U.S. patent application number 14/745175 was filed with the patent office on 2015-10-08 for particulate nanosorting stack.
The applicant listed for this patent is CALIFORNIA INSTITUTE OF TECHNOLOGY. Invention is credited to ADITYA RAJAGOPAL, AXEL SCHERER, THOMAS A. TOMBRELLO, SAMEER WALAVALKAR.
Application Number | 20150283513 14/745175 |
Document ID | / |
Family ID | 45888887 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150283513 |
Kind Code |
A1 |
WALAVALKAR; SAMEER ; et
al. |
October 8, 2015 |
PARTICULATE NANOSORTING STACK
Abstract
Methods and devices for isolating and sorting nanoparticles are
disclosed herein. Nanopores of a desired size can be formed in
silicon dioxide membranes and used as filters to separate
nanoparticles. Devices are also provided herein for sorting
nanoparticles with multiple filters having various sized
nanopores.
Inventors: |
WALAVALKAR; SAMEER; (LOS
ANGELES, CA) ; RAJAGOPAL; ADITYA; (IRVINE, CA)
; SCHERER; AXEL; (WOODSTOCK, VT) ; TOMBRELLO;
THOMAS A.; (ALTADENA, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CALIFORNIA INSTITUTE OF TECHNOLOGY |
PASADENA |
CA |
US |
|
|
Family ID: |
45888887 |
Appl. No.: |
14/745175 |
Filed: |
June 19, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13250575 |
Sep 30, 2011 |
9089819 |
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14745175 |
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61388342 |
Sep 30, 2010 |
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61405019 |
Oct 20, 2010 |
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Current U.S.
Class: |
216/41 ;
216/56 |
Current CPC
Class: |
B01D 61/145 20130101;
B81C 1/00031 20130101; B82Y 40/00 20130101; G01N 33/48721 20130101;
B81C 1/00111 20130101; B81C 1/00158 20130101; B81C 1/00531
20130101; B81C 1/00119 20130101; H01L 21/0331 20130101; B81C
1/00539 20130101; B01D 61/027 20130101; B07B 1/00 20130101; B01D
67/0062 20130101; B01D 71/027 20130101; B07B 1/4618 20130101; B01D
69/06 20130101 |
International
Class: |
B01D 67/00 20060101
B01D067/00; B01D 69/06 20060101 B01D069/06; B07B 1/46 20060101
B07B001/46; B01D 71/02 20060101 B01D071/02 |
Claims
1. A method for manufacturing a nanopore, said method comprising:
providing a silicon substrate having a top side and a bottom side;
forming a first nanopillar on the substrate; oxidizing the
nanopillar to form a SiO.sub.2 layer around a silicon core;
removing a portion of the nanopillar from the substrate to expose
the silicon core; selectively removing the silicon core by etching
to form a pore in the substrate.
2. The method of claim 1, wherein selectively removing the silicon
core comprises: providing an oxide layer on the bottom side of the
substrate; etching the oxide layer on the bottom side directly
below the first nanopillar to expose the silicon substrate; and
etching the exposed silicon substrate, thereby removing the core
and forming a nanopore through the substrate.
3. The method of claim 1, further comprising forming a second
nanopillar on the substrate.
4. The method of claim 3, wherein the first nanopillar is about 50
nm from the second nanopillar.
5. The method of claim 3, wherein the first nanopillar and the
second nanopillar are about 1 to 100 nm in diameter and about 200
nm to 1 micron in height.
6. The method of claim 1, further comprising forming a plurality of
additional nanopillars on the substrate, wherein the additional
nanopillars form an array of nanopillars of the same diameter.
7. The method of claim 6, further comprising: oxidizing the array
of nanopillars to form a SiO.sub.2 layer around a silicon core for
each of the additional plurality of nanopillars; and selectively
removing the silicon core of each of the additional plurality of
nanopillars to form a corresponding array of nanopores comprising a
same diameter.
8. The method of claim 1, further comprising providing a film layer
over the SiO.sub.2 layer of the nanopillar subsequent to oxidizing
the nanopillar and prior to selectively removing the silicon
core.
9. The method of claim 1, wherein oxidizing the nanopillar to form
the SiO.sub.2 layer around the silicon core comprises forming a
cylinder comprising a concentric silicon core and SiO.sub.2
layer.
10. The method of claim 1, further comprising removing any
remaining portion of the nanopillar from the substrate.
11. The method of claim 1, further comprising: providing an oxide
layer on the bottom side of the substrate; and etching the oxide
layer on the bottom side to form a cavity in fluid communication
with the nanopore in the substrate.
12. The method of claim 1, wherein oxidizing the nanopillar
comprises a thermal oxidation process.
13. A method of fabricating an array of nanopores in a substrate,
the method comprising: forming an array of nanopillars on a first
surface of the substrate, wherein each of the nanopillars comprises
an oxide outer layer over an un-oxidized inner core; removing at
least a portion of each of the nanopillars from the first surface
to expose the un-oxidized inner core; and selectively etching the
un-oxidized inner core of each of the nanopillars to form the array
of nanopores in the substrate.
14. The method of claim 13, wherein forming the array of
nanopillars comprises: providing a patterned hard mask over the
first surface of the substrate; and subsequently etching the
substrate using the patterned hard mask to provide an array of
un-oxidized nanopillars.
15. The method of claim 14, further comprising oxidizing the
un-oxidized array of nanopillars to form the oxide outer layer over
the un-oxidized inner core.
16. The method of claim 13, wherein selectively etching the
un-oxidized inner core comprises etching through an entire
thickness of the substrate to provide an array of nanopores
extending through the entire thickness of substrate.
17. The method of claim 13, wherein the substrate and the
un-oxidized inner core comprise silicon, and wherein the oxidized
outer layer comprises silicon dioxide.
18. The method of claim 13, further comprising etching the
substrate to form a plurality of cavities on a second opposing
surface of the substrate, wherein each of the plurality of cavities
is in fluid communication with a corresponding nanopore of the
array of nanopores.
19. The method of claim 13, further comprising: forming an oxide
layer over a second opposing surface of the substrate; forming an
array of holes in the oxide layer, each of the holes in a position
corresponding to the position of a nanopillar in the array of
nanopillars on the first surface; and etching the substrate through
the array of holes to form an array of cavities.
20. The method of claim 13, wherein each nanopore of the array of
nanopores is 100 nm to 1 micron from an adjacent nanopore from
center to center.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/250,575, filed Sep. 30, 2011, entitled
"PARTICULATE NANOSORTING STACK," which claims priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Patent Application Ser. No.
61/405,019, entitled MEASURING DNA POLYMERASES USING IN-PLANE SELF
ALIGNED CAPACITORS, filed Oct. 20, 2010 and U.S. Provisional Patent
Application Ser. No. 61/388,342, entitled SEQUENCING OF
SINGLE-STRANDED DNA BY MEANS OF SMALL-SIGNAL CAPACITANCE
MEASUREMENT, filed Sep. 30, 2010, the full disclosures of each of
which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates generally to filters comprising
nanopores and methods of filtering nanoparticles.
[0004] 2. Description of the Related Art
[0005] Devices and methods for filtering nanoparticles can be used
in a variety contexts. For example, over the past decade there has
been much interest in the isolation and study of exosomes and other
biological particles (vesicles, viruses, DNA, etc.) with nanometer
sizes. Due in part to the unavailability of effective nanometer
scale filtration options, current techniques to isolate such
particles rely on chemical techniques or the use of ultra high
speed (greater than 100,000 g) centrifugation. Such techniques are
non-specific and physically damaging (such as centrifugation) or
must be tailored to each particle (such as chemical or antibody
binding approaches). In addition to biological nanoparticles,
improved nanometer scale filters will find use in many other
contexts where separation of particles by size is desired. These
include, for example, protein filtration, dialysis, water
filtration, as well as many industrial contexts.
SUMMARY OF THE INVENTION
[0006] Methods and apparatuses are provided herein for making and
using filters comprising nanopores. The filters find use, for
example, in sorting particles based on size.
[0007] According to some embodiments, a filter comprising two or
more nanopores, wherein each of the nanopores has a diameter of
about 5 nm or less is provided. In some embodiments the nanopores
have a diameter of 2 nm or less. The nanopores are open to a top
and bottom of the substrate, thus allowing fluid and particles
smaller than the nanopores to pass through.
[0008] According to some embodiments, devices for sorting particles
are provided. The devices comprise a first filter comprising two or
more nanopores each having a first diameter; a second filter
comprising two or more nanopores each having a second diameter,
wherein the first diameter is different than the second diameter,
and wherein the first diameter or the second diameter is about 5 nm
or less. In some embodiments the nanopores have a diameter of 2 nm
or less. The first and second filters are arranged in the device to
provide a flow path from the first filter to the second filter. The
first and second filter may be fluidly connected by a spacer that
provides a flow path from the first filter to the second filter.
For example, an elastomer layer may be present between the first
and second filter. In some embodiments, the device further
comprises a first microfluidic device configured to direct a sample
to the first filter and a second microfluidic device located after
the second filter and configured to collect the sample and
particles smaller than any of the pores in the filters.
[0009] According to some embodiments, methods for forming devices
are provided. The methods include providing a silicon substrate
having a top side and a bottom side, forming a first nanopillar on
the substrate, oxidizing the nanopillar to form a SiO2 layer around
a silicon core, removing a portion of the nanopillar from the
substrate to expose the silicon core, and selectively removing the
silicon core by etching to form a pore in the substrate.
[0010] In some embodiments, an oxide layer is provided on the
bottom side of the substrate. The oxide layer is etched directly
below the first nanopillar to expose the silicon substrate, and the
exposed silicon substrate is etched, thereby removing the core and
forming a nanopore through the substrate.
[0011] According to some embodiments, methods of separating
particles from a sample are provided. The methods include flowing
the sample comprising the particles through a first filter, wherein
the first filter comprises two or more nanopores with a diameter
smaller than the particles to be separated from the sample, and
wherein the nanopores are formed in a silicon dioxide layer and
have a diameter less than about 10 nm. In some embodiments, the
nanopores have a diameter of about 5 nm or less. In some
embodiments, the nanopores have a diameter of about 2 nm or
less.
[0012] According to some embodiments, methods of sorting
nanoparticles by size are provided. The methods include flowing a
sample comprising two or more nanoparticles through a first filter,
wherein the first filter comprises two or more nanopores formed in
a silicon dioxide layer, each nanopore having a first diameter and
subsequently flowing the sample through a second filter, wherein
the second filter comprises two or more nanopores formed in a
silicon dioxide layer, each nanopore having a second diameter
smaller than the first diameter. The two or more nanoparticles
comprise at least one first nanoparticle with a third diameter
larger than the first diameter and at least one second nanoparticle
with a fourth diameter larger than the second diameter. The second
diameter may be equal to or less than about 5 nm or less than about
2 nm. The nanopores may be formed in a silicon dioxide layer on a
substrate. The sample may be flowed with the aid of a pressure
fluid. In some embodiments, the method further comprises extracting
the sorted particles.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is an illustrative embodiment of a device for sorting
particles.
[0014] FIG. 2 is an illustrative embodiment of a spacer layer in
the device of FIG. 1.
[0015] FIG. 3 is a top view of the device of FIG. 1.
[0016] FIG. 4 shows a flow chart illustrating a method for forming
a device for filtering nanoparticles.
[0017] FIGS. 5A-5E illustrates cross-sections of a substrate during
various steps for forming a device for separating particles.
[0018] FIG. 6 shows an image of silicon nanopillars formed by the
methods disclosed herein.
[0019] FIG. 7 shows an image of an array of silicon nanopores
formed according to one embodiment.
[0020] FIG. 8 shows an image of an array of silicon nanopores
formed according to one embodiment.
[0021] FIG. 9 shows an image of an array of silicon nanopores
formed according to one embodiment.
[0022] FIG. 10A is an illustrative embodiment of a filter according
to one embodiment.
[0023] FIG. 10B is an illustrative embodiment of a filter according
to one embodiment.
[0024] FIG. 11 illustrates a cross-sectional view of a sorting
device according to one embodiment.
[0025] FIG. 12 illustrates a relationship between silicon core
diameter and oxidation temperature.
DETAILED DESCRIPTION
[0026] Disclosed herein are devices comprising nanopores, as well
as methods and apparatuses for forming and using the devices. The
devices can be used, for example, as filters, for example for
filtering and/or sorting nanoparticles based on size. The devices
can be formed using standard techniques developed in the
semiconductor industry. In some embodiments, nanometer scale pores
are formed in a silicon dioxide layer and can be used to separate
particles based on size. The pores are in fluid communication with
both the top and bottom sides of the silicon dioxide layer. Stacks
made of several filters allow for sorting a mixture of particles by
size.
[0027] In some embodiments, particles of a particular size are
separated from a sample by passing the sample through a filter
comprising an array of nanopores. In some embodiments all or
substantially all of the nanopores are of approximately the same
diameter. In some embodiments a mixture of particles can be sorted
by size by passing the mixture through a series of filters, each
having an array of nanopores of a particular size.
[0028] Methods are also disclosed herein for forming the nanopores
on substrates. In some embodiments, the nanopores can be sized to
allow only nanoparticles of a particular size through the
substrate. For example, nanopores can be sized such that only
particles less than 5 nm, 4 nm, 3 nm, 2 nm, or 1 nm can pass
through the substrate.
[0029] While described primarily herein in relation to their use as
filters, the skilled artisan will appreciate that the disclosed
devices comprising nanopores can be used in a wide variety of other
contexts, such as substrates for the growth of cells in
culture.
Nanoparticle Filters
[0030] Devices for filtering particles comprise a substrate and at
least one nanopore on the substrate. In some embodiments a filter
comprising nanopores is prepared from a silicon substrate. As
described below, during the process of forming the nanopores, the
substrate may be oxidized and a portion of the remaining silicon
(if any) may be removed. In some embodiments, all of the remaining
silicon is removed, leaving a silicon dioxide membrane comprising
nanopores. Thus, in some embodiments the filter may comprise, for
example, a silicon dioxide membrane. For example, as illustrated in
FIG. 10A, the filter may comprise a silicon dioxide membrane 1010
with at least one nanopore 1020. As illustrated, the nanopores are
in fluid communication with a top side 1030 and a bottom side 1040
of the membrane. However, in some embodiments the filter may
comprise a variety of materials, such as silicon and silicon
dioxide. For example, as illustrated in FIG. 10B, the filter may
comprise a silicon dioxide layer 1010, at least one nanopore 1020
and a silicon layer 1030. As illustrated, the nanopores are in
fluid communication with a top side 1030 of the substrate and an
internal cavity in the substrate 1050. In some embodiments the
nanopores are formed only in a silicon dioxide layer. In other
embodiments, the nanopores are formed in a silicon dioxide layer
and extend through an additional layer, such as an underlying or
overlying silicon layer.
[0031] In addition, the filters comprise other materials and
structures. For example, a filter may comprise integrated
microelectronic devices. In some embodiments the filter may
comprise metal layers that may serve, for example, as sensors for
detecting the passage of nanoparticles through the pores on the
substrate. In some embodiments, a filter may comprise additional
physical features. For example, in some embodiments a filter may
comprise materials that serve to physically separate areas of the
filter. For example, the filter may comprise one or more physical
barriers that separate areas of the filter comprising arrays of
nanopores of different sizes. In some embodiments filters may
comprise internal flowpaths in fluid communication with one or more
nanopores, wherein the pathways are arranged to allow a liquid to
flow to a desired portion of the filter. In some embodiments a
flowpath may be in fluid communication with one or more of the
nanopores.
[0032] In some embodiments the nanopores have a diameter equal to
or less than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nm. In some
embodiments nanopores have a diameter of about 1, 2, 3, 4, 5, 6, or
7 nm. Diameter, as used herein, refers to the average or median
width of the pore. In some particular embodiments, filters
comprising nanopores of about 2 nm or less are provided.
[0033] The size of the nanopores is preferably selected to allow
the passage of one or more nanoparticles of a particular size,
while excluding larger particles. In some embodiments, the diameter
of the at least one nanopore of a filter is about 2 nm or less. In
some embodiments, the nanopore can have an average or median
diameter of about 2 nm or less. In some embodiments, the nanopore
can have a diameter of less than about 1 nm, 2 nm, 3 nm, or 4
nm.
[0034] In some embodiments, filters comprise two or more nanopores,
where each nanopore has the same size. For example, a filter may
comprise 2, 3, 4, 5, 10, 100, 1000, 10000, 100000, 1000000 or more
nanopores of the same size. In some embodiments all of the
nanopores on a filter are the same size.
[0035] As discussed in more detail below, in some embodiments
filters comprising one or more arrays of nanopores are provided.
The arrays may comprise, for example, 2, 3, 4, 5, 10, 100, 1000,
10000, 100000, 1000000 or more nanopores. In some embodiments the
nanopores are regularly spaced in the arrays. In some embodiments
all of the nanopores in an array are the same size. In some
embodiments the nanopores in an array may have differing sizes.
[0036] In some embodiments, the nanopores are arranged in a regular
pattern on the filter.
[0037] The distance between nanopores can be selected as desired
for a given application. In some embodiments, the distance between
nanopores can be about 100 nm to 300 nm from center to center. In
some embodiments, the distance between nanopores can be about 300
nm to 500 nm from center to center. In some embodiments, the
distance between nanopores can be about 500 nm to 1 micron from
center to center. In some embodiments, the distance between
nanopores can be about 150 nm from center to center.
[0038] In some embodiments, all or substantially all of the
nanopores on the substrate are approximately the same diameter. For
example, a substrate may comprise two or more nanopores, where each
nanopore has approximately the same diameter. In some embodiments,
a filter comprises two or more nanopores of about 2 nm in
diameter.
[0039] In other embodiments, a single substrate may comprise
nanopores of different sizes. In some embodiments, nanopores of a
particular size are grouped together on the substrate. For example,
a first portion of a substrate may comprise nanopores of a first
diameter and a second portion of a substrate may comprise nanopores
of a second diameter, wherein the second diameter is different from
the first diameter. The areas of different diameter nanopores may
be separated from each other, for example to enable the passage of
a sample through a single size of nanopores. In some embodiments
the areas of different diameter nanopores are separated spatially.
In some embodiments the areas of different diameters are separated
physically, for example by a physical barrier.
[0040] As mentioned above, in some embodiments, multiple regions of
nanopores can be integrated on a single substrate. For example,
multiple nanopores, for example multiple arrays of nanopores, can
be provided in different, physically separated areas of a single
substrate. See, for example, the device illustrated in FIG. 11 and
described in more detail below. In this way, multiple samples can
be isolated or sorted on the same substrate using multiple
individual devices. Thus, many nanoparticle samples can be sorted
simultaneously in parallel using a single substrate comprising an
array of filtering devices. Alternatively, a single sample can be
passed through multiple sized nanopores by being flowed through
different regions of a substrate (as in the device illustrated in
FIG. 11).
[0041] Exemplary arrays of nanopores are illustrated in FIGS. 7, 8
and 9.
[0042] A filter comprising nanopores may have any desired
thickness. The thickness can be selected based on the particular
use and the desired structure of the filter. In some embodiments,
the filter has a total thickness of about 300 microns. In some
embodiments the filter has a total thickness of about 100 to 500
microns or more. However, in some embodiments the filter may have a
thickness of about 10 to about 300 nm, for example if the filter
comprises only a silicon dioxide membrane containing the nanopores.
In some embodiments the thickness of the silicon dioxide layer
comprising the nanopores is about 10 to 300 nm, or 10 to about 100
nm. In some embodiments, a filter comprising a silicon dioxide
layer with nanopores with a diameter or 2 nm or less may have a
silicon dioxide layer thickness of about 10 to 100 nm. In some
embodiments, a filter may have a silicon dioxide layer thickness of
about 300 nm.
Nanoparticle Sorting Devices
[0043] One or more filters comprising nanopores may be used to
separate particles by size. In some embodiments, a single filter
may be used to separate particles of a particular size or sizes
from a sample. For example, a single filter may comprise nanopores
of a single desired size. As discussed in more detail below, a
sample, typically a liquid comprising one or more particles, is
applied to the filter and particles smaller than the diameter of
the nanopores are able to pass through the filter. Particles that
pass through the filter, and thus that are smaller than the
nanopore diameter, may be collected. In addition, particles that
are retained and thus that are of a larger size than the nanopore
diameter may also be collected.
[0044] In other embodiments a filter may comprise nanonpores of a
two or more sizes. For example, a single filter device may comprise
nanopores of two, three, four, five or more different sizes. The
different sized nanopores may be located in specific regions of the
substrate. For example, in the device illustrated in FIG. 11, a
first portion of the substrate 1110 may comprise only nanopores of
a first size, while a second portion 1120 comprises nanopores of a
second size. A third area 1130 may comprise nanopores of a
different size from the first two areas 1110 and 1120, etc. . .
.
[0045] In other embodiments, devices for filtering nanoparticles
and/or sorting nanoparticles comprise two or more filters. Such a
device may be referred to as a filter stack, or simply a stack. In
some embodiments, the device comprises multiple filters, arranged
such that a sample passes sequentially through each filter. For
example, the device may comprise a flow path that allows a sample,
such as a liquid or other fluid sample, to pass through a series of
two or more filters in sequence. The stack may include a first
filter and a second filter separated by a spacer layer. The spacer
layer may provide a flow path that allows for at least a portion of
a sample that has passed through nanopores in the first filter to
flow to the second filter. In addition, the spacer layer may
provide access to the region between the first and second filter,
such that material (such as particles) that has flowed through the
nanopores of the first filter and not through the second filter can
be collected. Additional spacer layers and filters may be used
between other filter sets; for example three, four, five, six,
seven, eight, nine, ten or more filters may be utilized, with a
spacer layer between the first and second filter, second and third
filter etc. . . . Thus, in some embodiments each filter is
separated from filters above by a spacer layer.
[0046] A microfluidic device, such as a chamber formed of an
elastomer, may be disposed above the first filter to provide a
means of providing the sample to be filtered to the first filter.
For example, the microfluidic device may comprise a chamber that is
pierceable by a needle, such that a fluid sample can be injected
into the chamber. The chamber may be in fluid contact with the
nanopores on the first filter, such that a flow path is created
from the microfluidic device to the first filter. In addition, a
second microfluidic device may be disposed under the last filter in
a stack, and in fluid communication with at least a portion of the
nanopores in the last filter, such that fluid flowing through the
last filter is collected and can be removed.
[0047] In some embodiments, each filter in a device comprising
multiple filters may comprise different size nanopores from other
filters in the device. In other embodiments, two or more filters in
a stack may comprise the same size nanopores.
[0048] In some embodiments, a flow path is created such that a
fluid sample can be flowed through each of two or more filters
sequentially, where each filter has a smaller nanopore size than
the previous filter. Portions of the sample retained at each filter
(for example because the particles are two large to pass through
the nanopores of the subsequent filter), can be removed.
[0049] In other embodiments, a device may comprise multiple filters
but be arranged such that multiple samples pass through different
filters simultaneously.
[0050] Referring to FIG. 1, in some embodiments a stack device for
filtering particles 100 comprises: a first filter 110 comprising at
least one nanopore 111 and a second filter 120 comprising at least
one nanopore 121. The first filter 110 may also be referred to as
the top filter, and is typically the first filter that a sample
passing through the device will encounter.
[0051] In some embodiments, a diameter of the at least one nanopore
121 of the second filter is different than a diameter of the at
least one nanopore 111 of the first filter. In some embodiments the
nanopores of the second filter 121 are smaller than the nanopores
of the first filter 111.
[0052] In some embodiments a filtration device comprises at least
one spacer layer 160 separating the first filter 110 and the second
filter 120. In some embodiments the spacer layer 160 may serve not
only to separate the filters, but also to provide a flow path to
the second filter 120. That is, the spacer layer 160 may contain
and direct the sample to the second filter 120. The spacer layer
160 may be, for example, an elastomer layer. In some embodiments
the layer 160 is formed from PDMS. This layer 160 may be configured
to allow at least a portion of a sample that has passed through the
overlying layer to be removed during or after filtration. For
example, nanoparticles that were able to pass through the first
filter 110 but not the second filter 120 may be removed. In some
embodiments the spacer layer 160 may be penetrable, for example by
a needle, to allow removal of material from between filters. It may
also allow addition of material to the space between filters, such
as additional liquid or reagents. In some embodiments the spacer
layer 160 is between and adjacent to the first filter 110 and the
second filter 120, or between any two other filters in a stack.
[0053] The device 100 may comprise additional filters. The
illustrated device comprises a third filter 130 and a bottom filter
140. As represented by the dashed line 190, additional sets of
filters and spacers may be located between filter 130 and the
bottom filter 140. The filters may be arranged as illustrated such
that a sample passes sequentially through the first 110, second
120, third 130 and bottom 140 filters, as well as through any
intervening filters between filter 130 and bottom filter 140. That
is, there may be a flow path for the sample between all of the
filters in the stack.
[0054] In some embodiments, each of the filters may comprise
different size nanopores such that as a sample moves through the
series of filters, particles of different sizes may be separated
and collected. In some embodiments a sample is passed through
filters having sequentially smaller nanopores. Thus, in some
embodiments the diameter of the at least one nanopore 121 of the
second filter is different than the diameter of the at least one
nanopore 111 of the first filter. Similarly, the diameter of the
nanopores of each filter of the sequence of filters may be
different than the diameter of the nanopores of the other filters.
In some embodiments the filters have sequentially smaller pore
sizes. For example, the filters 110, 120, 130, and 140 may be
stacked from largest pore size to smallest pore size. The pore
sizes may be selected such that a mixture of different size
nanoparticles is separated by size, wherein each size of particle
may be collected where it encounters a filter with nanopores
smaller than that particle size.
[0055] A spacer layer 160 may be an elastomer layer between the
first filter 110 and the second filter 120. In some embodiments, an
elastomer layer separates each of the filters 110, 120, 130, and
140 from the filter above. For example, an elastomer layer 170 may
be located between the second filter 120 and the third filter 130.
An elastomer layer may also be located between the third filter 130
and the fourth filter 140, and any additional pairs of adjacent
filters.
[0056] In some embodiments, the diameter of the nanopore of at
least one of the filters is about 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2
nm, 1 nm or less. In some embodiments, an average or median
diameter of at least one nanopore of the last filter in a filter
stack (here nanopore 141) is about 2 nm, 1 nm or less.
[0057] In some embodiments, the diameter of the nanopore 121 of the
second filter 120 is smaller than a diameter of the nanopore 111 of
the first filter 110.
[0058] FIG. 2 illustrates one embodiment of a spacer layer 160. In
some embodiments, the spacer layer comprises a hole 220 and a
channel 210. In other embodiments the spacer does not comprise a
channel. The hole 220 is located under at least a portion of the
nanopores of the overlying filter and over at least a portion of
the nanopores on the underlying layer, providing a flow path from
the overlying filter to the underlying filter such that a sample
passing through the overlying filter is contained and contacts the
nanopores of the underlying filter. Channel 210 may be used to add
or remove material from the space between the filters. For example,
particles can be removed that have passed through the overlying
filter but not through the underlying filter. In addition, fluid
may be provided through the channel to assist flow of the sample
through the filters. The channel 210 may approach an outer edge of
the layer 160 leaving a thin membrane 240. In some embodiments the
membrane 240 may be about 2 mm or less. In some embodiments, the
membrane 240 is configured to be punctured with a needle, such as a
microfluidics needle.
[0059] In some embodiments, the spacer layer 160 is an elastomer
layer made from PDMS. In some embodiments, the elastomer layer is
formed by molding a UV activatable polymer. In some embodiments,
the elastomer layer is made from syphil, or SQ-8. Other materials
for forming the elastomer layer and method of manufacturing the
layer will be apparent to the skilled artisan.
[0060] Referring again to FIG. 1, in some embodiments, the device
100 may further comprise a first microfluidic device 180 overlying
the stack. The first microfluidic device 180 may be, for example,
configured to insert a carrier fluid and/or sample into the device
such that it contacts the nanopores of the first filter in the
stack. In some embodiments the device 180 is an elastomeric
material such that a needle can be used to inject a sample into the
space 185 above the first filter 110. The device 185 may be, for
example, a molded elastomeric material such as PDMS.
[0061] FIG. 3 illustrates a cut-away view of a microfluidic device
340. Here, a hole 320 is illustrated over an array of nanopores 330
on the underlying filter 340. The channel 350 is in fluid
communication with the hole 320, and in turn with space 370
(corresponding to space 185 in FIG. 1). Addition or removal of a
sample and/or other material to the space 370 through hole 320 can
thus be achieved by providing the sample to channel 350. Material
may be introduced into the channel through a port 360. The port 360
may be open to the environment. In other embodiments the port 360
may be accessible, for example by penetrating an overlying membrane
with a needle.
[0062] In some embodiments, the device further comprises a second
microfluidic device 190 located at the bottom of the stack, below
the final filter 140. The second microfluidic device 190 may be
configured to collect the sample and particles that have passed
through all of the filters of the stack 100. In some embodiments,
the sample that is collected in the microfluidic device 190 will
comprise only particles that are smaller than any of the pores in
the filters.
[0063] In other embodiments a device may comprise a single filter
(formed from one substrate) with nanonpores of two or more sizes.
For example, a filter may comprise two physically separated
filtration regions and the sample may be contacted with a first
filtration region of the filter comprising nanopores of a first
size, and subsequently contacted with a second filtration region of
the filter having nanopores of a second size. For example, two or
more regions having different sized nanopores may be in series in a
single flowpath on a substrate. In this way, a single filter can be
used to sort particles of various sizes.
[0064] FIG. 11 illustrates a cross-sectional view of a sorting
device with a series of filtration regions on a single substrate
according to one embodiment. The illustrated device 1100 comprises
a single substrate 1102, a first filtration region 1110, and a
second filtration region 1120.
[0065] The device 1100 may comprise additional filtration regions.
The illustrated device comprises a third filtration region 1130 and
a last filtration region 1140. As represented by the dashed line,
additional filtration regions may be located between filtration
region 1130 and the final filtration region 1140. The filtration
regions may be arranged as illustrated such that the filters are in
series in a single flow path (dotted line 1101 with arrows
indicating direction of flow). That is, each filtration region is
in fluid communication with the next filtration region, and a
sample may flow through each of the filtration regions in order.
The device may thus be arranged as illustrated such that a sample
passes sequentially through the first 1110, second 1120, third 1130
and last 1140 filtration regions, as well as through any
intervening filtration regions between filtration region 1130 and
last filtration region 1140.
[0066] In some embodiments, each of the filtration regions may
comprise different size nanopores such that as a sample moves along
the flow path 1101 through the series of filtration regions,
particles of different sizes may be separated and collected. In
some embodiments a sample is passed through filtration regions
having sequentially smaller nanopores. Thus, in some embodiments
the diameter of the nanopores of the second filtration region 1120
is different (typically smaller) than the diameter of the nanopores
of the first filtration region 1110. Similarly, the diameter of the
nanopores of each filtration region of the sequence of filtration
regions may be different than the diameter of the nanopores of the
other filtration regions. In some embodiments, the filtration
regions 1110, 1120, 1130, and 1140 may be arranged from largest
pore size to smallest pore size.
[0067] In some embodiments the sorting device comprises at least
one separation layer 1150 that physically separates the filtration
regions. In some embodiments the separation layer 1150 may serve
not only to physically separate the filtration regions, but also to
contain and direct the sample to the second filter 1120 by creating
a flow path. The layer 1150 may be, for example, an elastomer
layer. In some embodiments the layer 1105 is formed from PDMS.
[0068] The layer 1150 may be penetrable, for example by a needle.
This may allow at least a portion of a sample that has passed
through the previous filtration region to be removed during
filtration. It may also allow addition of material to the space
between filters, such as additional fluid or reagents. In some
embodiments the separation layer 1150 is above and adjacent to the
series of filters (first filter 1110, the second filter 1120,
etc).
[0069] In some embodiments, the layer 1150 comprises at least one
channel 1155, 1165. The channel 1155 may be used to add or remove
material from a space (or the flow path) between two or more
filtration regions. For example, particles can be removed that have
passed through the filtration region prior to the channel. In some
embodiments, the channel 1155 may approach an outer edge of the
layer 1150 leaving a thin membrane (not shown). In some embodiments
the membrane may be about 2 mm or less. In some embodiments, the
membrane is configured to be punctured with a needle, such as a
microfluidics needle. In other embodiments the spacer does not
comprise a channel.
[0070] In some embodiments the sorting device comprises a second
separation layer 1160. The second separation layer 1160 may be
below and adjacent to the series of filtration regions (first
filtration region 1110, the second filtration region 1120, etc).
The second filtration regions may also comprise channels 1165 that
may be used to add or remove material from a space (or the flow
path) between two or more filtration regions.
[0071] In some embodiments, at least one of the separation layers
1150, 1160 may comprise a microfluidic device covering at least a
portion of the series of filters before the first filtration region
1110. The microfluidic device may be, for example, configured to
insert a carrier fluid and/or sample into the device. In some
embodiments the separation layers comprise an elastomeric material
such that a needle can be used to inject a sample into the space
before the first filtration region 1110, such as into the flowpath
1101. The microfluidic device may be, for example, a molded
elastomeric material such as PDMS.
[0072] In some embodiments, at least one of the separation layers
1150, 1160 may comprise a second microfluidic device covering at
least a portion of the series of filtration regions after the final
filtration region 1140. The second microfluidic device may be
configured to collect the sample and particles that have passed
through the filtration regions. In some embodiments, the sample
that is collected in the second microfluidic device will comprise
only particles that are smaller than any of the pores in the
filtration regions.
Fabrication of Filters and Devices
[0073] Devices comprising nanopores can be formed by the methods
disclosed herein. In some embodiments, nanopores can be formed on
silicon substrates. In some embodiments nanopores preferably have a
diameter less than about 50 nm, less than about 25 nm or even less
than about 10 nm. For example, nanopores may be from about 0.1 to 1
nm, from about 1 to about 2 nm, from about 1 to about 3nm, from
about 1 to about 4 nm, from about 1 to about 5 nm, from about 1 to
about 6 nm, from about 1 to about 7 nm in diameter. In some
embodiments the nanopores may be about 1, 2, 3, 4, 5, 6, 7, 8, 9 or
10 nm in diameter. In some embodiments the nanopores may be less
than about 1 nm in diameter, but sufficiently large to allow fluid
passage. In some embodiments, multiple nanopores are formed on the
substrate. In some embodiments all of the nanopores have
approximately the same diameter. In other embodiments, nanopores of
different diameters may be formed. Nanopores can be spaced between
about 20-50 nm apart, thereby allowing fabrication of arrays of
nanopores on a substrate. In some embodiments a single array is
formed on a substrate. In other embodiments, multiple arrays may be
formed on a single substrate. Each array of nanopores may comprise
nanopores of a single size. In some embodiments, multiple arrays of
nanopores are formed on a substrate, where individual arrays
comprise nanopores of a size that is different from one or more
other arrays on the substrate. That is, a single substrate may
comprise one or more arrays of nanopores of a first size, and one
or more arrays of nanopores of a second size, where the first size
and second size are different. For example, a single substrate may
comprise one or more arrays of nanopores of about 5 nm and one or
more arrays of nanopores of about 2 nm. Microfluidic channels may
be used in some embodiments to move a sample through various arrays
on a single substrate. The substrates comprising nanopores may be
used, for example, as filters for separating and sorting
particles.
[0074] Briefly, a silicon substrate can be patterned to form
nanopores having a desired size. First, a silicon substrate can be
patterned and etched to leave raised silicon structures or
nanopillars having a desired size and shape. The silicon
nanopillars can then be oxidized in a controlled manner to form
silicon dioxide on the outer area of the silicon nanopillars while
leaving an un-oxidized portion of the nanopillar at the center of
the structure having a desired size (an un-oxidized nanopillar
core). A layer may optionally be deposited over the oxide, such as
an aluminum oxide layer, for example to strengthen the substrate or
to provide a layer to be used for a device or structure. Next, a
portion of the silicon nanopillars can be removed using chemical or
mechanical methods. A small portion of the silicon nanopillars may
be left close to the surface of the substrate. Next, the remaining
silicon portion of the silicon nanopillar (the silicon core) is
selectively etched to create a nanopore having a desired size. A
selective etch can also be used to etch a small internal cavity in
the back side of the silicon substrate that is in fluid
communication with a nanopore. Other layers, s can be deposited on
portions of the device to achieve a filter with the desired
properties. For example devices can be formed on the substrate, for
example to make electrical measurements of materials moving through
the nanopores. In other embodiments, physical barriers may be
deposited to separate particular section of the substrate from
other sections.
[0075] FIG. 4 is a flow chart describing processes for producing a
substrate comprising nanopores according to some embodiments. A
substrate comprising silicon is provided 400. One or more
nanopillars are formed 410 on the substrate, for example by masking
and etching the silicon substrate.
[0076] Various methods can be used to pattern the substrate and
form the nanopillars, including photo-lithography and electron beam
lithography. In some embodiments, the silicon wafer is patterned on
a polished surface using photo or electron beam lithography to form
nano-scale spots. Preferably, the patterns have a diameter of about
20 nm to about 50 nm. Next, a hard mask can be placed on the
patterned surface using a lift-off process. In some embodiments,
reactive sputter deposited aluminum oxide can be used as a hard
mask. Next an electron beam is used to remove the resist. Next, the
silicon can be etched using plasma etching techniques commonly
employed in the microelectronics industry. The hard mask is then
selectively removed leaving high-aspect-ratio silicon
nano-pillars.
[0077] The size of the nanopillars, their height, diameter, and
spacing may be selected to provide nanopores of a desired size and
pattern. In some embodiments, nanopillars are spaced at a distance
equal to twice the diameter of the nanopillars or greater. In some
embodiments the diameter of the nanopillar is from about 20 nm to
about 50 nm. For example, for nanopillars with a diameter of about
50 nm, the center to center distance between adjacent nanopillars
would be about 100 nm or greater.
[0078] In some embodiments the height of the nanopillars may be
from about 200 nm to about 2.5 microns. In some embodiments, the
height of the nanopillars is about 200 nm to about 250 nm. In some
embodiments the diameter of the nanopillars is about 15 to about
100 nm or greater. In some embodiments, the diameter of the
nanopillar is about 50 nm and the height is about 1 micron. Of
course, nanopillars of other sizes can be used, depending on the
process conditions and the size and arrangement of the nanopores to
be formed.
[0079] In some embodiments, forming the nanopillars comprises:
providing an area of resist to create a hole; depositing an oxide
to the fill the hole; removing the resist; and etching the feature
to create a nanopillar structure.
[0080] In some embodiments, the area of resist is circular. In some
embodiments, the circular area of resist has a diameter as small as
30 nm.
[0081] In some embodiments, the area of resist is developed away to
create a hole. The area of resist may be developed using
lithographic techniques.
[0082] In some embodiments, a hard mask is provided on the surface
of the substrate. In some embodiments, the hard mask may be
patterned by means of a lift off process. For example, an oxide is
deposited. The oxide may be sputtered. In some embodiments, the
oxide is deposited using a reactive sputtering technique. In some
embodiments, the holes are filled with the oxide. In some
embodiments, an organic solvent is used to lift off the resist. In
some embodiments, an electron beam is used to remove the resist
leaving disks of alumina. These disks can be etched using a mixed
mode "pseudo-Bosch" technique to create vertical nanopillars. In
some embodiments, the oxide is an aluminum oxide.
[0083] In some embodiments, the disks are etched using plasma
etching techniques. In some embodiments, the disks are etched using
a mixed mode "pseudo Bosch" technique.
[0084] In some embodiments, the hard mask is then removed
selectively. In some embodiments, removing the hard mask
selectively leaves high-aspect-ratio silicon nanopillar structures,
or nanopillars. In some embodiments, the nanopillar structures are
around 1 to 100 nm in diameter and around 1 micron in height.
[0085] In some embodiments, an array of nanopillars is formed, as
illustrated in FIG. 6.
[0086] The number, size, shape and pattern of the nanopillars can
be controlled by the patterning of the mask and the extent of the
etching process. In some embodiments the nanopillars are
cylindrical and the mask is patterned accordingly. The number, size
and arrangement of the nanopillars can be controlled by the masking
and etching process. The diameter of the nanopillars may be
determined based on the desired size of the nanopores to be formed
and the oxidation process to be used in the next step. The height
of the nanopillars can be controlled by controlling the extent of
the etching of the substrate. In some embodiments the nanopillars
can be made from materials other than silicon that can be treated
to form a core that can be selectively etched.
[0087] The silicon nanopillar is oxidized 420 to form a nanopillar
comprising a silicon dioxide shell and having a core of silicon.
The oxidation may be controlled, for example by controlling
temperature, pressure and the nature of the oxidant, in order to
obtain a silicon core of a desired size. The size of the silicon
core will determine the size of the nanopore formed.
[0088] Exposing the silicon nanopillars to an oxidizing environment
forms silicon dioxide from the silicon in the nanopillar. It will
be appreciated that thermal oxidation is common in the
microfabrication of metal oxide semiconductor field effect
transistors.
[0089] In some embodiments, the oxidation step can be carried out
in an oxygen furnace. Oxidation of the silicon nanopillar forms
silicon dioxide from the silicon on the outer area of the silicon
nanopillar, as well as on the other exposed portions of the silicon
substrate. The formation of silicon dioxide and expansion can cause
strain to the silicon core of the oxidized pillars. The oxidation
process can be self terminating because oxidation stops when the
strain becomes too high. FIG. 5B shows a cross-section of a silicon
substrate 500 with a silicon nanopillar 520 with a thin silicon
dioxide layer 530 formed on the silicon substrate 500 and silicon
nanopillar 520. In other embodiments silicon dioxide may only be
formed from the silicon nanopillar 530 and not on the silicon
substrate, for example by masking the substrate. FIG. 6 illustrates
nanopillars before the oxidation step.
[0090] In some embodiments, the oxidation conditions can be
selected to achieve a desired amount of oxidation and, as a result,
a desired width of the un-oxidized silicon at the core of the
nanopillar. For example, the silicon nanopillar can be oxidized to
a desired depth based on the oxygen furnace temperature. Applicants
have discovered that the amount of silicon remaining at the core of
the nanopillar is directly related to the temperature of the
oxidation step and not oxidation time. In some embodiments, the
nanopillar expand as they are oxidized. In some embodiments the
temperature during the oxidation step is from about 800.degree. C.
to about 950.degree. C. FIG. 12 is a graph illustrating the
diameter of the un-oxidized silicon in the nano-pillar versus the
oxidation temperature used during the oxidation step. FIG. 12
illustrates data for silicon nano-pillars having an initial
diameter of about 35 nm and about 50nm. The diameter of the silicon
nanopillar (prior to oxidation) can be selected along with the
oxidation temperature to achieve a desired nanopore size. In some
embodiments the temperature during the oxidation step is above
about 850.degree. C., above about 900.degree. C., above about
950.degree. C., or above about 1000.degree. C. The oxidation
temperature can be selected to reliably form concentric
silicon/silicon-dioxide cylinders (e.g. silicon cylinders or cores
surrounded by an oxide sheath) having a silicon core with a desired
width. In some embodiments, the silicon core of the nanopillar can
have a width of less than about 10 nm. In some embodiments, the
silicon core of the nanopillar can have a width of about 1 nm to
about 5 nm after the oxidation step. In some embodiments, the
silicon core of the nanopillar can have a width of about 1 nm to
about 3 nm after the oxidation step. In some embodiments, the
silicon core of the nanopillar after oxidation can have a width of
less than or equal to about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7
nm, 8 nm, 9 nm, or 10 nm.
[0091] In some embodiments, a thin layer is deposited over the
silicon dioxide (including the nanopillar). The thin layer may
serve to strengthen the area of the substrate in which the nanopore
is to be formed. For example, if silicon has been removed from the
back side of the substrate under the nanopillar, a thin layer
deposited over the oxide may strengthen the substrate during the
etching of the silicon core and formation of the nanopore. In some
embodiments the thin layer is an aluminum oxide layer. The aluminum
oxide layer may be deposited after oxidation and prior to removing
a portion of the nanopillar and etching the core.
[0092] Referring back to FIG. 4, a portion of the nanopillar is
removed 430, thus exposing the silicon core. The portion of the
nanopillar may be removed by physical or chemical methods. In some
embodiments the nanopillars are physically broken to reveal the
silicon core. In other embodiments a portion of the nanopillar is
removed by a mechanical polishing or other mechanical and/or
chemical method to expose the silicon core.
[0093] The silicon core of the nanopillar is then selectively
etched 440, thereby forming a pore in a top surface of the
substrate. In some embodiments the silicon core is etched from the
top side of the substrate comprising the nanopillar. In other
embodiments, the silicon substrate may be selectively etched from
the back side to remove the silicon core.
[0094] In some embodiments the selective etching is continued until
the pore goes through the entire thickness of the substrate.
[0095] The selective etching can remove silicon relative to silicon
dioxide or the other materials present on the substrate. In some
embodiments, a dry etch is used to selectively remove the silicon,
such as etching with XeF.sub.2 or other fluorine based etchants. In
some embodiments a plasma or a wet etch, such as EDP (an aqueous
solution of ethylene diamine and pyrocatechol), can be used for the
selective etching. The etchant can be exposed to the front or top
surface where the nanopillars were formed or the back of the
substrate. In some embodiments, the etching can result in the
formation of hollow silicon dioxide shells with the interior
defining a nanopore. The etching conditions, such as time,
temperature, and etchant can be selected to etch the nanopore and a
portion of the silicon substrate underneath the nanopillar to
create an internal cavity in the silicon substrate with a desired
volume. In some embodiments, an internal cavity can be etched in
the silicon substrate from the back of the substrate, such that the
internal cavity is in fluid communication with the nanopore. The
etching of the backside to form the internal cavity can be carried
out after forming the nanopore. However, in other embodiments the
etching is carried out after oxidation, but prior to etching the
silicon core of the nanopillar. In some embodiments the silicon
remaining on the back side of the substrate is completely
removed.
[0096] In some embodiments the remaining portion of the nanopillar
is etched back to the level of the silicon substrate.
[0097] In some embodiments, an oxide layer is provided on the back
side of the substrate. For example an aluminum oxide layer can be
deposited on the back side of the substrate. In other embodiments
the oxide layer is formed from the silicon in the substrate itself.
After forming an oxide layer on the back side of the substrate, if
desired, a hole can be patterned into the oxide layer using
lithographic techniques. The hole may be patterned directly below a
nanopillar. In some embodiments, the hole is square. The substrate
can then be etched through the hole on the backside. In some
embodiments, the hole is etched into the substrate using a first
etch to form an internal cavity. In some embodiments, the first
etch is an anisotropic cryogenic silicon etch.
[0098] The size of the nanopore is preferably sized to allow
molecules of a desired size to pass through, while retaining larger
molecules. The size of the nanopore is dependent on the size of the
un-oxidized silicon core remaining in the nanopillar after
oxidation. In some embodiments, the nanopore has a diameter or
width of about 5 nm or less. In some embodiments, the diameter or
width of the pore is from about 1 nm to about 5 nm or about 1 nm to
about 2 nm. In some embodiments, the nano-pore has a width of about
1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm.
[0099] Additional processing steps may be carried out, as necessary
to obtain the desired features on the substrate.
[0100] FIGS. 5A-5E illustrates a process of making nanopores in a
silicon substrate according to some embodiments. First, a silicon
substrate 500 is patterned and etched to leave raised silicon
structures or nanopillars 510 having a desired size and shape (FIG.
5A). Typically, the nanopillars are cylindrical. The diameter of
the nanopillars will be determined, in part, by the desired size of
the nanopores to be formed. In some embodiments the nanopillars are
about 35 nm in diameter. In some embodiments the nanopillars are
about 50 nm in diameter. In some embodiments the nanopillars can be
etched to about 15 nm. In some embodiments the nanopillars are
about 200 nm in height. As discussed below, each nanopillar
corresponds to a single nanopore. One or more nanopillars may be
formed at a time by the appropriate masking and etching. When one
or more arrays of nanopores are to be formed, nanopillars
corresponding to each nanopore are formed.
[0101] The silicon pillars are oxidized in a controlled manner to
form silicon dioxide 520 on the outer area of the silicon
nanopillars while leaving an un-oxidized portion 530 of the
non-pillar at the center of the structure (FIG. 5B). The unoxidized
portion may be referred to as the silicon core 580. The oxidation
process is controlled to produce a silicon core of the desired
diameter, as the size of the silicon core will determine the size
of the nanopore corresponding to the pillar. For example, if a 2 nm
nanopore is to be formed, oxidation is carried out under conditions
such that a 2 nm core of unoxidized silicon remains at the center
of the nanopillar. A small portion of the silicon nanopillars may
be left close to the surface of the substrate.
[0102] In some embodiments, a layer is deposited on the substrate
after oxidation and prior to removing a portion of the nanopillars
(not shown). For example, an aluminum oxide layer can be deposited
over the silicon dioxide. The layer may server to strengthen the
silicon dioxide layer during subsequent processing.
[0103] Next, a portion of the silicon nanopillars can be removed
using chemical or mechanical methods (FIG. 5C). A cavity 550 may
optionally be etched into the silicon substrate underneath the
nanopillar, such that it is in fluid communication with the
nanopillar (FIG. 5D). A mask 540 may be deposited and patterned on
the reverse side of the substrate from the pillar. The silicon may
be etched back to create the cavity 550 underlying the nanopillar.
In some embodiments, the silicon substrate is etched away
completely, leaving the the silicon core in a silicon oxide
substrate.
[0104] The silicon core of the nanopillar is selectively etched
through the substrate to create a nanopore 560 having a desired
size (FIG. 5E). A selective etch can also form a small internal
cavity 570 in the silicon substrate that is in fluid communication
with the nanopore.
[0105] Selective etching of the nanopillar forms a nanopore and,
optionally, an internal cavity in the substrate. The etchant can be
exposed to the front polished surface where the nanopillars were
formed or to the back of the substrate. In some embodiments, the
etching can result in the formation of hollow silicon dioxide
shells with the interior defining a nanopore. In some embodiments,
an internal cavity can be etched in the silicon substrate that is
in fluid communication with the nanopore. The etching conditions,
such as time, temperature, and etchant can be selected to etch the
nanopore and a portion of the silicon substrate to create an
internal cavity in the silicon substrate with a desired volume.
[0106] In some embodiments the nanopillars can be made from
materials other than silicon that can be selectively etched. For
example, the nanopillars can be made from germanium, tungsten,
titanium, or III-V materials, such as Gallium Aresenide, Indium
Arsenide, Aluminum Arsenide, Gallium Nitride, Indium Nitride,
Aluminum Nitride, and any alloys of the above listed materials[
[0107] The size of the nanopore is preferably sized to allow a
nanoparticle of a desired size to pass through. The size of the
nanopore is dependent on the size of the un-oxidized silicon
remaining in the nanopillar after oxidation. In some embodiments,
the nanopore has a diameter or width of about 5 nm or less. In some
embodiments, the diameter or width of the pore is from about 1 nm
to about 5 nm or about 1 nm to about 2 nm.
[0108] In some embodiments, one or more arrays of nanopillars is
formed on the substrate in order to create one or more arrays of
nanopores. In some embodiments, each nanopillar is about 50 nm or
more from adjacent nanopillars, if any. In some embodiments, the
distance between nanopores of the ultimate array can be about 300
nm to 500 nm from center to center. In some embodiments, the
distance between nanopores of an array can be about 500 nm to 1
micron from center to center. In some embodiments, the distance
between nanopores of the array can be about 150 nm from center to
center. The exact distances can be determined based on the use of
the substrate comprising the nanopores and can be formed using the
appropriate patterning techniques. As mentioned above, in some
embodiments, the distance between the nanopores (center to center)
is twice the diameter or greater than the diameter of the
nanopillars from which they were formed. For example, the distance
between nanopores (center to center) may be about 100 nm when
formed from nanopillars having a diameter of about 50 nm.
[0109] In some embodiments, the above-described processes can be
used to form an array of nanopores of a particular size on one
portion of a substrate while a second portion of the substrate
remains protected. Subsequently, the second portion of the
substrate may be patterned while protecting the first portion, such
that a second array of nanopores of a different size is formed on
the second portion of the substrate. Additional arrays may be
formed in this way, such that a substrate may contain 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 or more arrays of nanopores. In some embodiments
each array may comprise different sized nanopores from at least one
other array. In some embodiments, each array comprises nanopores of
a different size from each other array on the same substrate.
[0110] Multiple arrays of nanopores are illustrated in the
photomicrograph in FIG. 7. Individual nanopores are illustrated in
FIGS. 8 and 9.
[0111] Once formed, substrates comprising one or more nanopores can
be used in any of a wide variety of applications. In some
applications they are used as filters, to separate particles of a
particular size. Two or more filters may be stacked to form a
filtration device as described herein, or used by themselves. In
some embodiments, two or more filtration regions, each comprising a
particular size of nanopore, may be formed on a single substrate to
form an array of filtration devices as described herein. In other
embodiments, a series of two or more filters and/or filtration
regions may be formed on a single substrate to form a sorting
device as described herein.
Methods of Isolatin and Sorting Particles
[0112] Methods are disclosed herein for isolating and/or sorting
nanoparticles in a sample. The methods disclosed herein can be
used, for example, to separate particles by size. For example,
metal-based, lipid-based, polymer-based or biological particles may
be separated by the methods and devices disclosed herein. In some
embodiments, biological particles to be separated may include but
are not limited to exosomes, vesicles, proteins, viruses, and DNA.
In other embodiments, gold nanoparticles may be separated by the
methods and devices disclosed herein.
[0113] In some embodiments, a method of isolating particles
comprises flowing a sample comprising at least one particle through
a first filter, wherein the first filter comprises two or more
nanopores. In some embodiments the filter comprises nanopores in a
silicon and/or silicon dioxide substrate. The nanopores are sized
to allow particles of a desired size to pass through, while
particles larger than the nanopores do not. In some embodiments the
two or more nanopores each have a diameter of about 5 nm or less,
about 4 nm or less, about 3 nm or less or about 2 nm or less.
[0114] For example, in some embodiments a mixture of nanoparticles,
such as gold nanoparticles, are sorted by size. The mixture of
nanoparticles may comprise nanoparticles of known sizes, a known
range of sizes, such as 5 to 100 nm, or unknown sizes. The mixture
is passed through a series of arrays of nanopores from smallest to
largest such that the nanopores are separated by size. For example,
the mixture may be passed through a series of arrays of nanopores
with each array being 10 nm larger than the previous array, such
that the nanoparticles are separated into sizes by 10 nm
increments. Of course the particular size of the nanopore arrays
may be selected based on particular circumstances and the desired
level of separation.
[0115] In some embodiments the sample comprising the nanoparticles
is a fluid. In some embodiments the sample is a liquid. In some
embodiments, the sample is not a liquid. In some embodiments, the
sample comprises predominantly nanoparticles.
[0116] Samples comprising particles of different sizes may be
sorted and the sorted particles collected. A stack of two or more
filters may be assembled, for example as disclosed above, and used
to sort particles in a sample based on size. A sample carrying
particles of two or more sizes can be filtered through two or more
filters, each having nanopores of decreasing diameter. For example,
a sample comprising known to or suspected of comprising
nanoparticles of 2 and 5 nanometers could be passed through a first
filter comprising nanopores of 6 nm and a second filter comprising
nanopores of 3 nm in order to separate the 2 nm particles from the
5 nm particles. Particles of 5 nm would pass through the first
filter but not the second, and thus could be recovered from a space
between the first filter and the second, while the particles of 2
nm would pass through both filters and could thus be collected from
the sample that passed through the second filter.
[0117] In some embodiments, a sample comprising multiple
nanoparticles of unknown sizes is passed through a series of
filters in order to separate and collect particles of various
sizes. For example, the sample may be passed through a 10 nm
filter, an 8 nm filter, a 6 nm filter, a 4 nm filter and a 2 nm
filter. The filters may be arranged such that the sample passes
sequentially through the filters. After passing through each
filter, a portion of the sample can be removed. In this way,
particles of between 2 nm and 4 nm can be collected, particles
between 4 nm and 6 nm can be collected, particles between 6 nm and
8 nm can be collected and particles between 8 nm and 10 nm can be
collected. Of course, the sizes and numbers of the filters can be
selected to achieve the desired separation of particles.
[0118] In some embodiments, a method of sorting biological
particles comprises flowing a sample comprising at least one
particle through a filter or stack of filters. The flow of the
sample through the stack may be aided by utilizing a pressurized
fluid. For example, the sample may be mixed with or injected into a
pressurized fluid and applied to a filter. In other embodiments the
sample is applied to a filter and the filter is subsequently or
simultaneously contacted with a pressurized fluid to aid movement
of the sample across the filter. In other embodiments a pressurized
fluid is continuously applied to one or more filters.
[0119] In some embodiments, a first carrier fluid comprising at
least one nanoparticle is provided. A first filteris provided with
at least one nanopore of diameter larger than the size of the
nanoparticle. Preferably the filter comprises at least two
nanopores of the same diameter. A second filter is provided,
comprising at least one nanopore smaller than the diameter of the
nanoparticle. Preferably the second filter comprises at least two
nanopores of this size. In some embodiments the first and/or second
filters comprise 10, 100, 1,000, 10,000, 100,000 or more nanopores.
An insert or spacer layer may be disposed between the first filter
and the second filter to provide a flow path from the first filter
to the second filter. The spacer layer may comprise, for example, a
molded elastomer comprising a hole and a channel as described
above. A second carrier fluid is provided in the flow path between
the first and second filters, wherein the first carrier fluid is at
a higher pressure than the second carrier fluid, such that the
nanoparticle passes through the first filter but not the second.
Pressure is removed between the filters and a washing fluid is
flowed into the space between the first and second filters such
that particles on the second filter can be extracted, for example
with the aid of a needle.
[0120] In some embodiments a sample may be introduced to the top of
a device comprising multiple filters and sequentially filtered
through each of the filters in the device, where sequential filters
comprise nanopores of decreasing diameter. In some embodiments, the
sample is introduced through a microfluidic device configured to
insert a needle with the sample. Nanoparticles that collect at each
filter (because they are too big to pass through the nanopores of
that filter), may be collected. For example, microfluidic needles
can be inserted into the space separating the various filter layers
in order to withdraw nanoparticles that collect at each filter
stage. During the filtration portion of operation, a carrier
solution devoid of sample particulates may be provided into the
flow path to aid flow of the sample through the filters. A carrier
solution may also be used to remove particles that collect at one
or more of the filters. In some embodiments, the sample carrying
the particles to be filtered will be introduced into a top
microfluidic channel at a higher pressure than the fluid being
flowed into the flow path between filters, thus forcing the
particles through each filter and along the flow path towards the
filter beneath. In some embodiments, no pressure is applied to the
a bottom microfluidic channel underneath the last filter, thereby
making the path of highest pressure difference towards the bottom.
In some embodiments, when the sample to be filtered is exhausted,
the pressure to the flow path between the filters can be turned off
and a carrier fluid can be introduced to move the sorted particles
into an area where they can be collected.
[0121] The decreased size of the individual devices and pores can
allow for small volumes of liquid or sample to be sorted
efficiently.
EXAMPLES
Example 1
Fabricating Nanopores
[0122] A hard mask of aluminum oxide was sputtered onto a silicon
substrate and patterned to form nanodisks having a diameter of
about 35 nm spaced evenly apart. A mixture of
SF.sub.6/C.sub.4F.sub.8 was used to etch the silicon substrate
around the hard mask, thereby forming a number of silicon
nanopillars with diameters of about 35 nm. FIG. 6 shows silicon
nanopillars after removal of the hard mask. After removing the
aluminum oxide hard mask using hydrofluoric acid, the nanopillars
were oxidized in a furnace at a temperature of above 850.degree. C.
The methods in this example resulted in an un-oxidized silicon
nanopillar core having a diameter of about 2 nm. Mechanical
polishing was performed to remove portions of the nanopillars in
order to expose the un-oxidized silicon core at the base of the
nanopillars. XeF.sub.2 was used to etch the remaining un-oxidized
silicon cores to form nanopores with a diameter of about 2 nm.
XeF.sub.2 was also used to etch a portion of the backside of the
substrate to form internal cavities in fluid communication with the
nanopores.
Example 2
Fabricating Nanopores
[0123] A silicon substrate is patterned and etched to form
nanopillars having a diameter of about 100 nm. The etching is
performed as described in M D Henry et at 2009 Nanotechnology 20
255305. The nanopillars are oxidized at 900 C for 5 hours to form
silicon dioxide nanopillars comprising about 20 nm silicon cores.
Aluminum oxide is deposited over the silicon dioxide layer to
strengthen the membrane. Nanopillars are snapped off with a q-tip.
A hole is patterned on the back-side of the 400 micron thick wafer.
A cryogenic silicon etch is performed to go about 250 microns into
the wafer, following the masked hole. XeF2 is used to etch the
cores of silicon out of the nanopillars, thereby forming nanopores
in the substrate.
Example 3
Forming a microfluidic Device to Sort Particles
[0124] Starting with bare silicon, PMMA (poly methylmethacrylate)
was spun onto a wafer and baked at 180C to drive off the solvents
suspending the PMMA. The PMMA was irradiated with an electron beam
using an electron beam pattern generator (EBPG) at 100kV and 1.5
nanoAmp beam current. The locations where the electron beam sliced
through the PMMA were dissolved away in a 1:3 mixture of
Methyl-Isobutyl-Ketone (MIBK) and Isopropanol thus defining an
array of 150 nm holes and a separate array of 50 nm holes in the
PMMA. Aluminum oxide was sputter deposited onto the sample and into
the holes using a DC magnetron sputtering system at 400 W with a
1:5 mixture of O2:Ar process gas chemistry. The chip was then
placed in choloroform to dissolve away the PMMA. This removed the
aluminum oxide sitting on the PMMA but left the alumina that had
been deposited into the circular holes defined in the PMMA. Thus, a
series of 150 and 50 nm disks of aluminum oxide were formed.
[0125] These samples were etched in an inductively coupled plasma-
reactive ion etcher (ICP-RIE) with a gas chemistry of SF6 and C4F8,
thereby creating nanopillars. The alumina mask was removed with
hydrofluoric acid and the samples were oxidized at 850.degree. C.
for 1 hour forming a 15 nm layer of oxide on the surface and
decreasing the diameter of the pillars slightly. Then, 100 nm of
Aluminum oxide was sputtered onto the top surface of the chip to
lend structural support to the membrane and the pillars were broken
off with a q-tip.
[0126] Next, two large (400 micron) square holes were defined on
the backside of the chip using photolithography. These holes were
etched through the wafer using a combination of Cryogenic silicon
etching and XeF.sub.2 etching. Once the etch reached the silicon
dioxide layer on the front-side of the wafer the XeF.sub.2 etched
out the core of the oxidized nanopillars leaving a nanopore. Next,
microfluidic channels were placed on top and bottom to allow for
fluid to flow up through the 150 nm holes and down through the 50
nm holes. Any particles between 150 nm and 50 nm would be trapped
on the microfluidic channel on the top-side of the chip. The use of
150 and 50 nm was specific to the size of gold particles to be
sorted, as described below.
Example 4
Sorting Gold Nanoparticles
[0127] The microfluidic device described in Example 3 was used to
separate nanoparticles of gold. A sample comprising three sizes of
gold particles was applied to the device and passed through the 150
nm and 50 nm arrays of nanopores. The three sizes of particles were
such that the largest particles would not pass through the 150 nm
nanopore array, the middle sized particles would not pass through
the 50 nm nanopore array and the smallest particles would pass
through both arrays. Particles were collected at the 150 nm array
of nanopores and at the 50 nm array of nanopores. In addition,
particles that were small enough to pass through both arrays were
collected. In this way, the particles were sorted by size.
Example 5
Biological Nanoparticles
[0128] A hard mask is applied to a silicon substrate in a desired
pattern. The silicon substrate is then etched to create a raised
pattern in the shape of the hard mask. Next, the hard mask is
removed to leave raised silicon structures on the substrate. The
silicon substrate and raised silicon structures are then oxidized
such that a silicon core having a desired width remain within the
structures (nanopillars) after oxidation. Next, a portion of the
nanopillars is removed to expose the silicon core. The remaining
un-oxidized silicon nano-pillar core is etched to create a
nano-pore having a desired diameter.
[0129] At least two arrays of nanopores are formed on the substrate
as described above. Next, microfluidic channels are placed on top
and bottom to allow for fluid to flow up through the first set of
nanopores and down through the second set of nanopores. A sample of
biological particles is applied to the device. Any particles
between diameters of the first and second nanopores are trapped on
the microfluidic channel on the top-side of the chip.
[0130] Although certain embodiments of the disclosure have been
described in detail, certain variations and modifications will be
apparent to those skilled in the art, including embodiments that do
not provide all the features and benefits described herein. It will
be understood by those skilled in the art that the present
disclosure extends beyond the specifically disclosed embodiments to
other alternative or additional embodiments and/or uses and obvious
modifications and equivalents thereof. In addition, while a number
of variations have been shown and described in varying detail,
other modifications, which are within the scope of the present
disclosure, will be readily apparent to those of skill in the art
based upon this disclosure. It is also contemplated that various
combinations or subcombinations of the specific features and
aspects of the embodiments may be made and still fall within the
scope of the present disclosure. Accordingly, it should be
understood that various features and aspects of the disclosed
embodiments can be combined with or substituted for one another in
order to form varying modes of the present disclosure. Thus, it is
intended that the scope of the present disclosure herein disclosed
should not be limited by the particular disclosed embodiments
described above. For all of the embodiments described above, the
steps of any methods need not be performed sequentially.
* * * * *