U.S. patent application number 10/731866 was filed with the patent office on 2005-02-10 for nanosyringe array and method.
Invention is credited to Montemagno, Carlo D., Neves, Hercules.
Application Number | 20050034200 10/731866 |
Document ID | / |
Family ID | 26873913 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050034200 |
Kind Code |
A1 |
Montemagno, Carlo D. ; et
al. |
February 10, 2005 |
Nanosyringe array and method
Abstract
A nanosyringe is constructed using micro fabrication and nano
fabrication techniques on a silicon substrate. The nanosyringe
includes a membrane of silicon carbide. The position and operation
of individual nanosyringes, arranged in an array of nanosyringes,
can be independently controlled. A nanosyringe array can inject or
extract a fluid from one or more cells or other structures.
Microfluidic structures coupled to the nanosyringe allow external
pumping or extraction. A cell matrix or organelles of individual
cells can be non-destructively sampled in real time.
Inventors: |
Montemagno, Carlo D.; (Los
Angeles, CA) ; Neves, Hercules; (Moorpark,
CA) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Family ID: |
26873913 |
Appl. No.: |
10/731866 |
Filed: |
December 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10731866 |
Dec 9, 2003 |
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10178056 |
Jun 21, 2002 |
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6686299 |
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60300013 |
Jun 21, 2001 |
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Current U.S.
Class: |
427/2.28 ;
438/800; D24/112 |
Current CPC
Class: |
A61M 2037/0053 20130101;
Y10S 977/92 20130101; A61M 2037/003 20130101; Y10S 977/962
20130101; A61M 37/0015 20130101; Y10S 977/721 20130101; A61M
2205/0244 20130101 |
Class at
Publication: |
D24/112 ;
438/800; 427/002.28 |
International
Class: |
A61L 002/00; B05D
003/00; H01L 021/00 |
Claims
What is claimed is:
1. A method comprising: forming a column on a silicon substrate,
the column having an atomically sharp tip; depositing a membrane on
the column; removing a portion of the column after depositing the
membrane; and etching the membrane to form a nozzle end.
2. A device comprising: a membrane formed by depositing a conformal
layer of silicon nitride on a silicon post oriented perpendicular
to a silicon substrate, the membrane having an orifice; and a fluid
reservoir in fluidic communication with the orifice.
3. The device of claim 2 further comprising a cell retention
cavity.
4. A method comprising: transferring a pattern of dots onto a
silicon substrate; oxidizing the substrate to form an atomically
sharp silicon tip corresponding to each dot; creating a shaft for
each silicon tip; forming a pedestal at the base of each shaft;
coating the silicon tips with a membrane material; and removing a
portion of the silicon from each tip.
5. The method of claim 4 further comprising forming an opening in
each tip.
Description
RELATED APPLICATION
[0001] This application is a divisional under 37 C.F.R. 1.53(b) of
U.S. patent application Ser. No. 10/178,056 filed Jun. 21, 2002,
which claims priority under 35 U.S.C. 119(e) to U.S. Provisional
Patent Application Ser. No. 60/300,013 filed on Jun. 21, 2001,
which applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to nanosyringes, and in
particular to a nanosyringe array and method of making the
nanosyringe array.
BACKGROUND
[0003] Microinjection techniques have been used for a variety of
applications, including high-efficiency transformation, protein
injection, pathogen injection and organelle transfer. Transfer of
DNA to mammalian cell cultures and embryos using microinjection has
also been performed. More recently, microinjection has been
employed to develop transgenic animals for pharmacological studies
in the cardiovascular system, endocrine system, cancer and
toxicology. It has also been used to examine the role of the c-fos
gene as a mitogenic signal in mammalian cells by injection of
protein inhibitors and monoclonal antibodies that block mitogenic
activities.
[0004] Micropipettes are primarily constructed of tapered
borosilicate glass, quartz, or aluminosilicate needles with a
minimum diameter of between 50 and 100 nm. The primary
disadvantages of these pipettes include inherent damage inflicted
on host cells, the inability to accurately control injection rates,
the inability to inject more than one cell at a time, and the
inability to inject more than one sample into a given cell at one
time. Recently, a galinstan expansion femtosyringe was formed that
reduced the damage inflicted on host cells. In addition, heat
induced galinstan (also known as gallinstan, a liquid metal alloy
of gallium, indium and tin) was used to accurately control the rate
of injection. These femtosyringes permit the injection of
subcellular organelles such as vacuoles, mitochondria and
chloroplasts while maintaining the integrity of cell membranes.
Such femtosyringes are expensive to form, and do not facilitate the
injection of more than one cell at a time, nor do they provide the
ability to inject different substances simultaneously into the same
cell. Expensive needle puller equipment is also required to form
femtosyringes.
[0005] In one attempt to provide an array of microneedles, a
plurality of parallel hollow non-silicon microneedles are formed on
a planar surface of a substrate. Multiple arrays of these needles
can be coupled to form a three dimensional array with the
substrates still attached, or removed. Cross coupling channels
provide for free fluid flow. The array is used to increase the flow
rate of a fluid to be injected. Further, the size of the needles
constructed using this technique are much larger than those
required to permit the injection of subcellular organelles, and may
lead to unacceptable damage to cellular structures.
SUMMARY
[0006] A nanosyringe is constructed using micro and nano
fabrication techniques on a substrate. In one embodiment, the
nanosyringe is formed as a membrane of silicon carbide or silicon
nitride on a silicon substrate using photolithography or other
means. The nanosyringe comprises a tip for penetrating a host
without destroying the integrity of a host membrane. As used
herein, the term needle is interchangeable with the term
syringe.
[0007] In one embodiment, an array of nanosyringes comprises a
large number of independently controlled nanosyringes suitable for
injecting a large number of cells or other structures at a given
time, or injecting a variety of samples into a single cell at one
time or at staggered time intervals. In one embodiment, each
nanosyringe is independently controlled with respect to injection
properties. The spacing of the nanosyringes is adjusted based upon
a specific objective at the time of formation of the array. For
example, arrays with a large spacing of 5-10 .mu.m may be used for
injecting large numbers of cells. As another example, arrays with
smaller spacing, such as less than 50 nm between tips, may be used
for injection various samples into a single cell at specific rates,
time intervals and location. They are further used to increase the
flow rate of a sample to a cell. In one embodiment, a variety of
samples can be injected in varying amounts and at varying
times.
[0008] In one embodiment, the arrays are utilized to draw fluid or
remove samples from cells. An external pumping system coupled to
one or more nanosyringes allows non-destructive sampling of a cell
matrix or organelles of a cell as well as real time sampling and
analysis of physiological changes within an individual cell. In one
embodiment, a nanosyringe both injects a first fluid and extracts a
second fluid coincident with a single penetration of a host
membrane.
[0009] In one embodiment, sensor and detection capabilities, as
well as micro-pumps and valves are directly integrated into the
system using micro and nano fabrication techniques on a
semiconductor substrate. This provides the ability to
instantaneously sample a cell's cytoplasm following the addition of
a particular drug injected into the nucleus of that cell. Arrays of
nanosyringes are also formed for a variety of microfluidic systems
where precise delivery of liquids is desired. In one embodiment, a
system is provided to independently position individual
nanosyringes within a three axis coordinate system.
[0010] In one embodiment, a silicon carbide nanosyringe is
constructed using micro and nano fabrication techniques on a
silicon substrate. Each nanosyringe is independently controlled
with respect to injection properties. An external pump system
coupled to a nanosyringe array allows non-destructive sampling of
the cell's matrix and organelles, and real time sampling and
analysis of physiological changes within individual cells. Sensor
and detection capabilities, as well as micro-pumps and valves are
directly integrated into the system using micro and nano
fabrication techniques on a semiconductor substrate.
[0011] The present subject matter includes fabrication of thin,
suspended membranes supported by a silicon substrate. In various
embodiments, the membrane includes thin film materials such as
silicon nitride or silicon carbide. In one embodiment, the membrane
is formed using a non-planar (that is, not flat) surface. The
present subject matter includes membranes formed using a cylinder,
column or cone.
[0012] Other aspects of the invention will be apparent on reading
the following detailed description of the invention and viewing the
drawings that form a part thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] In the drawings, like numerals describe substantially
similar components throughout the several views. Like numerals
having different letter suffixes represent different instances of
substantially similar components.
[0014] FIG. 1 illustrates a scanning electron microscope micrograph
of an array of silicon tips used to form nanosyringes.
[0015] FIG. 2 illustrates a cross section of a nanosyringe.
[0016] FIG. 3 illustrates a perspective view of the nanosyringe of
FIG. 1.
[0017] FIG. 4 illustrates a perspective partial section view of a
self-aligned nanosyringe.
[0018] FIG. 5 illustrates a perspective partial section view of an
array of self-aligned nanosyringes.
[0019] FIG. 6 illustrates a view of a positionable nanosyringe
relative to a silicon substrate.
[0020] FIG. 7 depicts a method of fabricating a nanoneedle (or
nanosyringe) array.
[0021] FIG. 8 illustrates a scanning electron micrograph of a
Si.sub.3N.sub.4 needle.
DETAILED DESCRIPTION
[0022] In the following detailed description, reference is made to
the accompanying drawings which form a part hereof, and in which is
shown by way of illustration specific embodiments in which the
invention may be practiced. These embodiments are described in
sufficient detail to enable those skilled in the art to practice
the invention, and it is to be understood that the embodiments may
be combined, or that other embodiments may be utilized and that
structural, logical and electrical changes may be made without
departing from the spirit and scope of the present invention. The
following detailed description is, therefore, not to be taken in a
limiting sense, and the scope of the present invention is defined
by the appended claims and their equivalents. In the drawings, like
numerals describe substantially similar components throughout the
several views. Like numerals having different letter suffixes
represent different instances of substantially similar
components.
[0023] In various embodiments, the present subject relates to a
stationary nanosyringe, an array of self-aligned stationary
nanosyringes and an array of individually positionable
nanosyringes.
[0024] A method of making a nanosyringe or nanosyringe array
includes forming at least one silicon tip as shown at 110 in FIG.
1. In one embodiment, the tips are atomically sharp. An array of
silicon tips is shown in FIG. 1. The silicon tips 110 serve as a
sacrificial material for syringe fabrication. The tips 110 shown in
FIG. 1 are less than approximately 10 nm in diameter, and each has
a shaft 120 which is approximately 1 .mu.m in diameter. These sizes
referred to are merely one example. Other sizes, as well as
materials other than silicon, are well within the scope of the
invention as will be apparent from a description of the process
steps used to form the tips.
[0025] Silicon wafer 130 is initially oxidized at 1100.degree. C.
in a steam ambient to form a layer of silicon dioxide. A pattern of
dots approximately 500 nm in diameter are defined through a
lithographic process, such as photolithography using an i-line
stepper. The pattern is transferred onto the silicon dioxide layer
through fluorine-based reactive ion etching (RIE). This is followed
by chlorine-based inductively coupled plasma (ICP) RIE to transfer
the pattern onto the silicon substrate, resulting in an array of
dots.
[0026] The silicon layer is again oxidized. Localized stress
effects acting around the neck of the post or cylinder produces an
atomically sharp tip. Reactive ion etching is used again to remove
the oxide from the floor of the silicon layer. Then another
chlorine RIE is performed to further etch the silicon and create
the shaft 120 for each tip 110. The silicon dioxide is removed
using a 1:6 buffered hydrofluoric acid solution. Methods other than
those described herein can be used to wholly or partially remove
the silicon substrate.
[0027] In one embodiment, a faceted profile or pedestal at the base
of the shaft is formed by anisotropic wet etching. For example,
using (100) silicon, an anisotropic wet etchant such as potassium
hydroxide yields <111> cuts at an angle of 54.74.degree.
relative to the surface of the silicon substrate. The cut is
illustrated in FIG. 2 at 210A, which shows a cross section of a
finished nanosyringe indicated generally at 200A.
[0028] The angle of the cut is determined, in part, by the
orientation of the crystal planes of the silicon substrate. The
orientation of the crystal planes are expressed using Miller
indexes and relate to how the silicon crystal is sliced. Wet
anisotropic etching will etch the silicon at different rates
depending on the orientation of the crystal planes.
[0029] The membrane is next fabricated on the silicon tip, shaft
and faceted base followed, in one embodiment, by removal of the
silicon structure by a combination of wet and dry etching steps.
Selective removal of the silicon can result in structural
supporting members within or about the nanosyringe.
[0030] FIG. 3 provides a perspective of a nanosyringe. To obtain
the finished nanosyringe, the previously formed silicon tip is
coated with a conformal membrane material 220 such as silicon
nitride or silicon carbide. A portion of the silicon tip is removed
through a combination of wet and dry RIE etching to leave a core
open area, or syringe cavity, 230 for fluid flow. The syringe
cavities are connected in one embodiment to channels and reservoirs
for fluid dispensing. A base structural support area 240 remains
following the etching to provide support for the syringe membrane
material 220.
[0031] An opening, or nozzle, at the tip of the syringe 250 is
formed using a process similar to submicron nozzle fabrication. In
one embodiment, a polymer layer or other photoresist material is
used as an etch mask. The tip may be etched using traditional
etching methods.
[0032] In one embodiment, a localized stress effect can be used to
form a syringe nozzle. In this embodiment, a concentration of
stresses occurring within the membrane at a sharp radius portion of
the silicon nitride causes a fracture which forms a nozzle on the
syringe. The sharp, or small, radius portion of the membrane
separates from a larger radius portion.
[0033] In one embodiment, silicon carbide or other material is
etched back to form a syringe nozzle. In one embodiment, greater
uniformity in nozzle placement and dimension is achieved by an
etching process.
[0034] In one embodiment, low-pressure chemical vapor deposition
(LPCVD) is used to deposit the membrane on the silicon tip. In one
embodiment, LPCVD is used to deposit silicon nitride and localized
stresses are controlled by introduction of controlled amounts of
oxygen in the film (or membrane) during deposition, thus forming
silicon oxynitride.
[0035] In general, LPCVD provides a membrane pin-hole free and with
uniform physical properties. Stresses induced are generally
characterized as tensile and the range of levels stress tends to be
rather narrow.
[0036] In one embodiment, the process of membrane fabrication
includes plasma enhanced chemical vapor deposition (PECVD). As with
LPCVD, the deposited film is determined by a chemical reaction
between the source gases supplied to the reactor. Since the
resulting films are non-stoichiometric, a wide range of stress
values can be obtained, from tensile stress to compressive.
[0037] In one embodiment, the silicon surface of the tips is
converted to silicon carbide (SiC) by carbonization. Silicon
carbide conformally coats the silicon tip and when carbonized, the
membrane provides a sharper and stronger nanosyringe.
[0038] In one embodiment, the nanosyringe remains stationary and a
cell is moved into position such as by using a
microelectromechanical systems (MEMS) actuator driven stage. In one
embodiment, the cell is positioned using laser tweezers.
[0039] FIG. 4 shows nanosyringe 410 adapted for penetrating cell
420. A faceted cavity 430 is formed in the same manner as the
faceted pedestal in FIG. 2 using an anisotropic etch in place of
the chlorine RIE to remove silicon to form the shaft of the
syringe. Faceted cavity 430 is used to trap and position the cell
at the same time. In one embodiment, cell 420 is supported by flat
quartz slide 430. Syringe 410 is self-aligned within faceted cavity
430. In addition to, or in lieu of, the faceted cavity, other
structure may be formed in the silicon substrate to capture cell
420. For example, conical structures or posts may be used to
position cell 420 for penetration by nanosyringe 410.
[0040] In one embodiment, quartz slide 430 comprises a cell
culture, an array of syringes, each having a faceted cavity, is
lowered against the cell culture to trap and hold cells in place in
a position reachable by the syringes as shown 510 in FIG. 5. In one
embodiment, a channel for each syringe is provided in a backside of
the substrate at 515A, 515B and 515C. As previously indicated, each
channel provides an independent path to a reservoir. The paths may
be connected in some embodiments so that more than one syringe is
coupled to the same reservoir, or multiple channels and syringes
may have separate reservoirs. In further embodiments, more than one
syringe is formed within a faceted cavity to facilitate the
injection of multiple samples or substrates into a particular cell
at a specific time or time intervals.
[0041] Patterning the backside of the silicon wafer includes
leaving some silicon to make channels, pumps, and other structures
for coupling to other systems for fluid handling.
[0042] For example, in one embodiment, a fluid reservoir is
patterned into the silicon. The reservoir receives fluid by
capillary extraction or contains fluids for delivery using the
nanosyringe.
[0043] FIG. 6 illustrates a positionable nanosyringe adapted for
movement relative to a silicon substrate. MEMS actuators (including
electrostatic comb drives or piezo elements, for example) drive the
syringe laterally in the x-axis and y-axis to precisely position
the syringe with respect to the object to be injected.
[0044] In one embodiment, syringes and arrays are movable with
respect to the cavity. Incorporation of a flexible structure or
suspension mechanism for syringe displacement and inclusion of
actuators for moving the syringe in a plane, as well as up and
down, are utilized. In one embodiment, electrostatic actuators for
x-axis and y-axis displacement are constructed using the remaining
backside of the silicon substrate. In one embodiment, the
electrostatic actuator includes one or more comb drive actuators.
Out-of-plane z-axis motion is provided by similar actuators by
means of electrostatic levitation. In one embodiment, corrugated
structure 600 is disposed concentric to the syringe and acts as a
bellows to allow freedom of movement of the syringe.
[0045] In one embodiment, both independent steering and a
self-alignment faceted cavity are used with a particular
nanosyringe.
EXEMPLARY EMBODIMENT
[0046] The following describes one embodiment of the present
subject matter.
[0047] Standard <100> silicon wafers is used as the base
material for the array fabrication. The individual fabrication
steps are depicted in FIG. 7. Initially the wafers were thermally
oxidized at 1100.degree. C. in a steam ambient with trichlorethane
(TCA) to form a 0.7-.mu.m thick layer of SiO.sub.2 (FIG. 7A). The
wafers were then primed at 90.degree. C. in hexamethyldisilazane
(HMDS) vapor to promote photoresist adhesion, followed by
photoresist coating. A photolithography step was performed using a
10X i-line step-and-repeat system to form an array of 0.5 .mu.m
dots. The dot pattern is then transferred onto the silicon dioxide
layer by magnetron-assisted reactive ion etch, using CHF.sub.3 (30
sccm) at 1 kW until the open areas were free of SiO.sub.2.
[0048] This patterned silicon dioxide (FIG. 7B) then serves as an
etch mask to etch the underlying silicon to define standing silicon
posts. The posts were etched using a chlorine-based inductively
coupled plasma (Cl.sub.2=50 sccm, BCl.sub.3=2.5 sccm, ICP power=75
W) to obtain 1-.mu.m tall silicon posts (FIG. 7C). A second thermal
oxidation step is performed to turn these silicon posts into
atomically sharp tips. When the posts are thermally oxidized, a
stress effect around the base of the post causes an uneven
oxidation along the length of the post thus resulting in a
cone-like structure. This was performed in a steam ambient, with
TCA added, at 1100.degree. C., for about 30 minutes (FIG. 7D). The
initial oxide which remained on top of the tips can then be used as
an etch mask to define a shaft at the base of the tips. To do this,
a plasma etchback step is performed in CHF.sub.3 ambient to strip
the oxide surrounding the tips. This is followed by a chlorine ICP
etch step to define the shafts (FIG. 7E). The samples are then
immersed in a 1:6 buffered hydrofluoric acid solution to strip all
the remaining oxide, thus exposing the tips (FIG. 7F). FIG. 1
illustrates a typical silicon tip array obtained by this
process.
[0049] Subsequent steps form the needles. By way of overview, the
needles are formed by a suspended silicon nitride membrane
utilizing the tip array as a mold. Suspended silicon nitride
membranes are formed into a corrugated membrane, which conforms to
the shape of the tips. The samples are coated with a layer of
low-stress Si.sub.3N.sub.4 using low-pressure chemical vapor
deposition (LPCVD) as shown in FIG. 7G. The thickness of this layer
depends on the needle diameter and aperture desired.
Photolithography is performed on the backside of the wafers using
an infrared aligner. This defines the windows for the through-wafer
etch. The wafers are then immersed in a 50% wt. potassium hydroxide
solution at 90.degree. C. until the cores of the tips are
removed.
[0050] The needle apertures are created using a process similar to
submicron nozzle fabrication. In one embodiment, photoresist is
spun to completely cover the needles and an RIE etchback is
performed. FIG. 8 shows a completed needle.
ADDITIONAL EMBODIMENTS
[0051] In one embodiment, materials other than silicon and silicon
carbide are used for fabrication of a nanosyringe. For example,
thin metal films deposited using techniques such as, but not
limited to, chemical vapor deposition, physical vapor deposition,
electroplating, and electroless plating.
[0052] In one embodiment, a nanofabricated array of syringes is
adapted for transcutaneous injection of medicament or for drawing
blood or cell samples. In such an embodiment, a plurality of
nanosyringes, each fabricated of silicon carbide, are arranged on a
substrate having structural silicon reinforcement beams or members.
The depth of penetration of a nanofabricated array of syringes may
be sufficiently shallow to avoid disturbing nerve cells and
therefore offers a low pain method of drawing samples or delivering
medicine. The present subject matter may be used to inject fluids
into blood cells or to extract cell matrix or organelles for
further analysis.
[0053] In one embodiment, the nanosyringe structure is strengthened
by providing reinforcing members on the silicon substrate. For
example, in one embodiment, the silicon includes a network or grid
of reinforcement beams etched or otherwise formed into the backside
of the array or within the nanosyringe. In one embodiment, the
nanosyringe is fabricated of a membrane material suited for a harsh
environment.
[0054] In one embodiment, the methods and devices described herein
are applied to the fabrication and use of syringes that are larger
or smaller than nano scale dimensions. For example, in various
embodiments, the syringes are more properly described as
millisyringes, microsyringes, picosyringes or femtosyringes.
[0055] In one embodiment, one or more microfluidic devices or
actuators are coupled to a nanosyringe. For example, in one
embodiment, a flow valve is coupled to a nanosyringe. Other
microfluidic devices are also contemplated, including, but not
limited to pumps, reservoirs, sensors, fluid conduits or channels.
In one embodiment, a microfluidic device or actuator is fabricated
on the same silicon substrate as the nanosyringe. In one
embodiment, the nanosyringe is fabricated on a first silicon
substrate and a microfluidic device or actuator is fabricated on a
second silicon substrate and the first and second substrate are
subsequently bonded together.
[0056] In one embodiment, the membrane is fabricated of material
other than silicon carbide. Silicon carbide exhibits robust
performance in a harsh environment, has good mechanical hardness
and is relatively chemically inert. Polycrystalline SiC can be
deposited using PECVD. Also, 3C-SiC can be formed on silicon by
carbonization of the silicon surface. In either case, thin
conformal films can be directly deposited on silicon. Silicon can
be used as a sacrificial member because of the high chemical
selectivity between Si and SiC. The SiC film can be released by
etching using wet chemistries such as potassium hydroxide,
ethylene-diamine/pyrocatechol (EDP) or hydrofluoric acid. High
selectivity is also found in reactive ion etching which also allows
fabrication of mechanical supporting structures within the
nanofabricated device.
[0057] In one embodiment, the nanosyringe or nanosyringe array is
optically transparent. Transparency allows monitoring operation of
the nanosyringe via an optical microscope.
[0058] In one embodiment, the tip is formed on the silicon
substrate without a cylinder or shaft at the base. The membrane is
formed on the tip as described above. In one embodiment,
microfluidic devices or MEMS devices are coupled to the
nanosyringe, also as described above.
CONCLUSION
[0059] The above description is intended to be illustrative, and
not restrictive. Many other embodiments will be apparent to those
of skill in the art upon reviewing the above description.
* * * * *