U.S. patent application number 10/837731 was filed with the patent office on 2004-10-14 for methods for isolating constituents of a sample.
Invention is credited to Gilton, Terry L..
Application Number | 20040203167 10/837731 |
Document ID | / |
Family ID | 32680353 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040203167 |
Kind Code |
A1 |
Gilton, Terry L. |
October 14, 2004 |
Methods for isolating constituents of a sample
Abstract
Methods for separating one or more constituents from a sample
include drawing a sample through a matrix that is part of a column,
trench, or channel. The matrix may be formed from hemispherical
grain silicon or from pores formed in a semiconductor substrate.
The sample may be drawn through the column, trench or channel by
application of pressure thereto, by capillary action,
electrophoretically, or by any other suitable technique. As the
sample is drawn through the channel, a stationary phase within the
matrix may cause different constituents of the sample to flow
through the column, trench, or channel at different rates from one
another. Capture molecules may be applied to the matrix to bind
constituents of the sample, or analytes, for prolonged periods of
time, facilitating their detection.
Inventors: |
Gilton, Terry L.; (Boise,
ID) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
32680353 |
Appl. No.: |
10/837731 |
Filed: |
May 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10837731 |
May 3, 2004 |
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09442713 |
Nov 18, 1999 |
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6762057 |
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09442713 |
Nov 18, 1999 |
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09177814 |
Oct 23, 1998 |
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Current U.S.
Class: |
506/9 ;
506/13 |
Current CPC
Class: |
Y10T 436/25375 20150115;
B01D 63/088 20130101; G01N 30/6095 20130101; G01N 33/54353
20130101; B01D 2313/345 20130101; G01N 27/44791 20130101; G01N
30/466 20130101; B01L 2400/0487 20130101; B01L 3/502707 20130101;
B01D 61/18 20130101; B01D 63/081 20130101; B01L 3/5023 20130101;
G01N 30/6065 20130101; B01L 2400/0421 20130101; G01N 30/6073
20130101; B01L 2300/0816 20130101; B01L 2200/12 20130101 |
Class at
Publication: |
436/161 |
International
Class: |
G01N 030/02 |
Claims
1. A method of substantially isolating a constituent of a sample,
comprising: dispersing the sample in a mobile phase; applying the
sample to a first end of an elongate trench formed in a substrate,
at least a portion of the elongate trench being lined with
hemispherical grain silicon; and drawing the sample across a
flowfront at least partially through recessed locations of the
hemispherical grain silicon to facilitate separation of the
constituent from the sample.
2. The method of claim 1, further comprising: detecting the
constituent as is passes through or out of the elongate trench.
3. The method of claim 1, wherein dispersing comprises dissolving
the sample in a liquid mobile phase.
4. The method of claim 1, wherein drawing comprises applying a
differential pressure to at least the elongate trench.
5. The method of claim 1, wherein drawing is effected without
applying differential pressure to the elongate trench.
6. The method of claim 1, wherein drawing is effected by capillary
action.
7. The method of claim 1, wherein drawing comprises applying an
electrical current across a length of the elongate trench.
8. The method of claim 1, wherein applying comprises applying the
sample to the first end of an elongate trench having at least one
type of capture substrate immobilized to at least the hemispherical
grain silicon lining the elongate trench.
9. The method of claim 8, wherein applying comprises applying the
sample to the first end of an elongate trench having at least one
type of antibody immobilized to at least the hemispherical grain
silicon lining the elongate trench.
10. The method of claim 8, wherein applying comprises applying the
sample to the first end of an elongate trench having at least one
type of antigen immobilized to at least the hemispherical grain
silicon lining the elongate trench.
11. The method of claim 8, further comprising: detecting prolonged
binding of the constituent by the capture substrate.
12. The method of claim 8, wherein drawing is at least partially
induced by the at least one type of capture substrate.
13. A method of identifying the presence of a constituent in a
sample, comprising: providing the sample in a mobile phase;
applying the sample to a first end of an ultrasmall flow channel
comprising a matrix with passageways therethrough; drawing the
sample across a flowfront through the ultrasmall flow channel and
into contact with at least one type of capture molecule immobilized
to at least one of silicon and silicon dioxide at a selected
location along the ultrasmall flow channel; and detecting prolonged
binding of the constituent with the at least one type of capture
molecule.
14. The method of claim 13, wherein detecting comprises applying a
detection reagent to at least the selected location and analyzing
the detection reagent to determine at least one of a presence of
the constituent in the sample and an absence of the constituent
from the sample.
15. The method of claim 14, wherein analyzing comprises analyzing
the detection reagent to quantify an amount of the constituent
bound to the at least one type of capture molecule.
16. The method of claim 13, wherein detecting comprises determining
an electrical characteristic of the selected location and comparing
the electrical characteristic to an electrical characteristic of a
control.
17. The method of claim 13, further comprising: applying the at
least one type of capture molecule to at least the selected
location.
18. The method of claim 17, wherein applying the at least one type
of capture molecule is effected before applying the sample.
19. The method of claim 13, wherein applying comprises applying the
sample to the ultrasmall flow channel with the at least one type of
capture molecule comprising an antibody.
20. The method of claim 13, wherein applying comprises applying the
sample to the ultrasmall flow channel with the at least one type of
capture molecule comprising an antigen.
21. The method of claim 13, further comprising applying a
differential pressure to at least the ultrasmall flow channel to
effect drawing of the sample.
22. The method of claim 13, wherein drawing is effected without
applying differential pressure to the ultrasmall flow channel.
23. The method of claim 22, wherein drawing comprises capillary
action.
24. The method of claim 13, wherein drawing comprises applying an
electrical current across a length of the ultrasmall flow channel
device.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/442,713, filed Nov. 18, 1999, pending, which is a divisional of
application Ser. No. 09/177,814, filed Oct. 23, 1998, pending.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to chromatographs and other
apparatus for separating the constituents of a sample.
Particularly, the present invention relates to a miniaturized
separation apparatus which comprises a porous capillary column.
More specifically, the porous separation apparatus of the present
invention includes a sample column and a detector that is disposed
along the column to detect the presence of and identify each
constituent that passes by the detector. The porous capillary
column may comprise a matrix of porous silicon or hemispherical
grain silicon on the surface thereof. The present invention also
includes methods for manufacturing and using the inventive
separation apparatus.
[0004] 2. Background of Related Art
[0005] Various techniques have long been employed to separate the
constituents of a sample in order to facilitate the identification
and quantification of one or more of the constituents. Separation
techniques are useful for separating inorganic substances and
organic substances, such as chemicals, proteins, and nucleic acids.
Techniques that have been conventionally employed for separating
the constituents of a sample include various types of
chromatography and electrophoresis.
[0006] Chromatography is a process that is employed in analytical
chemistry in order to separate and identify the constituents of a
sample. The various types of chromatography that have been
conventionally employed include thin layer chromatography (TLC),
column chromatography, gel permeation chromatography, ion-exchange
chromatography, affinity chromatography, high performance liquid
chromatography (HPLC), and gas chromatography (GC).
[0007] Thin film chromatography is a well known technique wherein a
drop of a sample liquid is applied as a spot to a sheet of
absorbent material, which may be paper or a sheet of plastic or
glass covered with a thin layer of inert absorbent material, such
as cellulose or silica gel. Thin layer chromatographic techniques
typically employ a solvent mixture, such as water and an alcohol as
respective stationary and mobile phases. The solvent mixture
permeates the absorbent material from one edge and the capillary
action of the absorbent material moves the sample across the thin
layer. One of the solvents binds more tightly to the absorbent
material to act as a stationary phase, while the other acts as a
mobile phase. As the solvent mixture moves across the absorbent
material, the constituents of the sample are separated relative to
their solubility in each of the two solvents. Stated another way,
the sample constituents equilibrate according to their relative
solubilities in each of the solvents. Constituents which are the
most soluble in the stationary phase move very little, while
constituents which are more soluble in the mobile phase move at
higher rates and therefore travel greater distances across the
absorbent material.
[0008] Conventional column chromatography techniques employ a
vertical tube, or column, that is filled with a finely divided
solid, or a liquid stationary phase. As a sample is washed down
through the stationary phase, it is dissolved in and carried by a
mobile phase, which is typically liquid or gas. The various
constituents of the sample travel through the stationary phase at
different rates. Thus, each of the constituents of the sample spend
a different amount of time in the column. The constituents may be
collected in fractions as they exit the column and subsequently
identified or otherwise analyzed. Constituents of the sample which
remain in the stationary phase may be separately identified or
otherwise analyzed by sectioning the stationary phase.
[0009] Gel permeation chromatography techniques typically employ a
column with a stationary phase disposed therein. The stationary
phase includes an absorbent gel material with pores of
substantially uniform size. As the mobile phase and the sample that
is dissolved therein pass through the stationary phase, some of the
molecules that are smaller than the pores become entrapped therein
and therefore pass through the column more slowly. The passage of
intermediately sized molecules, which are of approximately the same
size as the pores, through the column is delayed some, as such
molecules enter some of the pores. Molecules that are larger than
the pores of the absorbent gel material pass through the stationary
phase most quickly, as none of the larger molecules become
entrapped in the pores.
[0010] Ion exchange chromatography is another variation of column
chromatography, wherein the stationary phase comprises positively
or negatively charged particles. Oppositely charged constituents of
a sample are attracted to the stationary phase, and therefore pass
through the column at a slower rate than uncharged constituents and
constituents which have the same charge as the charged particles of
the stationary phase.
[0011] In affinity chromatography, the solid phase comprises
particles which have substrate molecules or particles, such as
purified antibodies or purified antigens, covalently attached
thereto. The substrate binds to a specific constituent or group of
constituents in a sample. For example, if the stationary phase
comprises antibodies that are specific for a particular antigen, as
the sample and mobile phase pass through the column, only that
particular antigen will be bound by the stationary phase. The
remainder of the sample constituents will pass through the column
quickly. The column is subsequently washed to remove any residual
amount of the sample from the column. The column is then washed
with a dissociating solution, such as a concentrated salt solution,
an acidic solution, or a basic solution, in order to dissociate the
separated sample constituent from the stationary phase.
[0012] High performance liquid chromatography ("HPLC") is similar
to column chromatography. In HPLC, the stationary phase is
typically a liquid that is carried on very small particles, for
example 0.01 mm or less. Consequently, the stationary phase has a
very large surface area, and the mobile phase flows extremely
slowly therethrough. Thus, a high pressure pump is typically
employed in order to increase the rate at which the mobile phase
moves through the column.
[0013] Conventional gas chromatography methods typically employ a
liquid solid phase that is supported by a solid column and a mobile
phase that comprises a substantially inert gas, such as nitrogen,
argon, hydrogen, or helium. The sample is vaporized as it is
injected into the column. As with thin layer chromatography, column
chromatography, and HPLC, the constituents of the sample travel
across the stationary phase at different rates, and therefore exit
the column at different times. As the constituents of the sample
exit the column, the constituents are analyzed by a detector, such
as a katharometer, a flame ionizer, or an electron capture system,
which generates a chromatogram. The identity of each constituent
may then be determined by analyzing the chromatogram.
[0014] Gas chromatographs are ever-decreasing in size in order to
increase their portability. Some small, or miniature or micro gas
chromatographs, include columns, which are also referred to as
capillary columns, that are fabricated on a silicon substrate. U.S.
Pat. No. 5,583,281 (the "'281 patent"), which issued to Conrad M.
Yu on Dec. 10, 1996; U.S. Pat. No. 4,935,040 (the "'040 patent"),
which issued to Michel G. Goedert on Jun. 19, 1990; and U.S. Pat.
No. 4,471,647 (the "'647 patent"), which issued to John H. Jerman
et al. on Sep. 18, 1994, each disclose exemplary small silicon gas
chromatography columns. The capillary columns that are disclosed in
each of the '281, '040, and '647 patents include open channels, or
conduits, that are etched into the semiconductor substrate.
[0015] Similarly, U.S. Pat. No. 5,132,012 (the "'012 patent"),
which issued to Junkichi Miura et al. on Jul. 21, 1992, discloses a
liquid chromatograph that includes a capillary column formed in a
semiconductor substrate. The capillary column of the chromatograph
of the '012 patent comprises an open channel, or conduit.
[0016] U.S. Pat. No. 5,571,410 (the "'410 patent"), which issued to
Sally A. Swedberg et al. on Nov. 5, 1996, discloses a miniature gas
chromatography system which includes a capillary column that is
formed in a non-silicon substrate by laser ablation. The capillary
column of the chromatograph of the '410 patent comprises an open
channel, or conduit, with a substantially smooth surface.
[0017] The use of substantially smooth, open-channeled capillary
columns in miniature chromatographs is, however, somewhat
undesirable from the standpoint that open-channeled columns
typically have a surface area that is limited by the area of the
substantially smooth surface of the channel. The amount of
stationary phase material that may be disposed along a given length
of substantially smooth, open-channeled capillary columns is also
limited by the surface area of that length of the capillary column.
Thus, in order to effectively separate the various constituents of
a sample, the capillary column must be relatively long.
Consequently, the substrate on which the capillary column is formed
must have a sufficient surface area to facilitate fabricating the
capillary column thereon. Thus, the use of substantially smooth,
open-channeled capillary columns in miniature gas chromatographs
imposes minimum size limitations on such chromatographs.
[0018] Another technique for separating the various constituents of
a sample is typically referred to as electrophoresis.
Electrophoresis is a process whereby molecules having a net overall
electrical charge are migrated at a rate that depends on the
electrical charge, size and shape of the molecule. Electrophoresis
techniques typically employ a solid matrix through which the
constituents, or molecules, of the sample are migrated. A variation
of electrophoresis that is typically referred to as polyacrylamide
gel electrophoresis (PAGE) separates molecules based strictly on
their size. In PAGE, the molecules of the sample are typically
linearized and separated, or disassociated from themselves and from
other molecules, by means of sodium dodecyl sulfate (SDS), a
detergent that binds to the hydrophobic regions of proteins, and
2-mercaptoethanol, or .beta.-mercaptoethanol, which breaks
disulfide (S--S) linkages that occur between some amino acids of a
protein. The sample is then migrated through a polyacrylamide gel
cross-linked matrix, which has very small pores. The pore size of
the polyacrylamide gel may be adjusted in accordance with the
molecular size, or weight, range for which separation is
desired.
[0019] The preparation of polyacrylamide gels is a relatively long
process. Moreover, the acrylamide that is used to form the gel
matrix is a neurotoxin. Some of the other chemicals that may be
utilized in electrophoretic processes are also hazardous. In
addition, the amount of electric current that may be used to
separate the constituents of a sample in gel electrophoresis has
conventionally been limited, as too great a current will melt or
otherwise disrupt the structure of the gel.
[0020] Thus, a small separation apparatus is needed that may be
employed to conduct various types of sample separation, which is
smaller than conventional devices, and which separates samples
adequately. There are also needs for reduced equipment and
operational costs.
SUMMARY OF THE INVENTION
[0021] The separation apparatus, method of manufacturing the
separation apparatus, and methods of using the separation apparatus
of the present invention address each of the foregoing needs.
[0022] The sample separation apparatus of the present invention
includes a substrate with a capillary column thereon, the latter
comprising a rough surface, such as a matrix which defines a
plurality of pores therethrough or an open column with a rough
surface, which is also referred to as a matrix. The surface area of
the matrix of each capillary column facilitates the separation of
the constituents of a sample over a relatively short length of the
column compared to the required lengths of conventional smooth,
"open," etched or ablated columns to effectively separate the
constituents. Preferably, the capillary column, which is also
referred to as a porous capillary column, comprises porous silicon
or hemispherical grain silicon, and is formed on a silicon
substrate. Such a column, depending on the width and depth thereof,
may be useful for separating the constituents of a sample or
detecting constituents in a sample having a volume of as small as
about one femtoliter (1.times.10.sup.-15 liter). The separation
apparatus may also include a detector disposed proximate the
capillary column. Such a detector analyzes a characteristic of a
constituent as the constituent passes through the capillary column,
and thereby identifies or otherwise analyzes the constituent.
[0023] In a first variation of the apparatus of the present
invention, the sample separation apparatus may be employed as a
chromatography column. Accordingly, a stationary, or solid, phase
is disposed on the matrix of the capillary column. The type of
stationary phase that is selected for use in the sample separation
apparatus is dependent upon several factors, including without
limitation the chromatographic technique that will be employed with
the separation apparatus and the type of sample constituents that
are to be isolated. The types of stationary phase materials that
are useful in conventional chromatographic processes are also
useful in the first variation of the separation apparatus.
[0024] A second variation of the separation apparatus of the
present invention is useful for conducting electrophoretic
separation. Thus, size of the pores that are defined through the
porous silicon matrix or the amount of space between grains of
hemispherical grain silicon of the capillary column is determined
by the desirable rate of separation and the size of the sample
constituents for which separation is desired. The second variation
of the separation apparatus also includes first and second
electrodes positioned proximate respective first and second ends of
the capillary column. The first and second electrodes are
connectable to opposite electrical charges so as to facilitate the
generation of a current along a length of the capillary column, and
thereby facilitate the movement and separation of the sample
constituents along the column. Preferably, the second variation of
the separation apparatus also includes a control column adjacent
the capillary column and having substantially the same dimensions,
structure, and pore sizes or spacing as the capillary column. The
control column is useful for determining the molecular size or
weight of at least some of the various sample constituents.
[0025] In a third variation of the apparatus, the sample separation
apparatus may be employed to detect the presence or absence of
increased levels of a certain analyte. Accordingly, the third
variation includes a capture substrate disposed on at least a
portion of the rough surfaces of the capillary column. Preferably,
the capture substrate has a specific affinity for the measured, or
assayed, analyte.
[0026] A method of fabricating the sample separation apparatus of
the present invention includes selectively forming a capillary
column in a substrate.
[0027] When a silicon substrate is employed, various techniques
which are known in the art may be employed to define a porous
silicon capillary column therein. Known techniques may also be used
in order to form pores of a desired size. Known semiconductor layer
formation processes may also be employed to fabricate a detector
proximate the capillary column. Similarly, known processes are
useful for fabricating electrodes and other structures upon a
surface of the substrate.
[0028] Capillary columns that include hemispherical grain silicon
may also be selectively formed in a substrate by known techniques.
First, a trench, which defines the path of the capillary column, is
defined in a substrate by known patterning processes, such as mask
and etch techniques. The surface area of the surfaces of the trench
may then be increased by known methods, such as by forming
hemispherical grain silicon thereon.
[0029] A method of utilizing the inventive separation apparatus
includes disposing a sample proximate an end of the porous
capillary column and drawing the sample through the porous
capillary column to generate a flowfront of the sample and effect
the separation of a constituent from the sample. The sample may be
drawn along the capillary column by positive pressure, negative
pressure, capillary action, electric current, or any other known
technique that is employed to facilitate the movement of a sample
along a separation apparatus.
[0030] Variations of the inventive method employ the separation
apparatus of the present invention to effect various separation
techniques, including, without limitation, various types of
chromatographic separation, electrophoresis, and the isolation and
detection of one or more analytes from a sample.
[0031] Other advantages of the present invention will become
apparent to those of ordinary skill in the relevant art through a
consideration of the appended drawings and the ensuing
description.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0032] FIG. 1 is a perspective view of an embodiment of a sample
separation apparatus of the present invention;
[0033] FIG. 1a is a cross-section taken along line 1a-1a of FIG. 1,
which also illustrates a sealing element disposed over at least a
portion of the sample separation apparatus;
[0034] FIG. 1b is a perspective view of a variation of the sample
separation apparatus of FIG. 1, which illustrates an alternative
placement of detectors;
[0035] FIG. 2 is a perspective view of a variation of the sample
separation apparatus of FIG. 1 that is useful for performing
chromatography;
[0036] FIG. 2a is a perspective view of a variation of the sample
separation apparatus of FIG. 2 including a vacuum source
operatively connected to the capillary column;
[0037] FIG. 3 is a perspective view of another variation of the
sample separation apparatus of FIG. 1 that is useful for performing
electrophoresis;
[0038] FIG. 3a is a schematic representation of the sample
separation apparatus of FIG. 3, illustrating use of the sample
separation apparatus in association with an electrophoresis
apparatus;
[0039] FIG. 4 is a perspective view of another variation of the
sample separation apparatus of FIG. 1 that is useful for isolating
and detecting an analyte;
[0040] FIG. 5 is a cross-sectional view of a substrate that has
been patterned to define capillary column regions thereon;
[0041] FIG. 6 is an enlarged cross-sectional view taken along line
6-6 of FIG. 1 and illustrating the capillary columns;
[0042] FIG. 7 is a schematic representation of the use of an
anodization chamber to porify the capillary column regions of the
substrate of FIG. 5; and
[0043] FIG. 8 is an enlarged cross-sectional view of an alternative
rough capillary column, which includes hemispherical grain silicon
on the surface thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0044] With reference to FIG. 1, a first embodiment of a sample
separation apparatus 10 of the present invention is depicted.
Sample separation apparatus 10 includes a substrate 12 and
capillary columns 14 formed in the substrate. Capillary columns 14
each include a matrix 16 and a plurality of pores 18 formed through
the matrix. Pores 18 permit gases and liquids to flow along the
distance of capillary columns 14. Capillary columns 14 may also
include one or more reaction regions 20 along the longitudinal
extent thereof. Preferably, each of the reaction regions 20 along
each capillary column 14 are discrete from one another. Sample
separation apparatus 10 may also include one or more detectors 22
disposed proximate each capillary column 14.
[0045] Substrate 12 may be formed of silicon, gallium arsenide,
indium phosphide, or another material that can be treated to form
porous regions, such as capillary columns 14, and upon which
electrical devices, such as detector 22, can be formed.
Accordingly, capillary columns 14 may each comprise porous
silicon.
[0046] Alternatively, capillary columns 14 may be etched into a
surface of substrate 12, and the surfaces of capillary columns 14
roughened. An exemplary means of roughening the surfaces of
capillary columns 14 includes forming hemispherical grain silicon
thereon.
[0047] FIG. 1 illustrates a sample separation apparatus 10 that
includes four capillary columns 14. The length and porosity of each
column 14 depends, in part, upon the surface tension and viscosity
of the sample to be measured, and the desired degree of separation.
As depicted, each capillary column 14 includes three reaction
regions 20. Preferably, variations of sample separation apparatus
10 with more than one capillary column 14 include an equal number
of reaction regions 20 along each capillary column. Moreover, in
variations of sample separation apparatus 10 wherein the capillary
columns 14 each include more than one reaction region 20, the
positioning and spacing between corresponding reaction regions are
preferably substantially the same along each of the capillary
columns. Preferably, corresponding reaction regions 20 on different
columns 14 have substantially the same dimensions and pores 18, or
spacing between adjacent grains of hemispherical grain silicon,
which spaces are also referred to as "pores," of substantially the
same sizes and porosity.
[0048] Pores 18 may have cross-sectional diameters ranging from
about one nanometer (1 nm) or less to about 100 nm or greater. Due
to the small size of pores 18, the surface tension of many liquid
samples will cause such samples to travel very slowly along the
distance of capillary column 14 and create a flowfront. Gaseous
samples typically do not exhibit capillary action; thus, some
amount of force is required to facilitate the movement of gaseous
samples along capillary column 14. Accordingly, a migration
facilitator 24, such as a pump, vacuum, or current-generating
device, which is also referred to as a flow facilitator, may be
disposed proximate capillary column 14 in order to facilitate or
increase the migration rate of a sample 70 therealong.
[0049] Detectors 22 may be disposed adjacent capillary column 14 in
order to identify or otherwise analyze a constituent of sample 70
as the constituent passes thereby. Various embodiments of detector
22 include, but are not limited to, thermistors, field effect
transistors (FETs) that are capable of sensing various types of
chemicals, components that measure current as a voltage is applied
to sample 70, and other devices that are known to measure at least
one characteristic of a constituent of sample 70 or otherwise
facilitate identification of the constituent. U.S. Pat. No.
5,132,012 (the "'012 patent"), which issued to Junkichi Miura et
al. on Jul. 21, 1992, the disclosure of which is hereby
incorporated by reference in its entirety, discloses an exemplary
field effect transistor that may be employed as a detector 22 in
the present invention. U.S. Pat. No. 4,471,647 (the "'647 patent"),
which issued to John H. Jerman et al. on Sep. 18, 1984, the
disclosure of which is hereby incorporated by reference in its
entirety, discloses an exemplary thermal detector that may be
employed as a detector 22 in the sample separation apparatus of the
invention. Detector 22 may be positioned proximate an exit end 14b,
which is also referred to as a second end, of capillary column 14
to analyze the various constituents of sample 70 as they pass
thereby. Alternatively, as shown in FIG. 1b, detector 22 may be
positioned proximate a reaction region 20 of capillary column 14.
More than one detector 22 may be disposed proximate each capillary
column 14 to analyze sample 70 and the constituents thereof at
various positions of the capillary column.
[0050] Separation apparatus 10 may also include a processor 80 and
a memory device 82, each of a type known in the art. Processor 80
receives information about sample 70, or "sample information," from
one or more types of detectors 22 along column 14 and processes the
sample information to output same in a user-friendly format to a
display 84 external of sample separation apparatus 10. In
processing the sample information, processor 80 may compare the
sample information to known information that has been stored in
memory device 82, and thereby identify the sample or generate other
data regarding the sample information. The sample identity may then
be transmitted to display 84. Following the comparison of sample
information to known information, processor 80 may direct memory
device 82 to store information about the sample, including its
identity and associated data.
[0051] With reference to FIG. 1a, separation apparatus 10 may also
include a sealing element 11 disposed over a substantial portion of
the area of each capillary column 14 that is exposed on substrate
12. Sealing element 11 is preferably electrically insulative and
may be manufactured from silicon dioxide, glass (e.g., borosilicate
glass (BSG), phosphosilicate glass (PSG), borophosphosilicate glass
(BPSG), etc.), silicon nitride, polyimide, other electrically
non-conductive polymers, or any other electrically insulative
material.
[0052] Turning now to FIG. 2, a second embodiment of the sample
separation apparatus 10' of the present invention is shown, which
comprises a chromatography column. Accordingly, a stationary phase
17 may be disposed on matrix 16' of each capillary column 14'.
Stationary phase 17 comprises a material that is selected on the
basis of several factors, including without limitation the
chromatographic technique that will be employed and type of sample
constituents for which separation or isolation is desired.
Conventionally employed stationary phase materials may also be
employed as stationary phase 17.
[0053] Separation apparatus 10' may also include a migration
facilitator 24' which comprises a pump 26' that applies positive
pressure to facilitate the migration of a sample along each
capillary column 14'. Exemplary pumps 26' that are useful in
separation apparatus 10' are disclosed in U.S. Pat. No. 5,663,488
(the "'488 patent"), which issued to Tak Kui Wang et al. on Sep. 2,
1997, the disclosure of which is hereby incorporated by reference
in its entirety. Preferably, pump 26' is positioned proximate a
sample application end 14a', or first end, of each capillary column
14', and is in flow communication with the capillary column and to
facilitate movement of a sample 70' along each column 14'. A valve
25' may be disposed between pump 26' and each column 14' in order
to control the volume of gas or liquid that is forced into the
column by the pump in order to apply pressure to the column.
Exemplary valves 25' that are useful in the separation apparatus of
the present invention include the valves that are disclosed in U.S.
Pat. No. 4,869,282 (the "'282 patent"), which issued to Fred C.
Sittler et al. on Sep. 26, 1989, and U.S. Pat. No. 5,583,281 (the
"'281 patent"), which issued to Conrad M. Yu on Dec. 10, 1996, the
disclosures of each of which are hereby incorporated by reference
in their entirety.
[0054] Alternatively, as depicted in FIG. 2a, migration facilitator
24' may comprise a vacuum source 28', as known in the art, which
exerts a negative pressure on sample 70' in order to pull the
sample along each capillary column 14'. Such a vacuum source is
operatively attached to capillary column 14', and in flow
communication therewith, proximate an exit end 14b', or second end,
thereof. Preferably, the amount of negative pressure that is
generated by vacuum source 28' and applied to each capillary column
14' may be adjusted or varied.
[0055] FIG. 3 illustrates a third embodiment of the sample
separation apparatus 10" of the present invention, which is
particularly useful for conducting electrophoretic separation on a
sample 70". The degree to which the constituents of sample 70" are
separated depends upon the cross-sectional diameter of pores 18".
Accordingly, the greatest degree of separation occurs when the size
of pores 18" is approximately equivalent to the size of the various
constituents of sample 70" for which separation is desired, or the
"targeted" constituents. Thus, pores 18" of small cross-sectional
diameters separate the smaller constituents of sample 70". Pores
18" of larger cross-sectional diameters permit the migration and
separation of the larger sized constituents through each capillary
column 14". Thus, the cross-sectional diameter of pores 18"
preferably facilitates separation of the various targeted
constituents of sample 70".
[0056] Electrophoretic techniques typically employ an electric
current to move the constituents of sample 70". Thus, sample
separation apparatus 10" may include a migration facilitator which
comprises an electric current-generating component 30.
Current-generating component 30 includes a first electrode 32
disposed proximate a sample application end 14a", which is also
referred to as a first end, of each capillary column 14", and a
second electrode 34 that is positioned proximate exit end 14b" of
each capillary column 14". First and second electrodes 32 and 34,
respectively, are fabricated from an electrically conductive
material, and are connectable to opposite electrical charges so as
to facilitate the generation of a current along a length of the
capillary column. Thus, first and second electrodes 32 and 34,
respectively, facilitate the migration of the constituents of
sample 70" along their respective capillary columns 14" and the
separation of the constituents during migration.
[0057] Alternatively, with reference to FIG. 3a, a sample
separation apparatus 10" which lacks a current-generating component
may be utilized in association with a conventional electrophoresis
apparatus 60 that includes a chamber 62 with a cathode 64 extending
into one end thereof and an anode 65 extending into an opposite end
of the chamber.
[0058] Referring again to FIG. 3, separation apparatus 10" also
includes a control column 36" adjacent at least one of capillary
columns 14", which has substantially the same dimensions and a
matrix 38" and pores 40" having substantially the same
configurations and sizes as the matrix 16" and pores 18" of each
capillary column 14". Control column 36" is useful for separating a
control which includes markers 42a, 42b, 42c, etc. of known
molecular size and weight. Thus, as is known in the art, at least
some of the various constituents of the sample may be compared to
markers 42a, 42b, 42c, etc. in order to approximate the molecular
size or weight of these constituents.
[0059] Referring now to FIG. 4, a fourth embodiment of the sample
separation apparatus 100 of the present invention is illustrated.
Separation apparatus 100 includes a stationary phase, which is
referred to as capture substrate 117, which detects the presence
and approximate levels of a particular analyte or group of analytes
in the sample. Capture substrate 117 may include an antibody, an
antigen, or any other substrate material which separates a
constituent from a sample on the basis of affinity for the
constituent. Accordingly, sample separation apparatus 100 comprises
an assay device. Preferably, capture substrate 117 has a specific
affinity for the detected analyte or group of analytes. Capture
substrate 117 is disposed along a portion of each capillary column
114 and securely bound to matrix 116 so as to retain substantially
all of the capture substrate on the matrix as a sample passes
thereby. Capture substrate 117 is preferably bound to matrix 116 at
reaction region 120. Accordingly, detector 122 is preferably
positioned proximate reaction region 120 in order to detect whether
or not capture substrate 117 has bound an analyte.
[0060] Referring again to FIG. 1, capillary columns 14 may be
formed upon substrate 12 by processes that are known in the art,
including processes for forming porous silicon from silicon. FIGS.
5 through 7 illustrate an exemplary process for fabricating sample
separation apparatus 10. With reference to FIG. 5, substrate 12 is
appropriately patterned to define the desired number and shapes of
capillary column regions 40. As shown in FIG. 6, pores 18 are then
created in the defined capillary column regions 40, which is also
referred to as "porifying" of the capillary column regions, by
techniques that are known in the art, such as anodization in the
presence of hydrofluoric acid (HF).
[0061] Referring again to FIG. 5, patterning may include masking
and etching techniques that are known in the art, such as those in
which photoresists are employed. A photoresist 44 is disposed over
the surface of substrate 12 and defined by photolithography
processes, as known in the art, to define a mask 46 with openings
48 therethrough. Openings 48 expose various areas of substrate 12,
which are referred to as capillary column regions 40.
[0062] Patterning may also include the doping of substrate 12 with
dopants and by techniques that are known in the art in order to
provide the desired amount of porosity and porous silicon of a
desired morphology. As those in the art are aware, the ability to
form pores in silicon by anodization processes, as well as the size
and density of such pores and the rate at which pores are formed,
depend upon the presence or absence of dopant and the type and
concentration of dopant. For example, small pores may be formed in
P-doped silicon. Larger pores are more readily formed in P+doped
silicon. N+doped silicon typically resists the formation of pores
by anodization. Accordingly, patterning may also include repeated
masking and differential doping of substrate 12 in order to
facilitate the subsequent selective creation of a porous matrix
through the substrate. Such doping processes are disclosed in U.S.
Pat. No. 4,532,700 (the "'700 patent"), which issued to Wayne I.
Kinney et al. on Aug. 6, 1985, and U.S. Pat. No. 5,360,759 (the
"'759 patent"), which issued to Reinhard Stengl et al. on Nov. 1,
1994, the disclosures of both of which are hereby incorporated by
reference in their entirety.
[0063] Alternatively, patterning may include a mask and etch, as
known in the art, followed by damaging, or "roughing," the exposed
areas of substrate 12 to define capillary column regions 40, as
disclosed in U.S. Pat. No. 5,421,958 (the "'958 patent"), which
issued to Robert W. Fathauer et al. on Jun. 6, 1995, the disclosure
of which is hereby incorporated by reference in its entirety. It is
known in the art that porous silicon forms more readily on damaged,
or roughened, areas on the surface of a silicon substrate 12. As
the '958 patent discloses, the damaging of substrate 12, or the
creation of imperfections on same, may include, without limitation,
mechanically damaging substrate 12 and applying energetic beams to
substrate 12.
[0064] FIG. 7 schematically illustrates an anodization chamber 50
in which an exemplary process for porifying capillary column
regions 40 of substrate 12 (see FIG. 6) may occur. The porifying of
capillary column regions 40 in order to define capillary columns 14
(see FIGS. 1 and 6) in substrate 12 may be performed by
conventional processes, including processes for forming porous
silicon regions in semiconductor devices. Exemplary process for
forming porous silicon from a silicon substrate are disclosed in
each of the '700, '759, and '958 patents. Such porification
processes typically include positioning substrate 12 within an
anodization chamber 50, adjacent a partition 52, which separates
the anodization chamber into a first cell 54 and a second cell 55,
which are also referred to as "sections." An anode 56 extends into
first cell 54. Similarly, a cathode 57 extends into second cell 55.
Partition 52 includes an opening 53 therethrough, which is covered
by substrate 12 and sealed to prevent the passage of liquids
between first cell 54 and second cell 55. Thus, an upper surface
12a of substrate 12 is exposed to first cell 54, while an opposing
base surface 12b is exposed to second cell 55. First cell 54 is
filled with an anodizing solution 58, such as concentrated
hydrofluoric acid, while second cell 55 is filled with an
electrically conductive liquid 59, such as 50% isopropyl alcohol.
By means of anode 56 and cathode 57, an electric current is then
applied to anodization chamber 50. As current passes through
substrate 12, the areas of upper surface 12a that are exposed to
first cell 54 become porous.
[0065] The size of pores 18 is determined by, and may be varied by,
varying several factors, including, without limitation, the
concentration of any doped regions of the substrate, the presence
or absence of dopants, the type of dopants, the relative
concentrations of the various elements of the anodizing solution,
the duration of exposure to the anodizing solution, the current
density, the illumination, and the temperature of the anodizing
solution.
[0066] Other known processes for patterning capillary column
regions 40 on substrate 12 and porifying same, such as that
disclosed in U.S. Pat. No. 5,599,759 (the "'759 patent"), which
issued to Shinji Inagaki et al. on Feb. 4, 1997, the disclosure of
which is hereby incorporated by reference in its entirety, are also
useful for defining capillary columns 14 on substrate 12, and are
therefore within the scope of the fabrication process of the
present invention.
[0067] With reference to FIG. 8, as another alternative, capillary
columns 214 that include hemispherical grain silicon 216 on the
surfaces 215 thereof may be formed in selected regions of a
substrate 212 by known techniques. First, an elongate trench 213,
which defines the path of the capillary column, is defined in a
substrate by known patterning processes, such as mask and etch
techniques. The area of the surfaces of trench 213 may then be
increased by known methods, such as by forming hemispherical grain
silicon 215 thereon. Exemplary methods of forming hemispherical
grain silicon that may be employed to fabricate capillary columns
214 include those disclosed in U.S. Pat. No. 5,407,534, which
issued to Randhir P. S. Thakur on Apr. 18, 1995; U.S. Pat. No.
5,623,243, which issued to Hirohito Watanabe et al. on Apr. 22,
1997; U.S. Pat. No. 5,634,974, which issued to Ronald A. Weimer et
al. on Jun. 3, 1997; U.S. Pat. No. 5,721,171, which issued to
Er-Xuan Ping et al. on Feb. 24, 1998; and U.S. Pat. No. 5,726,085,
which issued to Darius Lammont Crenshaw et al. on Mar. 10, 1998,
the disclosures of each of which are hereby incorporated by
reference in their entirety. In general, a film of amorphous
silicon is formed in trench 213. Impurities are then seeded into
the amorphous silicon. Then, the material is annealed to cause
nucleation sites to grow at the seeding sites to thereby form the
rough textured hemispherical grain silicon 216. A solid phase 218,
such as a native oxide layer, may then be grown on the surface of
the hemispherical grain silicon 216. Finally, the entire structure
210 may be enclosed by a cover layer 220 or a suitable package.
[0068] The hemispherical grain silicon 216 provides a rough texture
on the interior surface of the capillary column 214. The surfaces
215 of capillary column 214 are characterized by hemispherical or
mushroom-shaped bumps, which form a porous, matrix-like structure.
The hemispherical grain silicon 216 provides at least about 1.6 to
2.2 times the surface area that would otherwise be provided by a
conventional surface etched in silicon. Silicon oxide may be
employed as solid phase 218. Silicon oxide is a suitable solid
phase material for separating or detecting a wide variety of
materials. Alternatively, materials with different absorption
characteristics, such as suitable resins, metals, or metal oxides,
may be employed as solid phase 218.
[0069] Referring again to FIGS. 1-1b, detector 22, processor 80,
memory device 82, valves 25, first electrode 37 or cathode 32 (FIG.
3), second electrode 34 or an anode (FIG. 3), and other components
that are carried upon substrate 12 may be fabricated upon the
substrate in a desired location by known semiconductor fabrication
processes. Such semiconductor fabrication processes include,
without limitation, layer deposition processes (e.g., sputtering
and chemical vapor deposition); oxidation processes; patterning
processes (e.g., masking and etching); and other conventional
semiconductor device fabrication processes.
[0070] A stationary phase (see FIGS. 1 through 4) may be applied to
matrix 16 as known in the art.
[0071] With continued reference to FIG. 1, a method of utilizing
the inventive sample separation apparatus 10 includes disposing a
sample proximate first end 14a of at least one capillary column 14.
A liquid sample 70 may then be drawn along the length of capillary
columns 14 by capillary action or with the assistance of migration
facilitator 24. A gaseous sample 70 may be drawn along the length
of capillary column 14 by means of migration facilitator 24. As
sample 70 is drawn through pores 18 that are defined by matrix 16,
one or more constituents of sample 70 is separated from the
remainder of sample 70. The mechanism by which the separation of a
constituent from sample 70 occurs depends upon the separation
technique that is performed, as explained in greater detail below.
The separated constituents may then be detected when they are in
close proximity to, or proximate, a detector 22.
[0072] Referring again to FIGS. 2 and 2a, when sample separation
apparatus 10' is employed in a chromatographic technique, one or
more constituents of a sample 70' are separated in accordance with
their relative solvencies in stationary phase 17, which is disposed
on matrix 16', and a mobile phase, which carries the sample along
the length of each capillary column 14'. When either gas
chromatography or HPLC is performed, the use of a pump 26' (see
FIG. 2) or a vacuum source 28' (see FIG. 2a) is preferred in order
to facilitate the migration of the sample along each capillary
column 14'. Pump 26' or vacuum source 28' may also be employed to
facilitate sample migration along capillary columns 14' during the
use of sample separation apparatus 10' to perform other
chromatographic techniques.
[0073] Turning again to FIG. 3, in order to separate one or more
constituents of a sample 70" by electrophoresis, the sample is
first dissolved in a conventional carrier solvent, which typically
includes a pH buffer solution of a desired pH, 2-mercaptoethanol,
SDS, and glycerol. The SDS imparts the constituents of sample 70"
with a negative net charge and facilitates the unraveling, or
linearization, of the constituents. The 2-mercaptoethanol breaks
covalent disulfide (S--S) bonds between some amino acids of some
protein constituents.
[0074] With continued reference to FIG. 3, a first variation of the
electrophoretic method of the present invention includes applying
sample 70" to first end 14a" of at least one capillary column 14".
Preferably, sample 70" is diluted in a pH-buffered solution, as
known in the art. An electric current is then applied to
current-generating component 30, in order to migrate sample 70"
along capillary columns 14". Preferably, first electrode 32 acts as
a cathode (i.e., electrons flow therefrom), while second electrode
34 acts as an anode (i.e., electrons flow thereto).
[0075] Alternatively, with reference to FIG. 3a, a second variation
of the electrophoretic method according to the present invention is
illustrated, wherein sample separation apparatus 10" may be
disposed in an electrophoresis apparatus 60 of the type that is
typically employed in gel electrophoretic techniques.
Electrophoresis apparatus 60 includes a chamber 62 with a cathode
64 extending into one end thereof, and an anode 65 extending into
the opposite end thereof. A buffer solution of any of the types
that are typically employed in electrophoresis, and having a
desired pH, is poured into chamber 62. Sample separation apparatus
10" is then positioned in electrophoresis apparatus 60, with first
end 14a" of capillary columns 14" proximate cathode 64 and second
end 14b" proximate anode 65. A sample 70" is applied to first end
14a", and an electric current of desired amperage is then applied
to cathode 64 and anode 65 in order to migrate the sample along the
length of at least one capillary column 14".
[0076] In both the first and second variations of the
electrophoretic method of the present invention, as the sample
migrates through pores 18, the constituents 72a", 72b", 72c", etc.
of sample 70" may be separated on the basis of size or net electric
charge. When separation of constituents 72" on the basis of size is
desired, sample 70" preferably includes a substance, such as SDS,
which imparts each of constituents 72" with the same net electrical
charge. Various constituents of the sample may then be detected
with a detector, by staining, spectrophotometrically,
radiographically, or by other detection or identification
techniques that are known in the art.
[0077] As an example of the use of sample separation apparatus 100,
which is illustrated in FIG. 4, a constituent, or an "analyte" 172,
of a sample 170 is isolated from the remainder of the sample.
Sample 170 is applied to first end 114a of at least one capillary
column 114. As sample 170 moves through column 114, each of the
constituents of the sample, including analyte 172, contact capture
substrate 117. If sample 170 includes any analytes 172 for which
capture substrate 117 has an affinity, these analytes are bound by
the capture substrate 117 and isolated from the remainder of the
sample as the sample contacts and passes by the capture substrate.
The presence or absence of capture substrate 117-bound analytes 172
may then be detected by detector 122, by staining,
spectrophotometrically, radiographically, or by other detection or
identification techniques that are known in the art. The
concentration or relative amounts of each isolated analyte 172 may
also be determined in such a manner.
[0078] As another example of the use of sample separation apparatus
100, to detect the presence of silver, capillary column 114 may be
provided with a free chloride source, such as calcium chloride or
sodium chloride. When an aqueous solution containing silver is
drawn into the capillary column 114, resultant precipitation of
silver chloride would reduce the chloride concentration in
capillary column 114. The resultant reduced ionic conductivity in
capillary column 114 may be measured by detector 122 and compared
to a conductivity profile stored in a memory element associated
with sample separation apparatus 100. For the purpose of
comparison, another capillary column 114' of sample separation
apparatus 100 may be provided with no free chloride source. As the
aqueous silver solution is drawn into the second capillary column
114', the ionic conductivity of the second capillary column 114'
may be measured by another detector. The ionic conductivity profile
of the second capillary column 114' may be compared to that of the
first capillary column 114 and to the conductivity profile. The
measured and stored data may then be processed to determine the
concentration of silver in the original sample.
[0079] Although the foregoing description contains many specifics,
these should not be construed as limiting the scope of the present
invention, but merely as providing illustrations of some of the
presently preferred embodiments. Similarly, other embodiments of
the invention may be devised which do not depart from the spirit or
scope of the present invention. The scope of this invention is,
therefore, indicated and limited only by the appended claims and
their legal equivalents, rather than by the foregoing description.
All additions, deletions and modifications to the invention as
disclosed herein which fall within the meaning and scope of the
claims are to be embraced within their scope.
* * * * *