel or other appropriate particulate
material to provide a large surface area. The large surface area
facilitates fluid interactions with the particulate material, and
the tightly packed, random spacing of the particulate material
forces the liquid to travel over a much longer effective path than
the length of the column. In particular, the components of the
fluid interact with the stationary phase (the particles in the
liquid chromatography column) as well as the mobile phase (the
liquid eluent flowing through the liquid chromatography column)
based on the partition coefficients for each of the components. The
partition coefficient is a defined as the ratio of the time an
analyte spends interacting with the stationary phase to the time
spent interacting with the mobile phase. The longer an analyte
interacts with the stationary phase, the higher the partition
coefficient and the longer the analyte is retained on the liquid
chromatography column. The components may be detected
spectroscopically after elution from the liquid chromatography
column by coupling the exit of the column to a post-column
detector.
[0017] Spectroscopic detectors rely on a change in refractive
index, ultraviolet and/or visible light absorption, or fluorescence
after excitation with a suitable wavelength to detect the separated
components. Alternatively, the separated components may be passed
from the liquid chromatography column into other types of
analytical instruments for analysis. The analysis outcome depends
upon the sequenced arrival of the components separated by the
liquid chromatography column and is therefore time-dependent.
[0018] The length of liquid transport from the liquid
chromatography column to the analysis instrument such as the
detector is preferably minimized in order to minimize diffusion and
thereby maximize the separation efficiency and analysis
sensitivity. The transport length is referred to as the dead volume
or extra-column volume. Capillary electrophoresis is a technique
that utilizes the electrophoretic nature 25 of molecules and/or the
electroosmotic flow of fluids in small capillary tubes to separate
components of a fluid. Typically a fused silica capillary of 100 um
inner diameter or less is filled with a buffer solution containing
an electrolyte. Each end of the capillary is placed in a separate
fluidic reservoir containing a buffer electrolyte.
[0019] A potential voltage is placed in one of the buffer
reservoirs and a second potential voltage is placed in the other
buffer reservoir. Positively and negatively charged species will
migrate in opposite directions through the capillary under the
influence of the electric field established by the two potential
voltages applied to the buffer reservoirs. Electroosmotic flow is
defined as the fluid flow along the walls of a capillary due to the
migration of charged species from the buffer solution. Some
molecules exist as charged species when in solution and will
migrate through the capillary based on the charge-to-mass ratio of
the molecular species. This migration is defined as electrophoretic
mobility. The electroosmotic flow and the electrophoretic mobility
of each component of a fluid determine the overall migration for
each fluidic component. The fluid flow profile resulting from
electroosmotic flow is flat due to the reduction in frictional drag
along the walls of the separation channel. This results in improved
separation efficiency over liquid chromatography where the flow
profile is parabolic resulting from pressure driven flow.
[0020] Capillary electrochromatography is a hybrid technique which
utilizes the electrically driven flow characteristics of
electrophoretic separation methods within capillary columns packed
with a solid stationary phase typical of liquid chromatography. It
couples the separation power of reversed-phase liquid
chromatography with the high efficiencies of capillary
electrophoresis. Higher efficiencies are obtainable for capillary
electrochromatography separations over liquid chromatography
because the flow profile resulting from electroosmotic flow is flat
due to the reduction in frictional drag along the walls of the
separation channel when compared to the parabolic flow profile
resulting from pressure driven flows. Furthermore, smaller particle
sizes can be used in capillary electrochromatography than in liquid
chromatography because no back pressure is generated by
electroosmotic flow. In contrast to electrophoresis, capillary
electrochromatography is capable of separating neutral molecules
due to analyte partitioning between the stationary and mobile
phases of the column part particles using a liquid chromatography
separation mechanism.
[0021] The separated product of such separation devices may be
introduced as the liquid sample to a device that is used to produce
electrospray ionization. The electrospray device may be interfaced
to an atmospheric pressure ionization mass spectrometer (API-MS)
for analysis of the electrosprayed fluid.
[0022] A schematic of an electrospray system 50 is shown in FIG. 1.
An electrospray is produced when a sufficient electrical potential
difference Vspray is applied between a conductive or partly
conductive fluid exiting a capillary orifice and an electrode so as
to generate a concentration of electric field lines emanating from
the tip or end of a capillary 52 of an electrospray device. When a
positive voltage Vspray is applied to the tip of the capillary
relative to an extracting electrode 54, such as one provided at the
ion-sampling orifice to the mass spectrometer, the electric field
causes positively-charged ions in the fluid to migrate to the
surface of the fluid at the tip of the capillary. When a negative
voltage Vspray is applied to the tip of the capillary relative to
an extracting electrode 54, such as one provided at the
ion-sampling orifice to the mass spectrometer, the electric field
causes negatively-charged ions in the fluid to migrate to the
surface of the fluid at the tip of the capillary.
[0023] When the repulsion force of the solvated ions exceeds the
surface tension of the fluid sample being electrosprayed, a volume
of the fluid sample is pulled into the shape of a cone, known as a
Taylor cone 56 which extends from the tip of the capillary. Small
charged droplets 58 are formed from the tip of the Taylor cone 56
and are drawn toward the extracting electrode 54. This phenomenon
has been described, for example, by Dole et al., Chem. Phys. 1968,
49, 2240 and Yamashita and Fenn, J. Phys. Chem. 1984, 88, 445 1.
The potential voltage required to initiate an electrospray is
dependent on the surface tension of the solution as described by,
for example, Smith, IEEE Trans. Ind Appl 1986, IA-22, 527-535.
Typically, the electric field is on the order of approximately 106
V/m The physical size of the capillary determines the density of
electric field lines necessary to induce electrospray.
[0024] One advantage of electrospray ionization is that the
response for an analyte measured by the mass spectrometer detector
is dependent on the concentration of the analyte in the fluid and
independent of the fluid flow rate. The response of an analyte in
solution at a given concentration would be comparable using
electrospray ionization combined with mass spectrometry at a flow
rate of 100 uL/min compared to a flow rate of 100 nL/min.
[0025] The process of electrospray ionization at flow rates on the
order of nanoliters per minute has been referred to as
"nanoelectrospray". Electrospray into the ionsampling orifice of an
API mass spectrometer produces a quantitative response from the
mass spectrometer detector due to the analyte molecules present in
the liquid flowing from the capillary.
[0026] Thus, it is desirable to provide an electrospray ionization
device for integration upstream with microchip-based separation
devices and for integration downstream with API-MS instruments.
[0027] Attempts have been made to manufacture an electrospray
device which produces nanoelectrospray. For example, Wilm and Mann,
Anal. Chem. 1996, 68, 1-8 describes the process of electrospray
from fused silica capillaries drawn to an inner diameter of 2-4 um
at flow rates of 20 nL/min. Specifically, a nanoelectrospray at 20
nL/min was achieved from a 2 um inner diameter and 5 gm outer
diameter pulled fused-silica capillary with 600-700 V at a distance
of 1-2 mm from the ion-sampling orifice of an API mass
spectrometer.
[0028] Ramsey et al., Anal. Chem. 1997, 69, 1174-1178 describes
nanoelectrospray at 90 nL/min from the edge of a planar glass
microchip with a closed separation channel 10 um deep, 60 um wide
and 33 mm in length using electroosmotic flow and applying 4.8 kV
to the fluid exiting the closed separation channel on the edge of
the microchip for electrospray formation, with the edge of the chip
at a distance of 3-5
[0029] mm from the ion-sampling orifice of an API mass
spectrometer. Approximately 12 nL of the sample fluid collects at
the edge of the chip before the formation of a Taylor cone and
stable nanoelectrospray from the edge of the microchip. However,
collection of approximately 12 nL of the sample fluid will result
in remixing of the fluid, thereby undoing the separation done in
the separation channel. Remixing causes band broadening at the edge
of the microchip, fundamentally limiting its applicability for
nanoelectrospray-mass spectrometry for analyte detection. Thus,
nanoelectrospray from the edge of this microchip device after
capillary electrophoresis or capillary electrochromatography
separation is rendered impractical. Furthermore, because this
device provides a flat surface, and thus a relatively small amount
of physical asperity, for the formation of the electrospray, the
device requires an impractically high voltage to initiate
electrospray, due to poor field line concentration.
[0030] Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.;
McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430
describes a stable nanoelectrospray from the edge of a planar glass
microchip with a closed channel 25 um deep, 60 um wide and 35-50
mm, in length and applying 4.2 kV to the fluid exiting the closed
separation channel on the edge of the microchip for electrospray
formation, with the edge of the chip at a distance of 3-8 mm from
the ion-sampling orifice of an API mass spectrometer. A syringe
pump is utilized to deliver the sample fluid to the glass microchip
electrosprayer at a flow rate between 100-200 nL/min The edge of
the glass microchip is treated with a hydrophobic coating to
alleviate some of the difficulties associated with nanoelectrospray
from a flat surface and which slightly improves the stability of
the nanoelectrospray. Electrospraying in this manner from a flat
surface again results in poor field line concentration and yields
an inefficient electrospray.
[0031] Desai et al. 1997 International Conference on Solid-State
Sensors and Actuators, Chicago, Jun. 16-19, 1997, 927-930 describes
a multi-step process to generate a nozzle on the edge of a silicon
microchip 1-3 um in diameter or width and 40 gm in length and
applying 4 kV to the entire microchip at a distance of 0.25-mm from
the ion-sampling orifice of an API mass spectrometer. This
nanoelectrospray nozzle reduces the dead volume of the sample
fluid. However, the extension of the nozzle from the edge of the
microchip exposes the nozzle to accidental breakage. Because a
relatively high spray voltage was utilized and the nozzle was
positioned in very close proximity to the mass spectrometer
sampling orifice, a poor field line concentration and a low
efficient electrospray were achieved.
[0032] In all of the above-described devices, edge-spraying from a
monolithic chip is a poorly controlled process due to the inability
to rigorously and repeatably determine the physical form of the
chip's edge. In another embodiment of edge-spraying, ejection
nozzles, such as small segments of drawn capillaries, are
separately and individually attached to the chip's edge. This
process is inherently cost-inefficient and unreliable, imposes
space constraints in chip design, and is therefore unsuitable for
manufacturing.
[0033] Thus, it is also desirable to provide an electrospray
ionization device with controllable spraying and a method for
producing such a device which is easily reproducible and
manufacturable in high volumes.
SUMMARY OF THE INVENTION
[0034] The present invention provides a silicon microchip-based
electrospray device for producing reproducible, controllable and
robust nanoelectrospray ionization of a liquid sample. The
electrospray device may be interfaced downstream to an atmospheric
pressure ionization mass spectrometer (API-MS) for analysis of the
electrosprayed fluid and/or interfaced upstream to a miniaturized
liquid phase separation device, which may have, for example, glass,
plastic or silicon substrates or wafers.
[0035] The electrospray device of the present invention generally
comprises a silicon substrate or microchip defining a channel
between an entrance orifice on an injection surface and a nozzle on
an ejection surface (the major surface) such that the electrospray
generated by the electrospray device is generally approximately
perpendicular to the ejection surface. The nozzle has an inner and
an outer diameter and is defined by an annular portion recessed
from the ejection surface. The annular recess extends radially from
the outer diameter. The tip of the nozzle is co-planar or level
with and does not extend beyond the ejection surface and thus the
nozzle is protected against accidental break-age. The nozzle,
channel and recessed portion are etched from the silicon substrate
by reactive-ion etching and other standard semiconductor processing
techniques.
[0036] All surfaces of the silicon substrate preferably have a
layer of silicon dioxide thereon created by oxidization to
electrically isolate the liquid sample from the substrate and the
ejection and injection surfaces from each other such that different
potential voltages may be individually applied to each surface and
the liquid sample. The silicon dioxide layer also provides for
biocompatibility. The electrospray 25 apparatus further comprises
at least one controlling electrode electrically contacting the
substrate through the oxide layer for the application of an
electric potential to the substrate.
[0037] Preferably, the nozzle, channel and recess are etched from
the silicon substrate by reactive-ion etching and other standard
semiconductor processing techniques. The injection--side
feature(s), through-substrate fluid channel, ejection- side
features, and controlling electrodes--are formed monolithically
from a monocrystalline silicon substrate. That is, they are formed
during the course of and as a result of a fabrication sequence that
requires no manipulation or assembly of separate components.
[0038] Because the electrospray device is manufactured using
reactive-ion etching and other standard semiconductor processing
techniques, the dimensions of such a device can be very small, for
example, as small as 2 um inner diameter and 5 um outer diameter.
Thus, a nozzle having, for example, 5 um inner diameter and 250 um
in height only has a volume of 4.9 pL (picoliter). In contrast, an
electrospray device from the flat edge of a glass microchip would
introduce additional dead volume of 12 nL compared to the volume of
a separation channel of 19.8 nL thereby allowing remixing of the
fluid components and undoing the separation done by the separation
channel. The micrometer-scale dimensions of the electrospray device
minimizes the dead volume and thereby increases efficiency and
analysis sensitivity.
[0039] The electrospray device of the present invention provides
for the efficient and effective formation of an electrospray. By
providing an electrospray surface from which the fluid is ejected
with dimensions on the order of micrometers, the electrospray
device limits the voltage required to generate a Taylor cone as the
voltage is dependent upon the nozzle diameter, surface tension of
the fluid and the distance of the nozzle from the extracting
electrode. The nozzle of the electrospray device provides the
physical asperity on the order of micrometers on which a large 25
electric field is concentrated. Further, the electrospray device
may provide additional electrode(s) on the ejecting surface to
which electric potential(s) may be applied and controlled
independent of the electric potentials of the fluid and the
extracting electrode in order to advantageously modify and optimize
the electric field. The combination of the nozzle and the
additional electrode(s) thus enhance the electric field between the
nozzle and the extracting electrode. The large electric field, on
the order of 106 V/m or greater and generated by the potential
difference between the fluid and extracting electrode, is thus
applied directly to the fluidic cone rather than uniformly
distributed in space.
[0040] The microchip-based electrospray ionization device of the
present invention provides minimal extra-column dispersion as a
result of a reduction in the extra-column volume and provides
efficient, reproducible, reliable and rugged formation of an
electrospray. The design of the ionization device is also robust
such that the electrospray device can be readily mass-produced in a
cost-effective, high-yielding process.
[0041] In operation, a conductive or partly conductive liquid
sample is introduced into the channel through the entrance orifice
on the injection surface. The liquid sample and nozzle are held at
the potential voltage applied to the fluid, either by means of a
wire within the fluid delivery channel to the electrospray device
or by means of an electrode formed on the injection surface
isolated from the surrounding surface region and from the
substrate. The electric field strength at the tip of the nozzle is
enhanced by the application of a voltage to the substrate and/or
the ejection surface, preferably approximately less than one-half
of the voltage applied to the fluid. Thus, by the independent
control of the fluid/nozzle and substrate/ejection surface
voltages, the electrospray device of the present invention allows
the optimization of the electric field lines emanating from the
nozzle. Further, when the electrospray device is interfaced
downstream with a mass spectrometry device, the independent control
of the fluid/nozzle and substrate/ejection surface voltages also
allows for the direction and optimization of the electrospray into
an acceptance region of the mass spectrometry device.
[0042] The electrospray device of the present invention may be
placed 1-2 mm or up to 10 mm from the orifice of an API mass
spectrometer to establish a stable nanoelectrospray at flow rates
as low as 20 nL/min with a voltage of, for example, 700 V applied
to the nozzle and 0-350 V applied to the substrate and/or the
planar ejection surface of the silicon microchip.
[0043] An array or matrix of multiple electrospray devices of the
present invention may be manufactured on a single microchip as
silicon fabrication using standard, well-controlled thin-film
processes not only eliminates handling of such micro components but
also allows for rapid parallel processing of functionally alike
elements. The nozzles may be radially positioned about a circle
having a relatively small diameter near the center of the chip.
Thus, the electrospray device of the present invention provides
significant advantages of time and cost efficiency, control, and
reproducibility. The low cost of these electrospray devices allows
for one-time use such that cross-contamination from different
liquid samples may be eliminated.
[0044] The electrospray device of the present invention can be
integrated upstream with miniaturized liquid sample handling
devices and integrated downstream with an API mass spectrometer.
The electrospray device may be chip-to-chip or wafer-to-wafer
bonded to silicon microchip-based liquid separation devices capable
of, for example, capillary electrophoresis, capillary
electrochromatography, affinity chromatography, liquid
chromatography (LC) or any other condensed-phase separation
technique. The electrospray device may be alternatively bonded to
glass and/or polymer-based liquid separation devices with any
suitable method.
[0045] In another aspect of the invention, a microchip-based liquid
chromatography device may be provided. The liquid chromatography
device generally comprises a separation substrate or wafer defining
an introduction channel between an entrance orifice and a reservoir
and a separation channel between the reservoir and an exit orifice.
The separation channel is populated with separation posts extending
from a side wall of the separation channel perpendicular to the
fluid flow though the separation channel. Preferably, the
separation posts do not extend beyond and are preferably coplanar
or level with the surface of the separation substrate such that
they are protected against accidental breakage during the
manufacturing process. Component separation occurs in the
separation channel where the separation posts perform the liquid
chromatography function by providing large surface areas for the
interaction of fluid flowing through the separation channel. A
cover substrate may be bonded to the separation substrate to
enclose the reservoir and the separation channel adjacent the cover
substrate.
[0046] The liquid chromatography device may further comprise one or
more electrodes for application of electric potentials to the fluid
at locations along the fluid path. The application of different
electric potentials along the fluid path may facilitate the fluid
flow through the fluid path.
[0047] The introduction and separation channels, the entrance and
exit orifices and the separation posts are preferably etched from a
silicon substrate by reactive-ion etching and other standard
semiconductor processing techniques. The separation posts are
preferably oxidized silicon posts which may be chemically modified
to optimize the interaction of the components of the sample fluid
with the stationary separation posts.
[0048] In another aspect of the invention, the liquid
chromatography device may be integrated with the electrospray
device such that the exit orifice of the liquid chromatography
device forms a homogenous interface with the entrance orifice of
the electrospray device, thereby allowing the on-chip delivery of
fluid from the liquid chromatography device to the electrospray
device to generate an electrospray. The nozzle, channel and
recessed portion of the electrospray device may be etched from the
cover substrate of the liquid chromatography device.
[0049] In yet another aspect of the invention, multiples of the
liquid chromatography--electrospray system may be formed on a
single chip to deliver a multiplicity of samples to a common point
for subsequent sequential analysis. The multiple nozzles of the
electrospray devices may be radially positioned about a circle
having a relatively small diameter near the center of the single
chip.
[0050] The radially distributed array of electrospray nozzles on a
multi-system chip may be interfaced with a sampling orifice of a
mass spectrometer by positioning the nozzles near the sample
orifice. The tight radial configuration of the electrospray nozzles
allows the positioning thereof in close proximity to the sampling
orifice of a mass spectrometer.
[0051] The multi-system chip thus provides a rapid sequential
chemical analysis system fabricated using microelectromechanical
systems (MEMS) technology. For example, the multi-system chip
enables automated, sequential separation and injection of a
multiplicity of samples, resulting in significantly greater
analysis throughput and utilization of the mass spectrometer
instrument for, for example, high-throughput detection of compounds
for drug discovery.
BRIEF DESCRIPTION OF THE DRAWING
[0052] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawings will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0053] FIG. 1 shows a schematic of an electrospray system;
[0054] FIG. 2 shows a perspective view of an electrospray device of
the present invention; along line 4-4;
[0055] FIG. 3 shows a plan view of the electrospray device of FIG.
2;
[0056] FIG. 4 shows a cross-sectional view of the electrospray
device of FIG. 3 taken
[0057] FIG. 5 shows a schematic of an electrospray system
comprising an electrospray device of the present invention;
[0058] FIG. 6 shows a plan view of an electrospray device having
multiple electrodes on the ejection surface of the device;
[0059] FIG. 7 shows a cross-sectional view of the electrospray
device of FIG. 6 taken along line 7-7;
[0060] FIG. 8 illustrates a feedback control circuit incorporating
an electrospray device of the present invention;
[0061] FIGS. 9-20G show an example of a fabrication sequence of the
electrospray device;
[0062] FIG. 21A shows a cross-sectional view of a piezoelectric
pipette positioned a distance from and for delivery of a fluid
sample to the entrance orifice of the electrospray device;
[0063] FIG. 21B shows a cross-sectional view of a capillary for
delivery of a fluid 25 sample to and prior to attachment to the
entrance orifice of the electrospray device;
[0064] FIG. 22 shows a schematic of a single integrated system
comprising an upstream fluid delivery device and an electrospray
device having a homogeneous interface with the fluid delivery
device; FIG. 23A shows an exploded perspective view of a chip-based
combinatorial chemistry system comprising a reaction well block and
a daughter plate;
[0065] FIG. 23B shows a cross-sectional view of the chip-based
combinatorial chemistry system of FIG. 23A taken along line M-2313;
FIGS. 24A and 24B are color photographs of a real Taylor cone
emanating from an integrated silicon chip-based nozzle;
[0066] FIGS. 24C and 24D are perspective and side cross-sectional
views, respectively, of the electrospray device and mass
spectrometry system of FIGS. 24A and 24B;
[0067] FIG. 24E shows a mass spectrum of I ug/mL PPG425 in 50%
water, 50% methanol containing 0.1% formic acid, 0.1% acetonitrile
and 2 mM ammonium acetate, collected at a flow rate of 333
nL/min;
[0068] FIG. 25A shows an exploded perspective view of a liquid
chromatography device for homogeneous integration with the
electrospray device of the present 15 invention; FIG. 25B shows a
cross-sectional view of the liquid chromatography device of FIG.
25A taken along line 25B-25B;
[0069] FIG. 26 shows a plan view of a liquid chromatography device
having an exit orifice forming an off-chip interconnection with an
off-chip device;
[0070] FIG. 27 shows a plan view of a liquid chromatography device
having an exit orifice forming an on-chip interconnection with
another on-chip device;
[0071] FIGS. 28-29 show cross-sectional views of liquid
chromatography devices having alternative configurations;
[0072] FIGS. 30-35 show plan views of liquid chromatography devices
having 25 alternative configurations;
[0073] FIGS. 36A-46C show an example of a fabrication sequence of
the liquid chromatography device;
[0074] FIG. 47 shows a cross-sectional view of a system comprising
a liquid chromatography device homogenously integrated with an
electrospray device; FIG. 48 shows a plan view of the system of
FIG. 47; and FIG. 49 shows a detailed view of the nozzles of the
system of FIG. 47.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0075] An aspect of the present invention provides a silicon
microchip-based electrospray device for producing electrospray
ionization of a liquid sample. The electrospray device may be
interfaced downstream to an atmospheric pressure ionization mass
spectrometer (API-MS) for analysis of the electrosprayed fluid.
Another aspect of the invention is an integrated miniaturized
liquid phase separation device, which may have, for example, glass,
plastic or silicon substrates integral with the electrospray
device. The descriptions that follow present the invention in the
context of a liquid chromatograph separation device. However, it
will be readily recognized that equivalent devices can be made that
utilize other microchip-based separation devices. The following
description is presented to enable any person skilled in the art to
make and use the invention. Descriptions of specific applications
are provided only as examples. Various modifications to the
preferred embodiment will be readily apparent to those skilled in
the art, and the general principles defined herein may be applied
to other embodiments and applications without departing from the
spirit and scope of the invention. Thus, the present invention is
not intended to be limited to the embodiments shown, but is to be
accorded the widest scope consistent with the principles and
features disclosed herein.
Electrospray Device
[0076] FIGS. 2-4 show, respectively, a perspective view, a plan
view and a crosssectional view of an electrospray device 100 of the
present invention. The electrospray apparatus of the present
invention generally comprises a silicon substrate or microchip or
wafer 102 defining a channel 104 through substrate 102 between an
entrance orifice 106 on an injection surface 108 and a nozzle 110
on an ejection surface 112. The channel may have any suitable
cross-sectional shape such as circular or rectangular. The nozzle
110 has an inner and an outer diameter and is defined by a recessed
region 114. The region 114 is recessed from the ejection surface
112, extends outwardly from the nozzle 110 and may be annular. The
tip of the nozzle 110 does not extend beyond and is preferably
coplanar or level with the ejection surface 112 to thereby protect
the nozzle 110 from accidental breakage.
[0077] Preferably, the injection surface 108 is opposite the
ejection surface 112. However, although not shown, the injection
surface may be adjacent to the ejection surface such that the
channel extending between the entrance orifice and the nozzle makes
a turn within the device. In such a configuration, the electrospray
device would comprise two substrates bonded together. The first
substrate may define a through-substrate channel extending between
a bonding surface and the ejection surface, opposite the bonding
surface. The first substrate may further define an open channel
recessed from the bonding surface extending from an orifice of the
through-substrate channel and the injection surface such that the
bonding surface of the second substrate encloses the open channel
upon bonding of the first and second substrates. Alternatively, the
second substrate may define an open channel recessed from the
bonding surface such that the bonding surface of the first
substrate encloses the open channel upon bonding of the first and
second substrates. In yet another variation, the first substrate
may further define a second through-substrate channel while the
open channel extends between the two through-substrate channels.
Thus. the injection surface is the same surface as the ejection
surface.
[0078] A grid-plane region 116 of the ejection surface 112 is
exterior to the nozzle 110 and to the recessed region 114 and may
provide a surface on which a layer of conductive material 119,
including a conductive electrode 120, may be formed for the
application of an electric potential to the substrate 102 to modify
the electric field pattern between the ejection surface 112,
including the nozzle tip 110, and the extracting electrode 54.
Alternatively, the conductive electrode may be provided on the
injection surface 108 (not shown).
[0079] The electrospray device 100 further comprises a layer of
silicon dioxide 118 over the surfaces of the substrate 102 through
which the electrode 120 is in contact with the substrate 102 either
on the ejection surface 112 or on the injection surface 108. The
silicon dioxide 118 formed on the walls of the channel 104
electrically isolates a fluid therein from the silicon substrate
102 and thus allows for the independent application and sustenance
of different electrical potentials to the fluid in the channel 104
and to the silicon substrate 102. The ability to independently vary
the fluid and substrate potentials allows the optimization of the
electrospray through modification of the electric field line
pattern, as described below. Alternatively, the substrate 102 can
be controlled to the same electrical potential as the fluid when
appropriate for a given application.
[0080] As shown in FIG. 5, to generate an electrospray, fluid may
be delivered to the entrance orifice 106 of the electrospray device
100 by, for example, a capillary 52 or micropipette. The fluid is
subjected to a potential voltage Vfluid via a wire (not shown)
positioned in the capillary 52 or in the channel 104 or via an
electrode (not shown) provided on the injection surface 108 and
isolated from the surrounding surface region and the substrate 102.
A potential voltage Vsubstrate may also be applied to the electrode
120 on the grid-plane 116, the magnitude of which is preferably
adjustable for optimization of the electrospray characteristics.
The fluid flows through the channel 104 and exits or is ejected
from the nozzle I10 in the form of very fine, highly charged
fluidic droplets 58. The electrode 54 may be held at a potential
voltage Vextract such that the electrospray is drawn toward the
extracting electrode 54 under the influence of an electric field.
As it is the relative electric potentials which affect the electric
field, the potential voltages of the fluid, the substrate and the
extracting electrode may be easily adjusted and modified to achieve
the desired electric field. Generally, the magnitude of the
electric field should not exceed the dielectric breakdown strength
of the surrounding medium, typically air.
[0081] In one embodiment, the nozzle 110 may be placed up to 10 mm
from the sampling orifice of an API mass spectrometer serving as
the extracting electrode 54. A potential voltage Vfluid ranging
from approximately 500-1000 V, such as 700 V, is applied to the
fluid. The potential voltage of the fluid Vfluid may be up to 500
V/um of silicon dioxide on the surface of the substrate 102 and may
depend on the surface tension of the fluid being sprayed and the
geometry of the nozzle 110. A potential voltage of the substrate
Vsubstrate of approximately less than half of the fluid potential
voltage Vfluid.about. or 0-350 V, is applied to the electrode on
the grid-plane 116 to enhance the electric field strength at the
tip of the nozzle 110. The extracting electrode 54 may be held at
or near ground potential Vextract . . . (0 V). Thus, a
nanoelectrospray of a fluid introduced to the electrospray device
100 at flow rates less than 1,000 nL/min is drawn toward the
extracting electrode 54 under the influence of the electric
field.
[0082] The nozzle 110 provides the physical asperity for
concentrating the electric field lines emanating from the nozzle
110 in order to achieve efficient electrospray. The nozzle 110 also
forms a continuation of and serves as an exit orifice of the
through-substrate channel 104. Furthermore, the recessed region 114
serves to physically isolate the nozzle 110 from the grid-plane
region 116 of the ejection surface 112 to thereby promote the
concentration of electric field lines and to provide electrical
isolation between the nozzle 110 and the grid-plane region 116. The
present invention allows the optimization of the electric field
lines emanating from the nozzle 110 through independent control of
the potential voltage Vfluid of the fluid and nozzle 110 and the
potential voltage Vsubstrate of the electrode on the grid-plane 116
of the ejection surface 112.
[0083] In addition to the electrode 120, one or more additional
conductive electrodes may be provided on the silicon dioxide layer
118 on the ejection surface 112 of the substrate 102. FIGS. 6 and 7
show, respectively, a plan view and a cross-sectional view of an
example of an electrospray device 100' wherein the conductive layer
119 defines three additional electrodes 122, 124, 126 on the
ejection surface 112 of the substrate 102. Because the silicon
dioxide layer 118 on the ejection surface 112 electrically isolates
the silicon substrate 102 from the additional electrodes 122, 124,
126 on the ejection surface 112 and because the additional
electrodes 122, 124, 126 are physically separated from each other,
the electrical potential applied to each of the additional
electrodes 122, 124, 126 can be controlled independently from each
other, from the substrate 102 and from the fluid. Thus, additional
electrodes 122, 124, 126 may be utilized to further modify the
electric field line pattern to effect, for example, a steering
and/or shaping of the electrospray. Although shown to be of similar
sizes and shapes, electrode 120 and additional electrodes 122, 124,
126 may be of any same or different suitable shapes and sizes.
[0084] To further control and optimize the electrospray, a feedback
control circuit 130 as shown in FIG. 8 may also be provided with
the electrospray device 100. The feedback circuit 130 includes an
optimal spray attribute set point 132, a comparator and voltage
control 134 and one or more spray attribute sensors 136. The
optimal spray attribute set point 132 is set by an operator or at a
determined or default value. The one or more spray attribute
sensors 136 detect one or more desired attributes of the
electrospray from the electrospray device 100, such as the
electrospray ion current and/or the spatial concentration of the
spray pattern. The spray attribute sensor 136 sends signals
indicating the value of the desired attribute of the electrospray
to the comparator and voltage control 134 which compares the
indicated value of the desired attribute with the optimal spray
attribute set point 132. The comparator and voltage control 134
then applies potential voltages Vfluid, Vsubstrate to the fluid and
the silicon substrate 102, respectively, which may be independently
varied to optimize the desired electrospray attribute. Although not
shown, the comparator and voltage control 134 may apply
independently controlled additional potential voltages to each of
one or more additional conductive electrodes.
[0085] The feedback circuit 130 may be interfaced with the
electrospray device 100 in any suitable fashion. For example, the
feedback circuit 130 may be fabricated as an integrated circuit on
the electrospray device 100, as a separate integrated circuit with
electrical connection to the electrospray device 100, or as
discrete components residing on a common substrate electrically
connected to the substrate of the electrospray device.
[0086] Dimensions of the electrospray device 100 can be determined
according to various factors such as the specific application, the
layout design as well as the upstream and/or downstream device to
which the electrospray device 100 is interfaced or integrated.
Further, the dimensions of the channel and nozzle may be optimized
for the desired flow rate of the fluid sample. The use of
reactive-ion etching techniques allows for the reproducible and
cost effective production of small diameter nozzles, for example, a
2 um inner diameter and 5 um outer diameter.
[0087] In one currently preferred embodiment, the silicon substrate
102 of the electrospray device 100 is approximately 250-600 um in
thickness and the cross sectional area of the channel 104 is less
than approximately 50,000 um. Where the channel 104 has a circular
cross-sectional shape, the channel 104 and the nozzle 110 have an
inner diameter of up to 250 gin, more preferably up to 145 gm; the
nozzle 110 has an outer diameter of up to 255 gm, more preferably
up to 150 um; and nozzle 110 has a height of (and the recessed
portion 114 has a depth of) up to 500 um. The recessed portion 114
preferably extends up to 1000 pin outwardly from the nozzle 110.
The silicon dioxide layer 118 has a thickness of approximately 1-4
um, preferably 1-2 um.
Electrospray Device Fabrication Procedure
[0088] The fabrication of the electrospray device 100 will now be
explained with reference to FIGS. 9-20B. The electrospray device
100 is preferably fabricated as a monolithic silicon integrated
circuit utilizing established, well-controlled thin-film silicon
processing techniques such as thermal oxidation, photolithography,
reactiveion etching (RIE), ion implantation, and metal deposition.
Fabrication using such silicon processing techniques facilitates
massively parallel processing of similar devices, is time- and
cost-efficient, allows for tighter control of critical dimensions,
is easily reproducible, and results in a wholly integral device,
thereby eliminating any assembly requirements. Further, the
fabrication sequence may be easily extended to create physical
aspects or features on the injection surface and/or ejection
surface of the electrospray device to facilitate interfacing and
connection to a fluid delivery system or to facilitate integration
with a fluid delivery sub-system to create a single integrated
system.
[0089] Injection surface processing: entrance to through-wafer
channel
[0090] FIGS. 9A-II illustrate the processing steps for the
injection side of the substrate in fabricating the electrospray
device 100 of the present invention. Referring to the plan and
cross-sectional views, respectively, of FIGS. 9A and 9B, a
double-side polished silicon wafer substrate 200 is subjected to an
elevated temperature in an oxidizing ambient to grow a layer or
film of silicon dioxide 202 on the injection side 203 and a layer
or film of silicon dioxide 204 on the ejection side 205 of the
substrate 200. Each of the resulting silicon dioxide layers 202,
204 has a thickness of approximately 1-2 um. The silicon dioxide
layers 202, 204 provide electrical isolation and also serve as
masks for subsequent selective etching of certain areas of the
silicon substrate 200.
[0091] A film of positive-working photoresist 206 is deposited on
the silicon dioxide layer 202 on the injection side 203 of the
substrate 200. An area of the photoresist 206 corresponding to the
entrance to a through-wafer channel which will be subsequently
etched is selectively exposed through a mask by an optical
lithographic exposure tool passing short-wavelength light, such as
blue or near-ultraviolet at wavelengths of 365, 405, or 436
nanometers.
[0092] As shown in the plan and cross-sectional views,
respectively, of FIGS. I0A and I0B, after development of the
photoresist 206, the exposed area 208 of the photoresist is removed
and open to the underlying silicon dioxide layer 202 while the
unexposed areas remain protected by photoresist 2061. The exposed
area 210 of the silicon dioxide layer 202 is then etched by a
fluorine-based plasma with a high degree of anisotropy and
selectivity to the protective photoresist 206' until the silicon
substrate 200 is reached. The remaining photoresist is removed in
an oxygen plasma or in an actively oxidizing chemical bath like
sulfuric acid (H2SO4) activated with hydrogen peroxide (H2O2)
[0093] As shown in the cross-sectional view of FIG. 11, an
injection side portion 212 of the through channel in the silicon
substrate 200 is vertically etched by another fluorine-based etch.
An advantage of the fabrication process described herein is that
the dimensions of the through channel, such as the aspect ratio
(depth to width), can be reliably and reproducibly limited and
controlled. In the case where the etch aspect ratio of the
processing equipment is a limiting factor, it is possible to
overcome this limitation by a first etch on one side of a wafer
followed by a second etch on a second side of the wafer. For
example, a current silicon etch process is generally limited to an
etch aspect ratio of 30:1, such that a channel having a diameter
less than approximately 10 gm through a substrate 200 having
customary thickness approximately 250-600 um would be etched from
both surfaces of the substrate 200.
[0094] The depth of the channel portion 212 should be at or above a
minimum in order to connect with another portion of the through
channel etched from the ejection side 205 of the substrate 200. The
desired depth of the recessed region 114 on the ejection side 205
determines approximately how far the ejection side portion 220 of
the channel 104 is etched. The remainder of the channel 104, the
injection side portion 212, is etched from the injection side. The
minimum depth of channel portion 212 is typically 50 gm, although
the exact etch depth above the minimum etch depth does not impact
the device performance or yield of the electrospray device.
Ejection surface processing: nozzle and surrounding surface
structure
[0095] FIGS. 12-20B illustrate the processing steps for the
ejection side 205 of the substrate 200 in fabricating the
electrospray device 100 of the present invention. As shown in the
cross-sectional view in FIG. 12, a film of positive-working
photoresist 214 is deposited on the silicon dioxide layer 204 on
the ejection side 205 of the substrate 200. Patterns on the
ejection side 205 are aligned to those previously formed on the
injection side 203 of the substrate 200. Because silicon and its
oxide are inherently relatively transparent to light in the
infrared wavelength range of the spectrum, i.e. approximately
700-1000 nanometers, the extant pattern on the injection side 203
can be distinguished with sufficient clarity by illuminating the
substrate 200 from the patterned injection side 203 with infrared
light. Thus, the mask for the ejection side 205 can be aligned
within required tolerances.
[0096] After alignment, certain areas of the photoresist 214
corresponding to the nozzle and the recessed region are selectively
exposed through an ejection side mask by an optical lithographic
exposure tool passing short-wavelength light, such as blue or
near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. As
shown in the plan and cross-sectional views, respectively, of FIGS.
13A and 13B, the photoresist 214 is then developed to remove the
exposed areas of the photoresist such that the nozzle area 216 and
recessed region area 218 are open to the underlying silicon dioxide
layer 204 while the unexposed areas remain protected by photoresist
214'. The exposed areas 216, 218 of the silicon dioxide layer 204
are then etched by a fluorine-based plasma with a high degree of
anisotropy and selectivity to the protective photoresist 2141 until
the silicon substrate 200 is reached.
[0097] As shown in the cross-sectional view of FIG. 14, the
remaining photoresist 214' provides additional masking during a
subsequent fluorine based silicon etch to vertically etch certain
patterns into the ejection side 205 of the silicon substrate 200.
The remaining photoresist 214' is then removed in an oxygen plasma
or in an actively oxidizing chemical bath like sulfuric acid
(H2SO4) activated with hydrogen peroxide (H2O2).
[0098] The fluorine-based etch creates a channel 104 through the
silicon substrate 200 by forming an ejection side portion 220 of
the channel 104. The fluorine based etch also creates an ejection
nozzle 110, a recessed region 114 exterior to the nozzle 110 and a
grid-plane region 116 exterior to the nozzle 110 and to the
recessed 114. The grid-plane region 116 is preferably co-planar
with the tip of the nozzle so as to physically protect the nozzle
110 from casual abrasion, stress fracture handling and/or
accidental breakage. The grid-plane region 116 also serves as
platform on which one or more conductive electrodes may be
provided.
[0099] The fabrication sequence confers superior mechanical
stability to the fabricated electrospray device by etching the
features of the electrospray device a monocrystalline silicon
substrate without any need for assembly. The fabric sequence allows
for the control of the nozzle height by adjusting the relative a of
injection side and ejection side silicon etching. Further, the
lateral extent and shape of the recessed region 114 can be
controlled independently of its depth, affects the nozzle height
and which is determined by the extent of the etch on the ejection
side of the substrate. Control of the lateral extent and shape of
the recessed region 114 provides the ability to modify and control
the electric field pattern between the electrospray device 100 and
an extracting electrode.
[0100] Oxidation for electrical isolation
[0101] As shown in the cross-sectional view of FIG. 15, a layer of
silicon dioxide 221 is grown on all silicon surfaces of the
substrate 200 by subjecting the silicon substrate 200 to elevated
temperature in an oxidizing ambient. For example, the oxidizing
ambient may be an ultra-pure steam produced by oxidation of
hydrogen for a silicon dioxide thickness greater than approximately
several hundred nanometers or pure oxygen for a silicon dioxide
thickness of approximately several hundred nanometers or less. The
layer of silicon dioxide 221 over all silicon surfaces of the
substrate 200 electrically isolates a fluid in the channel from the
silicon substrate 200 and permits the application and sustenance of
different electrical potentials to the fluid in the channel 104 and
to the silicon substrate 200.
[0102] All silicon surfaces are oxidized to form silicon dioxide
with a thickness is controllable through choice of temperature and
time of oxidation. The thickness of the silicon dioxide can be
selected to provide the desired degree electrical isolation in the
device, where a thicker layer of silicon dioxide provides greater
resistance to electrical breakdown. Metallization for electric
field control
[0103] FIGS. 16-20B illustrate the formation of a single conductive
electrode electrically connected to the substrate 200 on the
ejection side 205 of the substrate I to 200. As shown in the
cross-sectional view of FIG. 16, a film of positive-working
photoresist 222 is deposited over the silicon dioxide layer on the
ejection side 205 of the substrate 200. An area of the photoresist
222 corresponding to the electrical contact area between the
electrode and the substrate 200 is selectively exposed through
another mask by an optical lithographic exposure tool passing
short-wavelength light, such as blue or near-ultraviolet at
wavelengths of 365, 405, or 436 nanometers.
[0104] The photoresist 222 is then developed to remove the exposed
area 224 of the photoresist such that the electrical contact area
between the electrode and the substrate 200 is open to the
underlying silicon dioxide layer 204 while the unexposed areas
remain protected by photoresist 2221. The exposed area 224 of the
silicon dioxide layer 204 is then etched by a fluorine-based plasma
with a high degree of anisotropy and selectivity to the protective
photoresist 222' until the silicon substrate 200 is reached, as
shown in the cross-sectional view of FIG. 17. Referring now to the
cross-sectional view of FIG. 18, the remaining photoresist is then
removed in an oxygen plasma or in an actively oxidizing chemical
bath like sulfuric acid (H2SO4) activated with hydrogen peroxide
(H2O2). Utilizing the patterned ejection side silicon dioxide layer
204 as a mask, a high-dose implantation is made to form an
implanted region 225 to ensure a low-resistance electrical
connection between the electrode and the substrate 200. A
conductive film 226 such as aluminum may be uniformly deposited on
the ejection side 205 of the substrate 200 by thermal or
election-beam evaporation to form an electrode 120. The thickness
of the conductive film 226 is preferably approximately 3000 A,
although shown having a larger thickness for clarity.
[0105] The conductive film 226 may be created by any method which
does not produce a continuous film of the conductive material on
the side walls of the ejection nozzle 110. Such a continuous film
would electrically connect the fluid in the channel 104 and the
substrate 200 so as to prevent the independent control of their
respective electrical potentials. For example, the conductive film
may be deposited by thermal or electron-beam evaporation of the
conductive material, resulting in line-of-sight deposition on
presented surfaces. Orienting the substrate 200 such that the side
walls of the ejection nozzle 110 are out of the line-of-sight of
the evaporation source ensures that no conductive material is
deposited as a continuous film on the side walls of the ejection
nozzle 110. Sputtering of conductive material in a plasma is an
example of a deposition technique which would result in deposition
of conductive material on all surfaces and thus is undesirable.
[0106] One or more additional conductive electrodes may be easily
formed on the ejection side 205 of the substrate 200, as described
above with reference to FIGS. 6 and 7. As shown in the
cross-sectional view of FIG. 19, a film of positive-working
photoresist 228 is deposited over the conductive film 226 on the
ejection side 205 of the substrate 200. Certain areas of the
photoresist 228 corresponding to the physical spaces between the
electrodes are selectively exposed through another mask by an
optical lithographic exposure tool passing short-wavelength light,
such as blue or near-ultraviolet at wavelengths of 365, 405, or 436
nanometers.
[0107] Referring now to the plan and cross-sectional views of FIGS.
20A and 20B, the photoresist 228 is developed to remove the exposed
areas 230 of the photoresist such that the exposed areas are open
to the underlying conductive film 226 while the unexposed areas
remain protected by photoresist 228'. The exposed areas 230 of the
conductive film 226 are then etched using either a wet chemical
etch or a reactive-ion etch, as appropriate for the particular
conductive material. The etch is either selective to the underlying
silicon dioxide layer 204 or the etch must be terminated on the
basis of etch rate and time of etch. Finally, the remaining
photoresist is then removed in an oxygen plasma.
[0108] The etching of the conductive film 226 to the underlying
silicon dioxide layer 204 results in physically and electrically
separate islands of conductive material or electrodes. As described
above, these electrodes can be controlled independently from the
silicon substrate or channel fluid because they are electrically
isolated from the substrate by the silicon dioxide and from each
other by physical separation. They can be used to further modify
the electric field line pattern and thereby effect a steering
and/or shaping of the electrosprayed fluid. This step completes the
processing and fabrication sequence for the electrospray device
100.
[0109] As described above, the conductive electrode for application
of an electrical potential to the substrate of the electrospray
device may be provided on the injection surface rather than the
ejection surface. The fabrication sequence is similar to that for
the conductive electrode provided on the ejection side 205 of the
substrate 200.
[0110] FIGS. 20C-20G illustrate the formation of a single
conductive electrode electrically connected to the substrate 200 on
the injection side 203 of the substrate 200.
[0111] As shown in the cross-sectional view of FIG. 20C, a film of
positive-working photoresist 232 is deposited over the silicon
dioxide layer on the injection side 203 of the substrate 200. An
area of the photoresist 232 corresponding to the electrical contact
area between the electrode and the substrate 200 is selectively
exposed through another mask by an optical lithographic exposure
tool passing short-wavelength light, such as blue or
near-ultraviolet at wavelengths of 365, 405, or 436 nanometers.
[0112] The photoresist 232 is then developed to remove the exposed
area 234 of the photoresist such that the electrical contact area
between the electrode and the substrate 200 is open to the
underlying silicon dioxide layer 202 while the unexposed areas
remain protected by photoresist 232'. The exposed area 234 of the
silicon dioxide layer 202 is then etched by a fluorine-based plasma
with a high degree of anisotropy and selectivity to the protective
photoresist 232' until the silicon substrate 200 is reached, as
shown in the cross-sectional view of FIG. 20D.
[0113] Referring now to the cross-sectional view of FIG. 20E, the
remaining photoresist is then removed in an oxygen plasma or in an
actively oxidizing chemical bath like sulfuric acid (H2SO4)
activated with hydrogen peroxide (H2O2). Utilizing the patterned
injection side silicon dioxide layer 202 as a mask, a high-dose
implantation is made to form an implanted region 236 to ensure a
low-resistance electrical connection between the electrode and the
substrate 200. A conductive film 238 such as aluminum may be
uniformly deposited on the injection side 203 of the substrate 200
by thermal or electron beam evaporation to form an electrode
1201.
[0114] In contrast to the formation of the conductive electrode on
the ejection surface of the electrospray device, sputtering, in
addition to thermal or electron-beam evaporation, may be utilized
to form the conductive electrode on the injection surface. Because
the nozzle is on the ejection rather than the injection side of the
substrate, sputtering may be utilized to form the electrode on the
injection side as the injection side electrode layer does not
extend to the nozzle to create a physically continuous and thus
electrically conductive path with the nozzle.
[0115] With the formation of the electrode on the injection surface
of the electrospray device, sputtering may be preferred over
evaporation because of its greater ability to produce conformal
coatings on the sidewalls of the exposed area 234 etched through
the silicon dioxide layer 202 to the substrate 200 to ensure
electrical continuity and reliable electrical contact to the
substrate 200.
[0116] For certain applications, it may be necessary to ensure
electrical isolation between the substrate 200 and the fluid in the
electrospray device by removing the conductive film from the region
of the surface adjacent to the entrance orifice 106 on the
injection side 203. The extent of the conductive film 238 which
should be removed is irrespective of etching method and may be
determined by the specific method utilized in creating the
interface between the upstream fluid delivery system/sub-system and
the injection side of the electrospray device. For example, a
diameter of between approximately 0.2-2 mm of the conductive film
238 may be removed from the region surrounding the entrance orifice
106. As shown in the cross-sectional view of FIG. 20F, another film
of positive-working photoresist 240 is deposited over the
conductive film 238 on the injection side 203 of the substrate 200.
An area of the photoresist 240 corresponding to the region adjacent
to the entrance orifice 106 on the in injection side 203 is
selectively exposed through another mask by an optical lithographic
exposure tool passing short-wavelength light, such as blue or
near-ultraviolet at wavelengths of 365, 405, or 436 nanometers.
[0117] The photoresist 240 is then developed to remove the exposed
area 242 of the photoresist such that the region adjacent to the
entrance orifice 106 on the injection side 203 is open to the
underlying conductive film 238 while the unexposed areas remain
protected by photoresist 240'. The exposed area 242 of the
conductive film 238 is then etched by, for example, a
chlorine-based plasma with a high degree of anisotropy and
selectivity to the protective photoresist 2401 until the silicon
dioxide layer 203 is reached, as shown in the cross-sectional view
of FIG. 20G. The specific technique for etching the conductive film
238 may be determined 25 by the specific conductive material
deposited. For example, aluminum may be etched either in a wet
chemical bath using standard aluminum etchant or in a plasma using
reactive-ion etching (RIE) and chlorine-based gas chemistry.
Utilization of standard wet aluminum etchant to etch an aluminum
film may be preferred as such wet etching may facilitate the
removal of any undesired conductive material deposited in the
channel 104 via the entrance orifice 106. Further, although
chlorine-based reactive-ion etching may be utilized, such etching
may lead to aluminum corrosion if removal of the photoresist is
delayed.
[0118] Forming the electrode on the injection surface for
application of an electric potential to the substrate of the
electrospray device may provide several advantages. For example,
because the ability to uniformly coat photoresist on a surface is
limited by nonplanar surface topology, coating photoresist on the
much flatter injection side results in a more uniform and
continuous photoresist film than coating photoresist on the
ejection side. The uniformity and continuity of the photoresist
film directly and positively impact the reliability and yield, at
least in part because failure of photoresist coverage would allow
subsequent etching of silicon dioxide in undesired locations during
the etching of exposed areas 224, 234.
[0119] Another advantage of forming the electrode on the injection
surface is the greater flexibility and reliability in the
conductive material deposition step because the interior surfaces
of the nozzle are not coated by the conductive material deposited
onto the injection surface rather than onto the ejection surface of
the electrospray device. As a result, sputtering may be utilized as
a deposition technique to ensure conformal coating of the
conductive material and electrical continuity from the surface to
the substrate contact. Further, the provision of the electrode on
the injection surface does not preclude the deposition and
patterning of additional conductive electrodes on the ejection side
to further modify the electric field line pattern to effect, for
example, a steering and/or shaping of the electrospray, as such
additional electrodes do not required electrical contact to the
substrate.
[0120] The ability to form the electrode on the injection surface
may also be advantageous in certain applications where physical
constraints, such as in packaging, may dictate the need for
injection-side rather than ejection-side electrical connection. The
above described fabrication sequence for the electrospray device
100 be easily adapted to and is applicable for the simultaneous
fabrication of a single monolithic system comprising multiple
electrospray devices including multiple channels and/or multiple
ejection nozzles embodied in a single monolithic substrate.
Further, the processing steps may be modified to fabricate similar
or different electrospray devices merely by, for example, modifying
the layout design and/or by changing the polarity of the photomask
and utilizing negative-working photoresist rather than utilizing
positive-working photoresist.
[0121] Further, although the fabrication sequence is described in
terms of fabricating a single electrospray device, the fabrication
sequence facilitates and allows for massively parallel processing
of similar devices. The multiple electrospray devices or systems
fabricated by massively parallel processing on a single wafer may
then be cut or otherwise separated into multiple devices or
systems.
Interface or Integration of the Electrospray Device
[0122] Downstream Interface or Integration of the Electrospray
Device
[0123] The electrospray device 100 may be interfaced or integrated
downstream to a sampling device, depending on the particular
application. For example, the analyte may be electrosprayed onto a
surface to coat that surface or into another device for purposes of
conveyance, analysis, and/or synthesis. As described above with
reference to FIG. 5, highly charged droplets are formed at
atmospheric pressure by the electrospray device 100 from
nanoliter-scale volumes of an analyte. The highly charged droplets
produce gas-phase ions upon sufficient evaporation of solvent
molecules which may be sampled, for example, through an orifice of
an atmospheric pressure ionization mass spectrometer (API-MS) for
analysis of the electrosprayed fluid.
[0124] Upstream Interface or Integration of the Electrospray
Device
[0125] Referring now to FIGS. 21-23, fluid may be delivered to the
entrance orifice of the electrospray device in any suitable manner
by upstream interface or integration with one or more fluid
delivery devices, such as piezoelectric pipettes, micropipettes,
capillaries and other types of microdevices. The fluid delivery
device may be a separate component to form a heterogeneous
interface with the entrance orifice of the electrospray device.
Alternatively, the fluid delivery device may be integrated with the
electrospray device to form a homogeneous interface with the
entrance orifice of the electrospray device.
[0126] FIGS. 21A and 21B illustrate examples of fluid delivery
devices forming heterogeneous interfaces with the entrance orifice
of the electrospray device. Preferably, the heterogeneous interface
is a non-contacting interface where the fluid delivery device and
the electrospray device are physically separated and do not
contact. For example, as shown in the cross-sectional view of FIG.
21A, a piezoelectric pipette 300 is positioned at a distance above
the injection surface 108 of the electrospray device 100A. The
piezoelectric pipette 300 deposits a flow of microdroplets, each
approximately 200 pL in volume, into the channel 104 through the
entrance orifice 106A. Preferably, the electrospray device 100A
provides an entrance well 302 at the entrance orifice 106A for
containing the sample fluid prior to entering the channel 104
particularly when it is desirable to spray a volume of fluid
greater than the volume of the through-substrate channel 104 and
continual supply of fluid is not feasible such as when using the
piezoelectric pipette 300. The entrance well 302 preferably has a
volume of 0.1 nL to 100 nL. Furthermore, to apply an electric
potential to the fluid, an entrance well electrode 304 may be
provided on a surface of the entrance well 302 parallel to the
injection surface 108. Alternatively, a wire (not shown) may be
positioned in channel 104 via the entrance orifice 106A.
Preferably, some fluid is present in the entrance well 302 to
ensure electrical contact between the fluid and the entrance well
electrode 304.
[0127] Alternatively, the heterogeneous interface may be a
contacting interface where a fluid delivery device is attached by
any suitable method, such as by epoxy bonding, to the electrospray
device to form a continuous sealed flow path between the upstream
fluid source and the channel of the electrospray device. For
example, FIG. 21B shows a cross-sectional view of a capillary 306
prior to attachment to the entrance orifice 106 of the electrospray
device 100B. The injection surface 108 of the electrospray device
100B may be adapted to facilitate attachment of the capillary 306.
Such features can be easily designed into the mask for the
injection side of the substrate and can be simultaneously formed
with the injection side portion of the channel during the etching
performed on the injection-side,
[0128] For example, where the inner diameter of the capillary 306
is greater than that of the channel 104 and the entrance orifice
106, the electrospray device 100B preferably defines a region 308
recessed from the injection surface 108 to form a mating collar for
mating and affixing with the capillary 306. Thus, capillary 306 may
be positioned and attached in the recessed region 308 such that the
exit orifice 310 portion of the, capillary 302 is positioned around
the entrance orifice 106. Further, the electrospray device 100B may
optionally provide an entrance well 312 at the entrance orifice
106B for containing the sample fluid prior to entering the channel
104. Although not shown, if the outer diameter of the capillary is
less than that of the channel and the entrance orifice, the
capillary may be inserted into and attached to the entrance orifice
of the electrospray device.
[0129] Referring now to the schematic of FIG. 22, rather than a
heterogeneous interface, a single integrated system 316 is provided
wherein an upstream fluid delivery device 318 forms a homogeneous
interface with the entrance orifice (not shown) of an electrospray
device 100. The system 316 allows for the fluid exiting the
upstream fluid delivery device 318 to be delivered on-chip to the
entrance orifice of the electrospray device 100 in order to
generate an electrospray.
[0130] The single integrated system 316 provides the advantage of
minimizing or eliminating extra fluid volume to reduce the risk of
undesired fluid changes, such as by reactions and/or mixing. The
single integrated system 316 also provides the advantage of
eliminating the need for unreliable handling and attachment of
components at the microscopic level and of minimizing or
eliminating fluid leakage by containing the fluid within one
integrated system.
[0131] The upstream fluid delivery device 318 may be a monolithic
integrated circuit having an exit orifice through which a fluid
sample can pass directly or indirectly to the entrance orifice of
the electrospray device 100. The upstream fluid delivery device 318
may be a silicon microchip-based liquid separation device capable
of, for example, capillary electrophoresis, capillary
electrochromatography, affinity chromatography, liquid
chromatography (LC) or any other condensed-phase separation
methods. Further, the upstream fluid delivery device 318 may be a
silicon, glass, plastic and/or polymer based device such that the
electrospray device 100 may be chip-to-chip or wafer-to-wafer
bonded thereto by any suitable method. An example of a monolithic
liquid chromatography device for utilization in, for example, the
single integrated system 316, is described below.
[0132] Electrospray Device for Sample Transfer of Combinatorial
Chemistry Libraries Synthesized in Microdevices The electrospray
device may also serve to reproducibly distribute and deposit a
sample from a mother plate to daughter plate(s) by nanoelectrospray
deposition. Electrospray device(s) may be etched into a microdevice
capable of synthesizing combinatorial chemical libraries. At the
desired time, the nozzle may spray a desired amount of the sample
from the mother plate to the daughter plate(s). Control of the
nozzle dimensions, applied voltages, and time of spraying may
provide a precise and reproducible method of sample deposition from
an array of nozzles, such as the generation of sample plates for
molecular weight determinations by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOFMS). The capability of transferring analytes, from a
mother plate to daughter plates may also be utilized to make other
daughter plates for other types of assays, such as proteomic
screening.
[0133] FIGS. 23A and 23B show, respectively, an exploded
perspective view and a cross-sectional view along line 23B-23B, of
a chip-based combinatorial chemistry system 320 comprising a
reaction well block or titer plate 322 and a receiving or daughter
plate 324. The reaction well block 322 defines an array of
reservoirs 326 for containing the reaction products from a
combinatorially synthesized compound.
[0134] The reaction well block 322 further defines channels 328,
nozzles 330 and recessed portions 332 such that the fluid in each
reservoir 326 may flow through a corresponding channel 328 and exit
through a corresponding nozzle 330 in the form of an electrospray.
The reaction well block 322 may define any number of reservoir(s)
in any desirable configuration, each reservoir being of a suitable
dimension and shape. The volume of a reservoir 326 may range from a
few nanoliters up to several microliters and more preferably ranges
between approximately 200 nL to I uL.
[0135] The reaction well block 322 may serve as a mother plate to
interface to a microchip-based chemical synthesis apparatus such
that the electrospray function of the reaction well block 322 may
be utilized to reproducibly distribute discreet quantities of the
product solutions to a receiving or daughter plate 324. The
daughter plate 324 defines receiving wells 334 which correspond to
each of the reservoirs 326. The distributed product solutions in
the daughter plate 324 may then be utilized to screen the
combinatorial chemical library against biological targets.
[0136] Illustration of an Electrospray Device Generating an
Electrospray Spray
[0137] FIGS. 24A and 24B show color images of a real Taylor cone
emanating from an integrated silicon chip-based nozzle. FIGS. 24C
and 24D are perspective and side cross-sectional views,
respectively, of the electrospray ray device and mass spectrometry
system shown in FIGS. 24A and 24B. Mi. 24A shows a chip-integrated
electrospray device comprising a nozzle and a recessed portion or
annulus, and a Taylor cone, liquid jet and plume of highly-charged
electrosprayed droplets of methanol containing 10 ug/mL
polypropylene glycol 425 (PPG425) containing 0. formic acid. FIG.
24B shows an ion-sampling orifice of a mass spectrometer in
addition to the electrospray device.
[0138] The electrospray device 100 is interfaced upstream with a
pipette 521. As shown in the upper right corner of each of FIGS.
24A and 24B and in FIGS. 24C 24D, the tip of the pipette 521 is
press-sealed to the injection side of the electrospray device 100.
The electrospray device 100 has a 10 um diameter entrance orifice
on the injection side, a 30 um inner diameter and a 60 um outer
diameter nozzle, a I pin. nozzle wall thickness and a 150 um nozzle
depth. The recessed portion or the annulus extends 300 um from the
outer diameter of the nozzle. The voltage applied to the fluid
Vfluid introduced to the electrospray device and thus the nozzle
voltage 900 V. The voltage applied to the substrate Vsubstrate and
thus the electrospray device is 0 V. The voltage applied to the
mass spectrometer which also serves as an extracting electrode
Vextract is approximately 40 V. The liquid sample was pumped using
a syringe pump at a flow of 333 nL/min through the pipette tip
pressed-sealed against the injection side of the electrospray
device. The nozzle is approximately min from the ion-sampling
orifice 62 of the mass spectrometer 60. The ion-sampling orifice 62
of the mass spectrometer 60 generally defines the acceptance region
of the mass spectrometer 60. The mass spectrometer for acquiring
the data was the LCT Time-Of-Flight mass spectrometer of Micromass,
Inc.
[0139] FIG. 24E shows a mass spectrum of I ug/mL PPG425 in 50%
water, 50% methanol containing 0.1% formic acid, 0.1% acetonitrile
and 2 mM anu-nonium acetate. The data were collected at a flow rate
of 333 nL/min. filling of the portion of the channel 412 between
the reservoir 410 and the filling electrode 430. After filling the
reservoir 410 with an appropriate volume of the sample fluid, any
suitable method may then be utilized to drive the fluid from the
reservoir 410 into the separation channel 412. For example, the
fluid may be driven from the filled reservoir 410 through the
separation channel 412 by applying hydrostatic pressure to the
reservoir 410 via the entrance orifice 406.
[0140] Alternatively or additionally, the fluid may be driven
through the separation channel 412 by applying a suitable
electrokinetic potential voltage difference between the reservoir
electrode 426 and the exit electrode 428 to generate
electrophoretic or electroosmotic fluidic motion. Preferably, the
electric potential difference is approximately 1000 V/cm of
separation channel length. Of course, any other suitable methods of
inducing fluidic motion may be utilized. Pressure-driven and
voltagedriven flow effect different separation efficiencies. Thus,
depending upon the application, one or both may be utilized.
[0141] Fluid then exits from the separation channel 412 through the
exit orifice 414 to, for example, a capillary 434, which has an
off-chip interconnection with the exit orifice 414, as shown in
FIG. 26. Alternatively, as shown in FIG. 27, the liquid
chromatography device 400 may perform separation on the fluid from
reservoir 410 such that selected analytes from the separation
performed by posts 416 passes through unpopulated channel 436 to
another on-chip device 438, such as for analysis and/or mixing,
while the remainder of the fluid is directed to the waste reservoir
439. The unpopulated channel 436 may be a mere continuation of the
separation channel 412 of the liquid chromatography device 400 or a
channel separate from the separation channel 412.
[0142] Two or more fluid samples may be driven through the liquid
chromatography device 400 by successively filling the reservoir and
driving the fluid through the separation channel 412. For example,
in certain applications, it may be desirable or necessary to first
coat the surfaces of the separation posts 416 with one or more
reagents and then pass an analyte sample over the conditioned
separation posts 416.
[0143] Various modifications may be made to the liquid
chromatography device describe above. For example, as shown in FIG.
28, rather than defining the entrance orifice and the introduction
channel in the substrate, the liquid chromatography device 400' may
provide an introduction channel 404' in the cover 420' such that
the entrance orifice 406' is defined on an exterior surface of the
cover 420'. Further, the cover 420' may define an exit channel 413
between an exit orifice 414' defined on an exterior surface of the
cover 4201 and a separation channel 412' which terminates within
the substrate 402'.
[0144] In another variation, an additional introduction channel 440
and entrance orifice 442 may be defined in the substrate 402", as
shown in FIG. 29, or in the cover (not shown). The additional
introduction channel 440 introduces fluid to the separation channel
412" such that the fluid from the additional introduction channel
440 intersects the path of fluid flow from the reservoir 410
through the unpopulated portion 43211 of the separation channel
412". The fluid reservoir 410 may be utilized as a buffer for an
eluent and the additional introduction channel 440 may be utilized
to introduce the fluid sample to the separation channel 412".
Further, the additional entrance orifice 442 may be utilized to
introduce several fluid samples in succession into the separation
channel 412". For example, in certain applications, it may be
necessary to first coat the surfaces of the separation posts 416
with one reagent and then pass an analyte over the conditioned
surfaces of the separation posts 416.
[0145] Referring now to FIGS. 30-35, although the liquid
chromatography device has been described as comprising a single
reservoir and a single separation channel, the monolithic liquid
chromatography device may be easily adapted and modified to
comprise multiples of the liquid chromatography device and/or
multiple entrance orifices, exit orifices, reservoirs and/or
separation channels. In each of the variations, any or all of the
reservoir(s), separation channel(s), and separation posts may have
different dimensions and/or shapes.
[0146] For example, multiple reservoir-separation channel
combinations may be provided on a single chip. In particular, as
shown in FIG. 30, a reservoir 410A may feed into a separation
channel 412A having separation posts 416A and another reservoir
410B may feed into another separation channel 412B having
separation posts 416B.
[0147] In another variation as shown in FIG. 31, a single reservoir
410C may feed multiple separation channels 412C, 412D. Each of
separation channels 412C, 412D may have therein separation posts
416C, 416D, respectively, which may have the same or different
properties, such as number, size and shape. Another channel 412E
may be provided as a null channel completely unpopulated by
separation posts. The output from the null channel 412E may be
utilized as a basis of comparison to the output from the separation
channel(s) populated by separation posts. Alternatively, all of the
channels 412C, 412D, 412E may be separation channels having
separation posts.
[0148] Referring now to FIG. 32, fluid from multiple reservoirs
410E and 410F may feed into a single separation channel 412F via
connecting channels 444E, 444F, respectively. The connecting
channels 444E, 444F are preferably unpopulated by separation posts
to facilitate the mixing of the fluid samples from the reservoirs
410E, 410F prior to passage through the separation channel 412F.
The mixing of samples may be utilized to Condition the primary
sample of interest prior to separation or to effect a reaction
between the samples prior to passage through the populated portion
of the separation channel 412F. Alternatively, fluid such as a
conditioning fluid from one reservoir 410E may flow through the
separation channel 412F in order to condition the surfaces of the
separation posts 416F prior to the passage of the other sample such
as an analyte sample from the other reservoir 410F. Although the
separation posts 416F are shown as having different cross-sections,
separation posts 416F may have the same size and cross-sectional
shape.
[0149] Alternatively, in addition to having fluid from multiple
reservoirs feed into a single separation channel via connecting
channels, fluid from another reservoir may be introduced to the
fluid flow along the separation channel, before and/or after the
fluid has passed through the populated portion of the separation
channel. For example, FIG. 33 shows that the fluid from multiple
reservoirs 410G, 410H may be fed into a single separation channel
412G via connecting channels 444G, 444H, respectively, and fluid
from another reservoir 4101 may be introduced to the fluid flow
along the separation channel 412G after the fluid has passed the
separation posts 416G. FIG. 34 shows that the fluid from multiple
reservoirs 410J, 410K may be fed into a single separation channel
412J via connecting channels 444J, 444K, respectively, and fluid
from another reservoir 410L may be introduced to the fluid flow
along the separation channel 412J prior to the fluid passing the
separation posts 416J.
[0150] For devices having multiple reservoirs and/or multiple
channels, separate electrodes may be provided for each reservoir
and/or for each channel, for example, in the unpopulated portion of
the channel upstream from the separation posts and/or near the exit
of the channel. Such provision of separate electrodes allow for the
separate and independent control of the fluidic flow for filling
each reservoir and/or for driving the fluid through the separation
channel.
[0151] The electric control may be simplified by having one common
reservoir electrode, one common filling electrode, and/or one exit
electrode among the multiple reservoirs and/or multiple channels.
For example, each of the multiple reservoirs may be separately
filled by applying a first voltage to the common reservoir
electrode and a second voltage, different from the first voltage,
to the filling electrode corresponding to the reservoir to be
filled while applying the first voltage to each of the other
filling electrodes. As is evident, the multiple reservoirs may be
simultaneously filled by applying a first voltage to the common
reservoir electrode and a second, different voltage to each of the
filling electrodes. Similarly, fluid may be separately driven
through each of the multiple channels by applying a third voltage
to the common reservoir electrode while applying a fourth voltage,
different from the third voltage, to the exit electrode
corresponding to the channel through which fluid is to be driven
and the third voltage to each of the other exit electrodes.
[0152] In yet another variation shown in FIG. 35, in addition to a
sample reservoir 410M and separation posts 416M, a plurality of
posts 416L may be provided in a channel 412M upstream from the
separation posts 416M for providing additional functionality such
as solid-phase extraction (SPE) for sample pretreatment. The SPE
posts 416L may be the same, similar to or different from the
separation posts 416M simply by varying the layout design. The SPE
posts 416L may provide surface functionality different from that of
the separation posts 416M. Alternatively, rather than providing a
sample reservoir, an introduction channel (not shown) may be
utilized to introduce a fluidic sample directly in the channel 412M
by allowing direct injection of the sample therein. Further,
reservoirs 410N, 410P may be provided to contain fluidic buffers
necessary for sample pretreatment upstream of the posts 416L. For
example, an eluent reservoir may be provided for eluting analytes
and a wash reservoir may be provided for sample cleanup.
[0153] After the fluid samples pass the SPE posts 416L, waste
products from, for example, the solid-phase extraction process may
be directed into a waste reservoir 41OQ. In particular, during the
SPE process, voltage differences may be applied between or amongst
reservoirs 410M, 410N, 410P, and 410Q such that a portion of the
fluid from reservoirs 410M, 410N is directed to waste reservoir
410Q while the remaining portion of the fluid from reservoir 410M
remain on the SPE posts 416L. Material may then be washed off of
the SPE posts 416L by directing fluid from, for example, reservoir
410P through channel 412M for separation of the extracted material
by separation posts 416M. Additional reservoirs 410R, 410S
downstream of the waste reservoir 410Q and upstream of the
separation posts 416M may be provided to contain gradient elution
of analytes in one reservoir and a diluent in the other reservoir.
Gradient elution facilitates chromatography by changing the mobile
phase composition, i.e. the polarity to facilitate analyte
interactions with the stationary phase, and thus facilitate
separation of the analytes. In addition, the diluent provides the
correct polarity of the solution for the next separation.
Liquid Chromatography Device Fabrication Procedure
[0154] The fabrication of the liquid chromatography device of the
present invention will now be explained with reference to FIGS.
36A-46B, The liquid chromatography device is preferably fabricated
as a monolithic silicon micro device utilizing established,
well-controlled thin-film silicon processing techniques such as
thermal oxidation, photolithography, reactive-ion etching (RIE),
ion implantation, and metal deposition. Fabrication using such
silicon processing techniques facilitates massively parallel
processing of similar devices, is time- and cost-efficient, allows
for tighter control of critical dimensions, is easily reproducible,
and results in a wholly integral device, thereby eliminating any
assembly requirements. Manipulation of separate components and/or
sub-assemblies to build an liquid chromatography device with high
reliability and yield is not desirable and may not be possible at
the micrometer dimensions required for efficient separation.
[0155] Further, the fabrication sequence may be easily extended to
create physical aspects or features to facilitate interfacing,
integration and/or connection with devices having other
functionalities or to facilitate integration with a fluid delivery
subsystem to create a single integrated system. Consequently, the
liquid chromatography device may be fabricated and utilized as a
disposable device, thereby eliminating the need for column
regeneration and eliminating the risks of sample
crosscontamination.
[0156] Referring to the plan and cross-sectional views,
respectively, of FIGS. 36A and 36B, a silicon wafer separation
substrate 500, double-side polished and approximately 250-600 um in
thickness, is subjected to an elevated temperature in an oxidizing
ambient to grow a layer or film of silicon dioxide 502 on the
reservoir side 503 and a layer or film of silicon dioxide 504 on
the back side 505 of the separation substrate 500. Each of the
resulting silicon dioxide layers 502, 504 has a thickness of
approximately 1-2 um. The silicon dioxide layers 502, 504 provide
electrical isolation and also serve as masks for subsequent
selective etching of certain areas of the separation substrate
500.
[0157] A film of positive-working photoresist 506 is deposited on
the silicon dioxide layer 502 on the reservoir side 503 of the
separation substrate 500. Certain areas of the photoresist 506
corresponding to the reservoir, separation channel and separation
posts which will be subsequently etched are selectively exposed
through a mask by an optical lithographic exposure tool passing
short-wavelength light, such as blue or near-ultraviolet at
wavelengths of 365, 405, or 436 nanometers.
[0158] Referring to the plan and cross-sectional views,
respectively, of FIGS. 37A and 37B, after development of the
photoresist 506, the exposed areas 508, 509, 510 of the photoresist
corresponding to the reservoir, separation posts and channel,
respectively, are removed and open to the underlying silicon
dioxide layer 502 while the unexposed areas remain protected by
photoresist 506'. The exposed areas 508, 509, 510 of the silicon
dioxide layer 502 are then etched by a fluorine-based plasma with a
high degree of anisotropy and selectivity to the protective
photoresist 5061 until the silicon separation substrate 500 is
reached. The remaining photoresist is removed in an oxygen plasma
or in an actively oxidizing chemical bath like sulfuric acid
(H2SO4) activated with hydrogen peroxide (H2O2).
[0159] As shown in the cross-sectional view of FIG. 38, the
reservoir 410, the separation channel 412, and the separation posts
416 in the separation channel 412 are vertically formed in the
silicon separation substrate 500 by another fluorine-based etch.
Preferably, the reservoir 410 and the separation channel 412 have
the same depth controlled by the etch time at a known etch rate.
The simultaneous formation of the reservoir 410 and the channel 412
ensures uniform depth such that there are no discontinuities in the
fluid-constraining surfaces to impede the fluid flow. The depth of
the reservoir 410 and the channel 412 is preferably between
approximately 5-um and more preferably approximately 10 gm. The
etch can reliably and reproducibly be executed to produce an aspect
ratio (etch depth to width) of up to 30:1. Although not shown, any
other reservoirs and/or channels, populated or unpopulated, may
also be formed by this etch sequence.
[0160] A film of positive-working photoresist is then deposited
over the silicon dioxide layer 502 and the exposed separation
substrate 500 on the reservoir side 503 of the separation substrate
500. An area of the photoresist corresponding to the introduction
channel which will be subsequently etched is selectively exposed
through a mask by an optical lithographic exposure tool passing
short-wavelength light, such as blue or near-ultraviolet at
wavelengths of 365, 405, or 436 nanometers. After development of
the photoresist, the exposed area of the photoresist corresponding
to the introduction channel is removed and open to the underlying
separation substrate 500 while the unexposed areas remain protected
by the photoresist.
[0161] As shown in the plan and cross-sectional views of FIGS. 39A
and 39B, respectively, the exposed area of the separation substrate
500 is then vertically etched by a fluorine-based plasma with a
high degree of anisotropy and selectivity to the protective
photoresist until the silicon dioxide layer 504 on back side 505 is
reached. Thus, a portion of the introduction channel 404 is formed
through the separation substrate 500. The remaining photoresist is
removed in an oxygen plasma or in an actively oxidizing chemical
bath like sulfuric acid (H2SO4) activated with hydrogen peroxide
(H2O2). The silicon dioxide layer 504 on the back side 505 may then
be removed by, for example, an unpatterned etch in a fluorine-based
plasma.
[0162] Alternatively, as shown in FIGS. 40A and 40B, the
introduction channel 404 may be formed by etching from both the
reservoir side 503 and the back side 505 of the substrate 500.
After performing a vertical etch though a portion of the substrate
500 to form a portion of the introduction channel 404 in a manner
similar to that described above, a film of positive-working
photoresist 512 is deposited on the silicon dioxide layer 504 on
the back side 505 of the separation substrate 500. Patterns on the
back side 505 may be aligned to those previously formed on the
reservoir side 503 of the separation substrate 500. Because silicon
and its oxide are inherently relatively transparent to light in the
infrared wavelength range of the spectrum, i.e. approximately
700-1000 nanometers, the extant pattern on the reservoir side 503
can be distinguished with sufficient clarity by illuminating the
separation substrate 500 from the patterned reservoir side 503 with
infrared light. Thus, the mask for the back side 505 can be aligned
within required tolerances. Upon alignment, an area of the
photoresist 512 corresponding to the entrance orifice and the
introduction channel which will be subsequently etched is
selectively exposed through a mask by an optical lithographic
exposure tool passing short-wavelength light, such as blue or
near-ultraviolet at wavelengths of 365, 405, or 436 nanometers.
[0163] After development of the photoresist 512, the exposed area
514 of the photoresist corresponding to the entrance orifice is
removed to expose the underlying silicon dioxide layer 504 on the
back side 505 of the separation substrate 500 while the unexposed
areas remain protected by the photoresist 512. The exposed area 514
of the silicon dioxide layer 504 is then etched by a fluorine-based
plasma with a high degree of anisotropy and selectivity to the
protective photoresist 512 until the substrate 500 is reached. The
remaining photoresist provides additional masking during a
subsequent fluorine-based silicon etch to vertically etch the
backside portion of the introduction channel. Thus, a
through-substrate introduction channel 404 is complete. The
remaining photoresist is removed in an oxygen plasma or in an
actively oxidizing chemical bath like sulfuric acid (H2SO4)
activated with hydrogen peroxide (H2O2) Preferably, the
introduction channel 404 has the same diameter as the entrance
orifice. A practical limit on etch aspect ratio of 30:1 constrains
the diameter of the entrance orifice being etched to be
approximately 10 um or greater for substrates of approximately 300
um thickness. Preferably, the entrance orifice 406 and the
introduction channel 404 are approximately 100 um in diameter due
to practical considerations. For example, the etch aspect ratio
imposes a minimum diameter, and the diameter is preferably
sufficiently large to enable ease of filling the reservoir 410 yet
sufficiently small to ensure a fluid surface tension to prevent the
fluid from leaking out of the reservoir 410.
[0164] Alternatively, both the introduction channel and the
entrance orifice may be formed by etching from the back side 505 of
the separation substrate 500. This may be preferable as it may be
difficult to satisfactorily coat the separation posts 416 with
photoresist. Further, this may be desirable depending on the
application of the device, e.g. the external sample delivery
system, the desired chip handling devices, the interfacing with
other devices, chip-based or non-chip based, and/or the packaging
considerations of the chip. Referring to the cross-sectional view
of FIG. 41, after the reservoir, separation channel and the
separation posts are etched in the separation substrate 500 (shown
in FIG. 38), a film of positive-working photoresist 516 is
deposited on the silicon dioxide layer 504 on the back side 505 of
the separation substrate 500. Patterns on the back side 505 may be
aligned to those previously formed on the reservoir side 503 of the
separation substrate 500 by illuminating the separation substrate
500 from the patterned reservoir side 503 with infrared light, as
described above. Upon alignment, an area of the photoresist 516
corresponding to the entrance orifice which will be subsequently
etched is selectively exposed through a mask by an optical
lithographic exposure tool passing short wavelength light, such as
blue or near-ultraviolet at wavelengths of 365, 405, or 436
nanometers.
[0165] After development of the photoresist 516, the exposed area
518 of the photoresist 516 corresponding to the entrance orifice is
removed to expose the underlying silicon dioxide layer 504 on the
back side 505 of the separation substrate 500. The exposed area 518
of the silicon dioxide layer 504 is then etched by a fluorine-based
plasma with a high degree of anisotropy and selectivity to the
protective photoresist 512 until the silicon separation substrate
500 is reached. The remaining photoresist is left in place to
provide additional masking during the subsequent etch through the
silicon separation substrate 500.
[0166] Referring now to the cross-sectional view of FIG. 42, the
introduction channel 404 is vertically formed through the silicon
separation substrate 500 by another fluorine-based etch. The
introduction channel 404 is completed by etching through the
separation substrate 500 until the reservoir 410 is reached. Thus,
the introduction channel 404 extends through the separation
substrate 500 between the entrance orifice 406 on the back side 505
of the separation substrate 500 and the reservoir 410. The
remaining photoresist is removed in an oxygen plasma or in an
actively oxidizing chemical bath like sulfuric acid (H2SO4)
activated with hydrogen peroxide (H2O2)
[0167] Oxidation for surface passivation and fluid isolation As
shown in the cross-sectional view of FIG. 43, a layer of silicon
dioxide 522 is grown on all silicon surfaces of the substrate 500
by subjecting the silicon substrate 500 to elevated temperature in
an oxidizing ambient. For example, the oxidizing ambient may be an
ultra-pure steam produced by oxidation of hydrogen for a silicon
dioxide thickness greater than approximately several hundred
nanometers or pure oxygen for a silicon dioxide thickness of
approximately several hundred nanometers or less. The layer of
silicon dioxide 522 over all silicon surfaces of the separation
substrate 500 electrically isolates a fluid in the channel from the
silicon posts having a circular cross-sectional shape and a
diameter and inter-post spacing of approximately I um. FIG. 44C
shows separation posts in a portion of a separation channel, the
separation posts having a rectangular or square cross-sectional
shape with a dimension of 2 um and inter-post spacing of
approximately I um.
[0168] In a variation, the entrance orifice and the introduction
channel for filling the fluid reservoir may be formed in the cover
substrate 524 after a layer of silicon dioxide 525 is grown on all
surfaces of the cover substrate 524, rather than in the substrate
500. As shown in FIG. 45, the cover substrate 524 may be bonded to
the reservoir side 503 of the separation substrate 500. The
entrance orifice 406' and the introduction channel 404' may be
formed in the cover substrate 524 after alignment with respect to
the reservoir 410. The entrance orifice 406' and the introduction
channel 404' may be formed in the same or similar manner as
described above by utilizing lithography to define the entrance
orifice pattern and reactive-ion etching to create the entrance
orifice and the through-cover introduction channel. The cover
substrate 524 is again subjected to elevated temperature in an
oxidizing ambient to grow a layer of oxide on the surface of the
introduction channel 404'. Further, the introduction channel 404'
may be formed from one or two sides of the cover substrate 524. If
channel 404' is formed from two sides of the cover substrate, the
cover substrate 524 may be bonded to substrate 500 after forming
the channel 404' and after oxidation of the channel surface. One
advantage of defining the entrance orifice on the same side of the
completed liquid chromatography device as the reservoir and
separation channel is that the back side of the substrate 500 is
then free from any features and may then be bonded to a protective
package without special provision for filling the reservoir through
an entrance orifice defined on the back-side of the substrate.
[0169] Metallization for fluid flow control
[0170] FIGS. 46A and 46B illustrate the formation of a reservoir, a
filling, and an exit electrode as well as conductive lines or wires
connecting the electrodes to bond pads in the cover substrate 526,
preferably comprising glass and/or silicon. The cover substrate 526
shown in FIGS. 46A and 46B does not provide an entrance orifice or
an introduction channel although the metallization process
described herein may be easily adapted for a cover substrate
providing an entrance orifice and an introduction channel.
[0171] As shown in the plan and cross-sectional view of FIGS. 46A
and 46B, respectively, prior to the depositing of conductive
material on the cover substrate 526, all surfaces of the cover
substrate 526 are subjected to thermal oxidization in a manner that
is the same as or similar to the process described above to create
a film or layer of silicon dioxide 528. Such oxidization is not
performed where the cover substrate 526 comprises glass.
[0172] The silicon dioxide layer 528 provides a surface on which
conductive electrodes may be formed. The thickness of the silicon
dioxide layer 528 is controllable through the oxidation temperature
and time and the final thickness can be selected to provide the
desired degree of electrical isolation, where a thicker layer of
silicon dioxide provides a greater resistance to electrical
breakdown. The silicon dioxide layer 528 electrically isolates all
electrodes from the cover substrate 526 and isolates the fluid in
the reservoir and the channel of the liquid chromatography device
from the cover substrate 526. The ability to isolate the fluid from
the cover substrate 526 complements the electrical isolation
provided in the separation substrate through oxidation and ensures
the complete electrical isolation of the fluid from both the
separation substrate and the cover substrate 526. The complete
electrical isolation of the sample fluid from both substrates
allows for the application of electric potential differences
between spatially separated locations in the fluidic flow path
resulting in control of the fluid flow through the path.
[0173] The cover substrate 528 may be cleaned after oxidation
utilizing an oxidizing solution such as an actively oxidizing
chemical bath, for example, sulfuric acid (H2SO4) activated with
hydrogen peroxide (H2O2) The cover substrate 528 is then thoroughly
rinsed to eliminate organic contaminants and particulates. A layer
of conductive material 530 such as aluminum is then deposited by
any suitable method such as by DC magnetron sputtering in an argon
ambient. The thickness of the aluminum is preferably approximately
3000 A, although shown having a larger thickness for clarity.
Although aluminum is utilized in the fabrication sequence described
herein, any type of highly conductive material such as other
metals, metallic multi-layers, silicides, conductive polymers, and
conductive ceramics like indium tin oxide (ITO) may be utilized for
the electrodes. The surface preparation for satisfactory adhesion
may vary depending on the specific electrode material used. For
example, the silicon dioxide layer 528 provides a surface to which
aluminum electrodes may adhere as aluminum does not generally
adhere well to native silicon. A film of positive-working
photoresist 532 is then deposited over the surface of the
conductive material 530. Areas of the photoresist layer 532
corresponding to areas surrounding the electrodes (shown) and
conductive lines or wires and bond pads which will be subsequently
etched are selectively exposed through a mask by an optical
lithographic exposure tool passing short-wavelength light, such as
blue or 20 near-ultraviolet at wavelengths of 365, 405, or 436
nanometers.
[0174] After development of the photoresist 532, the exposed areas
of the photoresist are removed, leaving opening to the underlying
aluminum conductive layer 530 while the unexposed areas 534, 536,
538 corresponding to the reservoir, filling and exit electrodes,
respectively, as well as conductive lines or wires and bond pads
remain protected by the photoresist. The conductive electrodes and
the linesibond pads may be etched, such as by a wet chemical etch
or a reactive-ion etch, as appropriate for the particular
conductive material. The etch is selective to the underlying
silicon dioxide layer 528 or is terminated upon reaching the
silicon dioxide layer 528 as determined by the etch time and rate.
The remaining photoresist is removed in an oxygen plasma or in a
solvent bath such as acetone. The fabrication sequence thus results
in physically and electrically separate islands of conductive
electrodes, lines and bond pads according to the pattern designed
in the mask.
[0175] The cover substrate may be larger than the separation
substrate to allow access to the bond pads and/or directly to the
electrodes for the application of potential voltage(s) to the
electrode(s). As shown in FIG. 46C, the cover substrate 526' is
larger than the separation substrate such that the separation
substrate only extends to dashed line 540 relative to the cover
substrate 526'. Conductive leadthroughs such as connecting metal
lines 542, 544 and 546 extend from the reservoir, filling and exit
electrodes, 534, 536, 538, respectively, and enable the application
of potential voltage(s) to the electrode(s).
[0176] Alternatively, a metal lead may be formed from each
electrode to an otherwise unpatterned area of the separation
substrate such that a through-substrate access channel formed in
the cover substrate and filled with a conductive material by
chemical vapor deposition (CVD) allows access to the electrode(s).
As an alternative to chemical vapor deposition, the sidewalls of
the through-substrate access channel may be sloped, for example by
KOH etch, to facilitate continuous deposition of a conductive
material thereon, thereby providing an electrically continuous path
from the separation substrate to the top of the cover substrate
where potential voltages can be applied. In these variations, the
separation and the cover substrates may be of the same size.
[0177] Although the electrodes are preferably provided on a surface
of the cover substrate, the electrodes may be alternatively and/or
additionally provided on the separation substrate by appropriate
modifications to the above-described fabrication process. For
example, in such a variation, the side walls of the reservoir are
preferably not at a 90' angle relative to the bottom wall and can
be formed at least in part by, for example, a wet chemical
potassium hydroxide (KOH) etch. The sloped reservoir side walls
allow for the deposition of a conductive material thereon. In
another variation, the electrodes may also be formed by a damascene
process, known in the art of semiconductor fabrication. The
damascene process provides the advantage of a planar surface
without the step up and step down surface topography presented by a
bond line or pad and thus facilitates the bonding of the separation
and cover substrate, as described below.
[0178] The above described fabrication sequence for the liquid
chromatography device may be easily adapted to and is applicable
for the simultaneous fabrication of a monolithic system comprising
multiple liquid chromatography devices including multiple
reservoirs and/or multiple separation channels as described above
embodied in a single monolithic substrate. Further, although the
fabrication sequence is described in terms of fabricating a single
liquid chromatography device, the fabrication sequence facilitates
and allows for massively parallel processing of similar devices.
The multiple liquid chromatography devices or systems fabricated by
massively parallel processing on a single wafer may then be cut or
otherwise separated into multiple devices or systems.
[0179] Although control of the liquid chromatography device has
been described above as comprising reservoir, filling and exit
electrodes, any suitable combination of such and/or other
electrodes in electrical contact with the fluid in the fluid path
may be provided and easily fabricated by modifying the layout
design. Further, any or all of the electrodes may be additionally
or alternatively provided in the separation substrate. Electrodes
may be formed in the separation substrate by modifying the
fabrication sequence to include additional steps similar to or the
same as the steps as described above with respect to the formation
of the electrodes in the cover substrate.
[0180] Bonding cover substrate to separation substrate
[0181] As described above, the cover substrate is preferably
hermetically bonded by any suitable method to the separation
substrate for containment and isolation of the fluid in the liquid
chromatography device. Examples of bonding silicon to silicon or
glass to silicon include anodic bonding, sodium silicate bonding,
eutectic bonding, and fusion bonding.
[0182] For example, to bond the separation substrate to a glass
cover substrate by anodic bonding, the separation substrate and
cover substrate are heated to approximately 400.degree. C. and a
voltage of 400-1200 Volts is applied, with the separation substrate
chosen as the anode (the higher potential). Further, as the
required bonding voltage depends on the surface oxide thickness, it
may be desirable to remove the oxide film or layer from the back
side 505 of the separation substrate prior to the bonding process
in order to reduce the required bonding voltage. The oxide film or
layer may be removed by, for example, an unpatterned etch in a
fluorine-based plasma. The etch is continued until the entire oxide
layer has been removed, and the degree of over-etch is unimportant.
Thus, the etch is easily controlled and high-yielding.
[0183] Critical considerations in any of the bonding methods
include the alignment of features in the separation and the cover
substrates to ensure proper functioning of the liquid
chromatography device after bonding and the provision in layout
design for conductive lead-throughs such as the bond pads and/or
metal lines so that the electrodes (if any) are accessible from
outside the liquid chromatography device. Another critical
consideration is the topography created through the fabrication
sequence which may compromise the ability of the bonding method to
hermetically seal the separation and cover substrates. For example,
the step up and step down in the surface topography presented by a
metal line or pad may be particularly difficult to form a seal
therearound as the silicon or glass does not readily deform to
conform to the shape of the metal line or pad, leaving a void near
the interface between the metal and the oxide.
Integration of Liquid Chromatography and Electrospray Devices on a
Chip
[0184] The cross-sectional schematic view of FIG. 47 shows a liquid
chromatography-electrospray system 600 comprising a liquid
chromatography device 602 of the present invention integrated with
an electrospray device 620 of the present invention such that a
homogeneous interface is formed between the exit orifice 614 of the
liquid chromatography device 602 and the entrance orifice 622 of
the electrospray device 620. The single integrated system 600
allows for the fluid exiting the exit orifice 614 of the liquid
chromatography device 602 to be delivered on-chip to the entrance
orifice 622 of the electrospray device 620 in order to generate an
electrospray.
[0185] As shown in FIG. 47, the entrance orifice 606 and the
introduction channel 604 of the liquid chromatography device 602
are formed in the cover substrate 608 along with the electrospray
device 620. Alternatively, the liquid chromatography entrance
orifice and the introduction channel may be formed in the
separation substrate.
[0186] Fluid at the electrospray nozzle entrance 622 is at the exit
voltage applied to the exit electrode 610 in the separation channel
612 near the liquid chromatography exit orifice 614. Thus, an
electrospray entrance electrode is not necessary.
[0187] The single integrated system 600 provides the advantage of
minimizing or eliminating extra fluid volume to reduce the risk of
undesired fluid changes, such as by reactions and/or mixing. The
single integrated system 600 also provides the advantage of
eliminating the need for unreliable handling and attachment of
components at the microscopic level and of minimizing or
eliminating fluid leakage by containing the fluid within one
integrated system.
[0188] The integrated liquid chromatography-electrospray system 600
may be utilized to deliver liquid samples to the sampling orifice
of a mass spectrometer. The sampling orifice of the mass
spectrometer may serve as an extraction electrode in the
electrospray process when held at an appropriate voltage relative
to the voltage of the electrospray nozzle 624. The liquid
chromatography-electrospray system 600 may be positioned within 10
nun of the sampling orifice of the mass spectrometer for efficient
extraction of the fluid from the electrospray nozzle 624.
[0189] Multiple liquid chromatography-electrospray systems on a
single chip
[0190] Multiples of the liquid chromatography-electrospray system
600 may be formed on a single chip to deliver a multiplicity of
samples to a common point for subsequent sequential analysis. For
example, FIG. 48 shows a plan view of multiple liquid
chromatography-electrospray systems 600 on a single chip 650 and
FIG. 49 shows a detailed view of area A of systems 600 with the
separation channels shown in phantom and without the recessed
portions for purposes of clarity. As shown, the multiple nozzles
624 of the electrospray devices 620 may be radially positioned
about a circle having a relatively small diameter near the center
of the single chip 650. The dimensions of the electrospray nozzles
and the liquid chromatography channels limit the radius at which
multiple nozzles are positioned on the multi-system chip 650. For
example, the multi-system chip may provide 96 nozzles with widths
of up to 50 gm positioned around a circle 2 mm in diameter such
that the spacing between each pair of nozzles is approximately 65
gm.
[0191] Alternatively, an array of multiple electrospray devices
without liquid chromatography devices may be formed on a single
chip to deliver a multiplicity of samples to a common point for
subsequent sequential analysis. The nozzles may be similarly
radially positioned about a circle having a relatively small
diameter near the center of the chip. The array of electrospray
devices on a single microchip may be integrated upstream with
multiple fluid delivery devices such as separation devices
fabricated on a single microchip. For example, an array of radially
distributed exit orifices of a radially distributed array of micro
liquid chromatography columns may be integrated with radially
distributed entrance orifices of electrospray devices such that the
nozzles are arranged at a small radius near the orifice of a mass
spectrometer. Thus, the electrospray devices may be utilized for
rapid sequential analysis of multiple sample fluids. However,
depending upon the specific application and/or the capabilities of
the downstream mass spectrometer (or other downstream device), the
multiples of the electrospray devices may be utilized one at a time
or simultaneously, either all or a portion of the electrospray
devices, to generate one or more electrosprays. In other words, the
multiples of the electrospray devices may be operated in parallel,
staggered or individually.
[0192] The single multi-system chip 650 may be fabricated entirely
in silicon substrates, thereby taking advantage of well-developed
silicon processing techniques described above. Such processing
techniques allow the single multi-system chip 650 to be fabricated
in a cost-effective manner, resulting in a cost performance that is
consistent with use as a disposable device to eliminate
cross-sample contamination. Furthermore, because the dimensions and
positions of the liquid chromatographyelectrospray systems are
determined through layout design rather than through processing,
the layout design may be easily adapted to fabricate multiple
liquid chromatography-electrospray systems on a single chip.
[0193] Interface of a multi-system chip to mass spectrometer
[0194] The radially distributed array of electrospray nozzles 624
on a multi-system chip may be interfaced with a sampling orifice of
a mass spectrometer by positioning the nozzles near the sampling
orifice. The tight radial configuration of the electrospray nozzles
624 allows the positioning thereof in close proximity to the
sampling orifice of a mass spectrometer.
[0195] The multi-system chip 650 may be rotated relative to the
sampling orifice to position one or more of the nozzles for
electrospray near the sampling orifice. Appropriate voltage(s) may
then be applied to the one or more of the nozzles for electrospray.
Alternatively, the multi-system chip 650 may be fixed relative to
the sampling orifice of a mass spectrometer such that all nozzles,
which converge in a relatively tight radius, are appropriately
positioned for the electrospray process. As is evident, eliminating
the need for nozzle repositioning allows for highly reproducible
and quick alignment of the single multi-system chip and increases
the speed of the analyses.
[0196] One, some or all of the radially distributed nozzles 624 of
the electrospray devices 620 may generate electrosprays
simultaneously, sequentially or randomly as controlled by the
voltages applied to the appropriate electrodes of the electrospray
device 620.
[0197] While specific and preferred embodiments of the invention
have been described and illustrated herein, it will be appreciated
that modifications can be made without departing from the spirit of
the invention as found in the appended claims.
* * * * *