U.S. patent application number 10/439892 was filed with the patent office on 2003-11-06 for method of forming a membrane with nanometer scale pores and application to biofiltration.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Ferrari, Mauro, Hansford, Derek J..
Application Number | 20030205552 10/439892 |
Document ID | / |
Family ID | 29272687 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030205552 |
Kind Code |
A1 |
Hansford, Derek J. ; et
al. |
November 6, 2003 |
Method of forming a membrane with nanometer scale pores and
application to biofiltration
Abstract
A method of forming a membrane having nanometer scale pores
includes forming an etch stop layer on a substrate and forming a
base layer on the etch stop layer. Advantageously, a silicon
nitride etch stop layer is formed on a silicon substrate and the
base layer is a thermally grown oxide layer. Micron scale holes are
etched through the base layer and, advantageously, partially
through the underlying etch stop layer. A sacrificial base layer of
controlled thickness is formed on the base layer and lining the
holes. A thermally grown oxide is advantageously used as the
sacrificial base layer. A plug layer is then formed on the base
layer, on the sacrificial base layer and filling the holes.
Polysilicon is advantageously used as the plug layer. The plug
layer is planarized followed by the creation of an aperture in the
backside of the wafer. Release of the etch stop layer and the
sacrificial base layer results in a membrane having pores therein
with lateral dimensions determined by the thickness of the
sacrificial base layer, typically less than about 50 nm. Such
membranes are shown to be favorably used in biofiltration and
bioseparation applications.
Inventors: |
Hansford, Derek J.;
(Columbus, OH) ; Ferrari, Mauro; (Dublin,
OH) |
Correspondence
Address: |
MICHAELSON AND WALLACE
PARKWAY 109 OFFICE CENTER
328 NEWMAN SPRINGS RD
P O BOX 8489
RED BANK
NJ
07701
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
29272687 |
Appl. No.: |
10/439892 |
Filed: |
May 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10439892 |
May 16, 2003 |
|
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|
09715840 |
Nov 17, 2000 |
|
|
|
60166049 |
Nov 17, 1999 |
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Current U.S.
Class: |
216/2 |
Current CPC
Class: |
B01D 67/0062 20130101;
B01D 67/0058 20130101; B01D 71/022 20130101; B01D 2325/04 20130101;
B01D 2325/08 20130101; B01D 2325/02 20130101; B01D 69/02 20130101;
B01D 67/0072 20130101; B01D 71/02 20130101 |
Class at
Publication: |
216/2 |
International
Class: |
C23F 001/00 |
Claims
What is claimed is:
1. A method of forming a porous membrane comprising: a) forming an
etch stop layer on a substrate; and, b) forming a base layer on
said etch stop layer; and, c) patterning and etching holes through
said base layer and not completely through said etch stop layer;
and, d) forming a sacrificial base layer on said base layer and
lining said holes, wherein said sacrificial base layer has a
nanometer scale thickness, substantially uniform across the wafer;
and, e) forming a plug layer on said sacrificial base layer and
filling said holes, wherein said plug layer is selectively
removable in comparison with said sacrificial base layer without
doping; and, f) planarizing said plug layer; and, g) patterning and
etching an aperture through the backside of said substrate and
through said etch stop layer, exposing thereby said plug layer and
said sacrificial base layer; and, h) selectively removing said
sacrificial base layer, forming thereby nanometer scale pores
through said base layer.
2. A method as in claim 1 further comprising the use of protective
layers: immediately following step f; f.sub.1) forming protective
layers on the frontside and backside of said wafer; and,
immediately following step g; g.sub.1) removing remaining portions
of said backside protective layer and said frontside protective
layer; and.
3. A method as in claim 1 further comprising the use of support
ridges: immediately following step a; a.sub.1) forming support
ridges on said substrate.
4. A method as in claim 1 further comprising the use of anchor
points: immediately following step d; d.sub.1) forming anchor
points in said sacrificial base layer; and.
5. A method as in claim 1 wherein said substrate is a silicon
wafer.
6. A method as in claim 5 wherein said etch stop layer is silicon
nitride.
7. A method as in claim 6 wherein said sacrificial base layer is
thermally formed oxide.
8. A method as in claim 7 wherein said plug layer is
polysilicon.
9. A method as in claim 8 wherein said selective removal of said
sacrificial base layer is with HF or SF.sub.6/oxygen plasma.
10. A method as in claim 1 wherein said nanometer scale pores are
less than about 50 nanometers in lateral extent.
11. A biocompatible membrane produced according to the method of
claim 1 having nanometer scale pores.
12. A membrane as in claim 11 wherein said membrane is derived from
silicon compounds and has sub-fifty nanometer scale pores
therein.
13. A membrane as in claim 12 wherein said membrane has a glucose
diffusion test of at least 1 mg/dl and an albumin diffusion test of
at most 0.1 g/dl over approximately 330 minutes.
14. A method of separating biological substances comprising
filtering a mixture of said biological substances through a
membrane as in claim 11 having nanometer scale pores therein.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/715,840, filed Nov. 17, 2000 which derives
from provisional patent application No. 60/166,049 filed Nov. 17,
1999, the entire disclosures of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Invention
[0003] This invention relates generally to the field of membranes
having nanometer scale pores that can be used in biofiltration and
bioseparation applications. More particularly, this invention
relates to the formation of such membranes with microfabrication
techniques.
[0004] 2. Description of the Prior Art
[0005] There is a revolution occurring in biological research with
emphasis rapidly shifting towards the view of biology in terms of
complex physical and chemical interactions. Interdisciplinary
research between engineers, biologists, physicists, and clinicians
is becoming prevalent. A rapidly developing field of research is
the use of microfabrication to make mechanically, electrically,
and/or chemically interactive structures for biological research
and applications, known collectively as BIOMEMs (BIOlogical
MicroElectroMechanical devices). By using microfabrication
techniques derived from techniques developed to process
semiconductors, MEMs structures can be fabricated with spatial
features having typical dimensions ranging from the sub-micron
range up to several millimeters. These multi-scale structures
correspond well with hierarchical biological structures, from
proteins and sub-cellular organelles to tissues and organs. This
structural hierarchical correlation between biological structures
and fabricated structures allows scientists to investigate
biological structures on their respective size scales and interact
in more appropriate and responsive manners to the structures within
the body and within biological fluids.
[0006] It would be desirable to use standard microlithography to
produce structures that can be used for basic biological research,
diagnostic, and therapeutic applications. However, conventional
lithographic techniques have feature size limitations that prevent
their use for fabricating structures that can physically interact
with molecules of biological interest, such as proteins,
nucleotides, and various physiological nutrients. To interact
directly with these molecules, features must be fabricated with
typical sizes less than approximately 50 nm (nm=nanometer=10.sup.-9
meter), which is not projected to be attainable by state of the art
lithography until the year 2008. Furthermore, because of the
fabrication techniques typically used for MEMS structures, and the
potential for contamination they introduce, state of the art
equipment will typically not be used to fabricate structures
intended for biological applications, leading to a further delay in
the fabrication of direct interaction structures.
[0007] An example of structures having several uses in biological
and other research fields is a membrane having nanometer scale
("nanoscale") pores therein that would be useful for
biofiltrations, separations, and other purposes. In particular, a
uniform size distribution of pore openings without the presence of
over-sized pores, would prevent even small amounts of undesirably
large material from passing through the membrane.
[0008] Previous techniques to fabricate membranes having nanoscale
pores (or "nanopore membranes") include the work of Chu et al (U.S.
Pat. No. 5,770,076), the work done by the IBM Corporation ("Process
for Producing a Precision Filter", IBM Technical Disclosure
Bulletin, Vol. 32, No. 4A, September 1989), among others. However,
these approaches typically include one or more disadvantages.
Typically, boron or other doping is used to produce etching
selectivity among various elements of the structure that, when
selectively etched, produce the nanoscale pores. However, boron
doping can be subject to imprecise diffusion and, hence, imprecise
geometric control of the membrane structure. Boron doping can also
introduce undesirable mechanical stress into the membrane.
Fabrication techniques described herein produce nanoscale pores
without the necessity of doping with boron or any other dopant.
[0009] Other membranes having nanoscale pores are fabricated having
angled paths through the membrane, including right angle turns as
in the filters of Chu et al (supra). While such pores can be useful
for many practical applications, the introduction of turns and
curves increases the likelihood of fouling or clogging by the
filtered material. Thus, a direct path for the nanoscale pores
through the membrane is desired, and such paths are produced by the
techniques described herein.
[0010] In view of the foregoing, a need exists in the art for a
technique for fabricating membranes with features less than
approximately 50 nm, without doping and including direct paths
through the membrane. Ideally, such a technique would rely upon
standard lithography processing techniques and would yield a device
that is compatible with biological research, diagnostic, and
therapeutic applications.
SUMMARY OF THE INVENTION
[0011] Accordingly and advantageously, the invention provides a
method of forming a membrane having nanometer scale pores therein.
An etch stop layer is formed on a substrate and a base layer is
then formed on the etch stop layer. Advantageously, pursuant to
some embodiments of the invention, a silicon nitride etch stop
layer is formed on a silicon substrate and the base layer is a
thermally grown oxide layer. Micron scale holes are etched through
the base layer and, advantageously, partially through the
underlying etch stop layer. That is, holes are etched completely
through the base layer and, perhaps into, but not completely
through, the etch stop layer.
[0012] A sacrificial base layer is formed on the base layer and
lining the holes. Removal of the sacrificial base layer will form
the nanometer scale pores. Thus, the thickness of the sacrificial
base layer determines the lateral extent of the pores and should be
accurately and carefully controlled for uniformity of pore size. A
thermally grown oxide is advantageously used as the sacrificial
base layer.
[0013] A plug layer is then formed on the base layer, on the
sacrificial base layer and filling the holes in those regions
unoccupied by sacrificial base layer. Polysilicon is advantageously
deposited as the plug layer.
[0014] The plug layer is planarized (and optionally polished),
followed by the creation of an aperture in the backside of the
wafer. Protective layers are typically used on both sides of the
wafer to protect the plug and sacrificial base layers during
aperture formation, then removed. Release of the etch stop layer
and the sacrificial base layers results in a membrane having pores
therein with lateral dimensions determined by the thickness of the
sacrificial base layer, typically less than about 50 nm. The pores
fabricated in membranes pursuant to embodiments of the present
invention traverse the membrane directly, without bends or right
angle turns, thereby reducing the tendency to clog or foul during
biofiltration or bioseparation.
[0015] Unlike many prior art procedures for the fabrication of
nanopore filters, the present techniques do not use doping
(typically boron doping) of any layer to produce etching
selectivity. Absence of doping typically results in better
dimensional control and fewer mechanical stresses within the
membrane.
[0016] Membranes produced pursuant to embodiments of the present
invention are capable of achieving a glucose diffusion test result
of at least 1 mg/dl and an albumin diffusion test result of at most
0.1 g/dl. Further, the present membranes exhibit no significant
biofouling or agglomeration of protein in the pores, in contrast to
commercially available membranes.
[0017] These and other advantages are achieved in accordance with
the present invention as described in detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] To facilitate understanding, identical reference numerals
have been used, where possible, to designate identical elements
that are common to the figures. The drawings are not to scale and
the relative dimensions of various elements in the drawings are
depicted schematically and not to scale.
[0019] The techniques of the present invention can readily be
understood by considering the following detailed description in
conjunction with the accompanying drawings, in which:
[0020] FIG. 1 depicts in cross sectional view a substrate with an
etch stop layer formed thereon.
[0021] FIG. 2 depicts in cross sectional view a base layer formed
on the etch stop layer of FIG. 1.
[0022] FIG. 3 depicts in cross sectional view micrometer scale
holes etched through the base layer of FIG. 2 and partially through
the etch stop layer.
[0023] FIG. 4 depicts in cross sectional view the formation of a
sacrificial base layer on the structure of FIG. 3.
[0024] FIG. 5 depicts in cross sectional view anchor points formed
in the sacrificial base layer of FIG. 4.
[0025] FIG. 6 depicts in cross sectional view a plug layer formed
on top of and filling the micrometer scale holes of FIG. 5.
[0026] FIG. 7 depicts in cross sectional view the plug layer of
FIG. 6 after planarization.
[0027] FIG. 8A depicts in cross sectional view a protective layer
formed on both sides of the wafer of FIG. 7.
[0028] FIG. 8B depicts in cross sectional view the result of
patterning and etching an aperture in the substrate of FIG. 8A.
[0029] FIG. 9A depicts in cross sectional view the result of
removing the protective layers from both sides of the wafer of FIG.
8B.
[0030] FIG. 9B depicts in cross sectional view a membrane having
nanometer scale pores therein following release of the exposed etch
stop layer and sacrificial base layer of FIG. 9B.
[0031] FIG. 10 summarizes typical processing steps used in
fabrication of the structures depicted in FIGS. 1-9B.
[0032] FIG. 11 depicts in schematic, cut-away view a device used to
test the membrane of the invention.
[0033] FIG. 12 is a graphical comparison of glucose diffusion
through three different nanopore membranes, WHATMAN
(.diamond-solid.), MILLIPORE (bare line) and the micromachined
nanopore membrane of the invention (.box-solid.)
[0034] FIG. 13 is a graphical comparison of the diffusion of
glucose and albumin through a typical nanopore membrane of the
invention.
[0035] FIG. 14 is a graphical comparison of glucose diffusion
through a typical nanopore membrane of the invention incubated in
pure glucose and mixed glucose/albumin solutions.
[0036] FIG. 15 is a graphical comparison of diffusion through
MILLIPORE membranes incubated in pure glucose and mixed
glucose/albumin solutions
[0037] FIG. 16 compares albumin diffusion through WHATMAN filters,
MILLIPORE filters and a typical micromachined nanopore filter of
the invention.
DETAILED DESCRIPTION
[0038] After considering the following description, those skilled
in the art will clearly realize that the teachings of the invention
can be readily utilized in the fabrication of porous membranes
having nanometer scale pores therein (also referred to herein as
"micromachined" or "microfabricated" membranes), and the use of
such membranes for filtration and separation. Typical procedures
for fabrication of micromachined membranes pursuant to some
embodiments of the present invention are illustrated in the
accompanying figures.
[0039] FIG. 1 depicts in cross sectional view (not to scale) a
typical substrate 20 having an etch stop layer 22 thereon. To be
concrete in our description, we describe the detailed processing
steps advantageously employed pursuant to some embodiments of the
present invention for the fabrication of porous membranes deriving
from silicon-based structures. However, the techniques described
herein can also be employed in connection with the fabrication of
porous membranes deriving from other materials. For biofiltration
or separation, such other materials are advantageously selected to
be biocompatible, such as metals (e.g., titanium), ceramics (e.g.,
silica, silicon nitride, polymers (e.g., polytetrafluoroethylene,
polymethylmethacrylate, polystryenes, silicones), among others.
[0040] Substrate 20 is typically a silicon wafer. Since the etch
stop layer 22 is typically thin, it is advantageous in some
embodiments to etch support ridges in the surface of substrate 20
before depositing the etch stop layer 22 on top of the support
ridges. When used, such support ridges (not depicted in FIG. 1)
occur between the substrate 20 and etch stop layer 22. The support
ridges help provide mechanical rigidity to the subsequently formed
membrane structure.
[0041] Etch stop layer 22 is employed in dual roles as an etch stop
layer that is subsequently removed (or sacrificed) to expose the
nanometer scale pores of the membrane. It thus functions as a
sacrificial etch stop layer, buried beneath subsequent layers and
may be precisely referred to as a "buried sacrificial etch stop
layer." However, for economy of language, we refer to layer 22
herein simply as an etch stop layer, understanding thereby that
multiple functions are performed.
[0042] Advantageously, pursuant to some embodiments of the present
invention, etch stop layer 22 is low stress silicon nitride
("nitride") typically deposited on substrate 20 by means of low
pressure chemical vapor deposition. In some embodiments, the
nitride etch stop layer has a thickness of approximately 0.4 .mu.m
[.mu.m=micrometer (micron)=10.sup.-6 meter].
[0043] Several advantages of the present invention derive from the
multiple functions performed by etch stop layer 22. For example,
the buried, sacrificial nature of the etch stop layer facilitates
three-dimensional control of the pore structure. Prior art
techniques typically endeavor to control pore structure by
balancing the etching of two different layers. The buried,
sacrificial etch stop techniques employed in various embodiments of
the present invention facilitate the formation of pores less than
approximately 50 nm. Moreover, these pores can be uniformly formed
across the entire wafer.
[0044] Techniques employed in various embodiments of the present
invention achieve etching selectivity without the use of diffused
boron, or other doping material. Diffused boron used as an etch
stop typically results in an imprecise membrane depth due largely
to the imprecise geometrical properties of diffusion during various
processing steps occurring at elevated temperatures. Further,
diffused boron introduces mechanical stresses into the completed
membrane, advantageously avoided when boron diffusion is
unnecessary.
[0045] The buried, sacrificial etch stop layer employed in various
embodiments of the present invention can also provide improved
etching selectivity. That is, the etch stop layer is etched only
negligibly by the KOH etchant typically employed. In contrast,
boron-doped materials are typically etched to a rather greater
extent by KOH etchant.
[0046] Following deposition of the etch stop layer 22, a base
structural layer ("base layer") 24 is deposited, as depicted in
FIG. 2. In one embodiment, 5 .mu.m of polysilicon is used as the
base layer. In other embodiments, low stress silicon nitride may be
used as the base layer, in which case it operates as its own etch
stop layer.
[0047] Holes 26 are then patterned and etched in base layer 24 to
define the shape of the pores in the final membrane (FIG. 3). The
shape of holes 26 can be determined by masking techniques known in
the art. For example, the holes 26 may be etched through the
polysilicon base layer 24 by means of a chlorine plasma, with a
thermally grown oxide layer used as a mask. Masking and production
of holes 26 follow techniques known in the art and, thus, these
steps are not discussed in detail herein. Holes 26 typically have
lateral dimensions of the order of .mu.m, readily achievable with
known techniques of microfabrication.
[0048] Holes 26 should completely penetrate base layer 24.
Therefore, it is advantageous to employ a 10%-15% over-etch of base
layer 24, as depicted in FIG. 3. Nitride etch stop layer 22
typically acts as an etch stop layer for the plasma etching of
polysilicon base layer 24 such that complete punch-through of layer
22 is avoided. Complete punch-through is disadvantageous in that
material filling holes 26 would not then be shielded from backside
etching (typically with KOH etchant) applied from the substrate
side of the membrane structure. Therefore, control of hole etching
to avoid punch-through is desired.
[0049] A sacrificial base layer is grown on base layer 24, and
depicted as 28 in FIG. 4. Sacrificial base layer 28 is used to
define the nanometer scale pores in the final membrane, so
controlling the growth of this layer is an important factor in
producing membranes with reproducible pore sizes. For polysilicon
base layers, it is convenient to use a sacrificial silicon oxide
layer for the sacrificial base layer 28. Typically, the sacrificial
silicon oxide is grown by thermal oxidation of the base layer 24,
typically employing a growth temperature of between approximately
8500 to 9500 for approximately one hour with an annealing step of
approximately ten minutes.
[0050] Other techniques can be used to form the sacrificial base
layer 28. For example, a thermally evaporated tungsten film can be
used as a sacrificial base layer in connection with the fabrication
of polymer membranes, and selectively removed with hydrogen
peroxide. The primary characteristic of the sacrificial base layer
28 is the ability to control its thickness with high precision and
high uniformity across the entire wafer, and hence across the
entire membrane. A variable thickness occurring in layer 28
typically leads to varying pore openings in the final membrane.
Hence, a suboptimal membrane is the result, having at least some
large pores capable of passing larger than desired materials.
[0051] Thermal oxidation of polysilicon and nitride layers allows
the thickness of sacrificial base layer 28 to be controlled to a
precision of approximately 5% across the entire wafer. Limitations
on this thickness control typically arise from local
inhomogeneities in the base layer, such as variations of the
initial thickness of the native oxide (especially for polysilicon),
the grain size or density, and the impurity concentration.
[0052] A layer of material ("plug layer" or "plug material") is to
be deposited and fill holes 26 in preparation for removal of
sacrificial base layer 28 and the formation of nanometer pores.
However, it is advantageous in some embodiments of the present
invention to provide for direct contact between the plug material
and the base layer 24 to help maintain pore spacing between layers,
improve adhesion and mechanical strength, among other purposes.
Thus, it is convenient to etch anchor points or anchor openings 30
in sacrificial base layer 28. This is conveniently accomplished
pursuant to some embodiments of the present invention by making use
of the same mask used to define holes 26 shifted diagonally by
approximately 1 .mu.m. This produces anchor points in one or two
corners of each hole, thereby providing the desired mechanical
connection between structural layers.
[0053] A plug layer 32 is then deposited to fill holes 26 as well
as coat the regions between holes, as depicted in FIG. 6. It is
convenient to use approximately 1.5 .mu.m of polysilicon for the
plug layer 32 pursuant to some embodiments of the present
invention. Plug layer 32 is then planarized to the level of base
layer 24 as depicted in FIG. 7.
[0054] The method of planarization depends upon the material used
as plug material. For realtively hard microfabrication materials
(such as polysilicon and nitride), chemical mechanical
planarization is a convenient method. Other materials could be
planarized by means of a plasma etch, typically including a quick,
wet chemical smoothing or polishing. Plasma etching has the
potential advantage that it may be feasible to select a plasma and
etching conditions such that the base layer 24 is not affected. On
the other hand, plasma etching has the potential disadvantage of
the need for controlled etch timing to avoid deleterious etching of
the plug material 32 residing in holes 26.
[0055] The portion of the membrane containing nanometer scale pores
will derive from structure 40 in FIG. 7. At this point, it is
convenient to etch the lower (backside) portion of substrate 20 to
provide backside access to the membrane. To protect the upper
(frontside) of the membrane during backside etch, it is convenient
to apply a protective layer 34 to both the frontside of the wafer
34f and the backside of the wafer 34b, as depicted in FIG. 8.
Protective layer 34 should be impervious or substantially undamaged
by the etchant used to etch the silicon substrate 20 such that,
during etching of substrate 20, protective layer 34f on the
frontside of the membrane protects membrane structure 40 from
significant damage. For silicon etching by means of KOH with
polysilicon and nitride layers it is found that a thin layer of
nitride is conveniently used as protective layer 34, since nitride
is insignificantly etched by KOH and only slowly dissolves in HF.
For polymeric structural materials used to construct a membrane,
silicone is conveniently used as a protective layer since the
temperature of nitride deposition (typically around 835.degree. C.)
renders it impractical for use with polymers.
[0056] A backside etch window is patterned and etched in protective
layer 34b, exposing the silicon substrate 20 in desired areas.
Silicon substrate 20 is then etched, typically by placing the
entire structure in a KOH bath at a temperature of approximately
80.degree. C. for sufficient time for the silicon substrate 20 to
etch up to the level of etch stop layer 22 (as evidenced by the
smooth etch stop layer 22). FIG. 8B depicts the aperture 36 thereby
created in substrate 20.
[0057] Protective layer 34 is now removed from both frontside and
backside, resulting in the structure depicted in FIG. 9A. Thus,
another characteristic desired of protective layer 34 is that it be
removable without causing significant damage to the underlying
membrane or other structures, resulting in a substantially
undisturbed membrane as depicted in FIG. 9A.
[0058] At this point, the portion of the etch stop layer 22 exposed
by aperture 36 and the remaining portions of the sacrificial base
layer 28 are removed. For a nitride etch stop layer 22 and an oxide
sacrificial base layer 28, etching is advantageously performed by
an HF or SF.sub.6/oxygen plasma. However, other etchants may also
be employed and the removal of the etch stop layer and the
sacrificial base layer can be done in distinct processing steps. In
all such cases, the result is that each hole 26 now contains one
pore defined by the thickness of sacrificial base layer 28. The
pores thus having lateral dimensions in the range of nanometers are
depicted by 41 in FIG. 9B.
[0059] FIG. 10 summarizes the foregoing processing steps: Form an
etch stop layer on a substrate (step 50). Form a base layer on the
etch stop layer (step 52). Etch micrometer scale holes through the
base layer and, advantageously, partially into the etch stop layer
(step 54). Form a sacrificial base layer on the base layer and
lining the micrometer scale holes (step 56). Pattern anchor points
in the sacrificial base layer (step 58). Form a plug layer on the
base layer and filling the micrometer scale holes (step 60) and
planarize substantially to the level of the base layer (step 62).
Protective layers are formed on both the frontside and backside of
the wafer (step 64) followed by patterning and etching of the
backside protective layer and underlying substrate to form an
aperture (step 66). The protective layers are released or otherwise
removed, advantageously accomplished in such manner as to cause no
significant deleterious changes to other materials or structures
(step 68). The etch stop layer is removed (in the region exposed by
the aperture) and the sacrificial base layer is also removed from
the micrometer scale holes, creating thereby nanometer scale pores
(step 70).
[0060] The performance of the membrane 40 produced pursuant to some
embodiments of the present invention was analyzed in comparison
with two other types of membranes. In particular, a membrane 40
(with 24.5 nanometer pore size +/-0.9 nm) of the invention was
compared with porous alumina (i.e., a WHATMAN ANODISC membrane with
a pore size of 20 nm) and a mixed cellulose acetate and nitrate
membrane (i.e., a MILLIPORE ISOPORE with a pore size of 25 nm). All
membranes were examined in vitro by measuring relative
concentrations of glucose on both sides of the microfabricated
interface over time, using a mini diffusion chamber constructed
around the membranes, as shown in FIG. 11.
[0061] FIG. 11 depicts a chamber 80 with a first compartment 82 and
a second compartment 84 with fixed volumes of approximately 2 ml.
Sampling ports 86 are provided in each compartment. The
compartments are at least partially separated by the membrane under
consideration 90. Advantageously, the two compartments are sealed
with O-rings and are screwed together.
[0062] Glucose is measured on either side of the membrane 90 in the
diffusion chamber by means of a quantitative enzymatic assay (e.g.,
TRINDER, SIGMA) and calorimetric reading via a spectrophotometer.
Samples of approximately 0.1 ml were taken from the diffusion
chamber and approximately 10 .mu.l of that sample were added to
approximately 3 ml of glucose reagent in a cuvette, and were mixed
gently by inversion. Each tube was incubated for approximately 18
minutes at room temperature and then readings were taken at a
wavelength of 505 nm. The reagent is linear up to approximately 750
mg/dl. The diffusion chamber itself was attached to a motor for
stirring in order to minimize boundary layer effects (diffusion
resistance at the liquid/membrane interface). In order to ensure
wetting of the pores, the receptor cell was first filled with
phosphate buffer saline for about fifteen minutes before the
filling of the donor cell. The donor cell was filled with solutions
of glucose in phosphate buffer saline in varying concentrations.
These tests were carried out at 37.degree. C.
[0063] Albumin was measured on either side of the membrane using
the same diffusion chamber. Albumin diffusion and/or exclusion was
measured and quantified using Albumin BCP (bromocresol purple,
SIGMA.) A sample of approximately 0.1 ml was taken at time zero and
at the end of the diffusion period (time=330 minutes). An aliquot
of approximately 300 .mu.l was then added to approximately 3 ml of
the reagent and absorbance was read at 600 nm. Reagent plus
deionized water was used as the blank. The assay is linear up to
approximately 6 g/dl but is not accurate below approximately 1
g/dl.
[0064] FIGS. 12-15 depict the results of these tests. The results
demonstrate that glucose concentration increases and begins to
plateau at about 330 minutes. FIG. 12 shows the diffusion of
glucose from a pure glucose solution and a mixed solution of
glucose and albumin through 24.5 nm pore-sized silicon
membranes.
[0065] The presence of albumin does not seem to impede passage of
glucose through the membranes, nor slow down glucose transport
under the experimental conditions employed. FIG. 13 shows that no
detectable amounts of albumin diffuse through the microfabricated
membrane. The same membrane, however, shows glucose diffusion. The
microfabricated membranes are able to achieve complete exclusion of
albumin (to within the limits of detection), while allowing glucose
diffusion.
[0066] Comparing these diffusion rates with those of commercially
available membranes, it is seen in FIG. 14 that microfabricated
filters pursuant to some embodiments of the invention have glucose
diffusion properties comparable to the MILLIPORE and WHITMAN
membranes with similar pore size. However, when albumin diffusion
is measured for all three membranes, the nanopore micromachined
membranes of the invention have the greatest albumin exclusion, as
shown in the table of FIG. 16.
[0067] The foregoing results illustrate glucose diffusion test
results of at least 1 mg/dl in 330 minutes. The membrane has an
albumin diffusion test result of at most 0.1 g/dl in 330
minutes.
[0068] All of the membranes were evaluated before and after
diffusion experiments to determine if any structural or surface
changes had occurred. There were significant changes in membrane
morphology for both the WHATMAN and MILLIPORE membranes after being
incubated with glucose, albumin, and phosphate buffered saline for
over 24 hours at 37.degree. C. In contrast, the micromachined
silicon membrane of the invention showed the same appearance before
and after the tests. In fact, the microfabricated membrane pores
are free from biofouling and any agglomeration of the protein in
the pores. The MILLIPORE and WHATMAN membranes display
inhomogeneities and morphological changes after all diffusion
tests.
[0069] In sum, the microfabricated silicon membranes of the
invention were characterized in terms of glucose diffusion, albumin
exclusion and stability in biological environments. Results
indicated that glucose does indeed diffuse through the
microfabricated membranes at a rate comparable to commercially
available membranes. At the same time, albumin is excluded from
passage. In a mixed solution of glucose and albumin, it has been
shown that only glucose diffuses through the membranes. Although
several membranes, such as those by WHATMAN and MILLIPORE are
available for absolute filtration, these membranes do not have all
the desired membrane properties, such as stability,
bio-compatibility, and well-controlled perm-selectivity.
[0070] The filter technology of the invention alleviates several of
the problems associated with current commercially available
separation membranes. Through the use of controlled sacrificial
base layer deposition, membranes can be fabricated with sufficient
precision to guarantee high pore uniformity in sub-micron
dimensions. The thickness of the thermally grown oxide can be
controlled to +/-1 nm for nominal pore sizes as small as
approximately 18 nm. This is the size range needed to obtain
absolute protein exclusion and glucose diffusion for biosensor
applications. Moreover, this filter technology can bring in the
added advantages of stability, minimal protein adsorption through
established silicon surface modification techniques, reusability,
and sterilizability.
[0071] Although various embodiments which incorporate the teachings
of the present invention have been shown and described in detail
herein, those skilled in the art can readily devise many other
varied embodiments that still incorporate these teachings.
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