U.S. patent application number 10/894944 was filed with the patent office on 2004-12-30 for nanopump system.
Invention is credited to Martin, Francis J., Walczak, Robbie J..
Application Number | 20040262159 10/894944 |
Document ID | / |
Family ID | 26970673 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040262159 |
Kind Code |
A1 |
Martin, Francis J. ; et
al. |
December 30, 2004 |
Nanopump system
Abstract
A self-contained delivery device for delivery a selected volume
of stored electrolyte solution at selected time intervals is
disclosed. The device includes a housing having a delivery port,
and contained within the housing, a chamber containing an upstream
supply reservoir for holding a quantity of electrolyte solution, a
downstream delivery reservoir for receiving electrolyte solution
from the supply reservoir and, disposed between the two reservoirs,
a membrane having a plurality of flow-through channels extending
between the two reservoirs. A pair of electrodes placed in the
chamber on either side of the membrane are controlled by a
controller contained within the housing, for pumping selected
quantities of the electrolyte solution at selected time intervals.
The invention also includes a device for detecting a target nucleic
acid sequence contained in a solution of solution of nucleic acid
fragments. The device includes a chamber, and a membrane disposed
in said chamber and having a channel extending between an upstream
chamber region, where the said channel has a selected minimum
cross-sectional dimension in the range between 2 and 100 nm.
Attached to a wall portion of the channel, is a capture nucleic
acid having a sequence complementary to the target sequence.
Upstream and downstream electrodes disposed in the upstream and
downstream chamber regions, respectively, are in contact with
electrolyte solution placed in the corresponding chamber regions. A
controller in the device includes a power source operatively
connected to the electrodes for applying a selected voltage
potential across the channel, to move individual nucleic acid
sequences contained in the solution through the channel, where the
sequences can hybridize to complementary target sequences bound to
the channel wall portion.
Inventors: |
Martin, Francis J.; (San
Francisco, CA) ; Walczak, Robbie J.; (Hilliard,
OH) |
Correspondence
Address: |
CALFEE HALTER & GRISWOLD, LLP
800 SUPERIOR AVENUE
SUITE 1400
CLEVELAND
OH
44114
US
|
Family ID: |
26970673 |
Appl. No.: |
10/894944 |
Filed: |
July 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10894944 |
Jul 19, 2004 |
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10170774 |
Jun 13, 2002 |
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60298733 |
Jun 15, 2001 |
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60298450 |
Jun 15, 2001 |
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Current U.S.
Class: |
204/450 ;
204/230.2; 204/600 |
Current CPC
Class: |
F04B 19/006 20130101;
B01L 2300/0681 20130101; B01L 3/502753 20130101; B01L 2300/0896
20130101; B82Y 30/00 20130101; B01L 2400/0418 20130101; B01D 61/56
20130101; F05B 2250/84 20130101; B01L 3/50273 20130101; B01L
2400/0415 20130101; B82Y 15/00 20130101; F04B 17/00 20130101; B01D
57/02 20130101 |
Class at
Publication: |
204/450 ;
204/600; 204/230.2 |
International
Class: |
G01L 001/20; C25B
009/00; C07K 001/26 |
Claims
1-7. (canceled)
8. A device for detecting a target nucleic acid sequence contained
in a solution of solution of nucleic acid fragments and having a
selected pH, comprising a chamber, a membrane disposed in said
chamber and having a channel extending between an upstream chamber
region adapted to hold the electrolyte solution of such
different-length fragments, and a downstream chamber region adapted
to hold an electrolyte solution, where said channel has a selected
minimum cross-sectional dimension in the range between 2 and 100 nm
and a net surface charge within a given pH range that includes the
selected solution pH, attached to a wall portion of the channel, a
capture nucleic acid having a sequence complementary to the target
sequence, upstream and downstream electrodes disposed in said
upstream and downstream chamber regions, respectively, for
contacting solution placed in the corresponding chamber regions, a
controller including a power source operatively connected to said
electrodes for applying a selected voltage potential across said
channel, to move individual nucleic acid sequences contained in the
solution through said channel, where said sequences can hybridize
to complementary target sequences bound to the channel wall
portion.
9. The device of claim 8, wherein said controller further includes
a voltage regulator for regulating the voltage applied across said
channel, to effect selective release of the target sequence from
the capture nucleic acid, based on the degree of complementarity
between the target sequences and the capture nucleic acid.
10. The device of claim 8, wherein said membrane includes an array
of channels, each having attached to a wall portion thereof, a
capture nucleic acid with a selected sequence complementary to a
selected one of a plurality of different sequences.
11. The device of claim 10, wherein one of said electrodes includes
a plurality of electrode elements, each associated with one of said
membrane channels, and said controller is operatively connected to
each of said electrode elements to apply and regulate the voltage
applied across each of the channels.
12. The device of claim 8, wherein said controller is effective to
place across the electrodes, a voltage potential effective to move
nucleic acid fragments electrophoretically through said
channel.
13. The device of claim 8, wherein said channel has a minimum
dimension in the 2-25 nm range.
14. A method for detecting a target nucleic acid sequence contained
in an electrolyte solution of nucleic acid fragments and having a
selected pH, comprising placing the solution in a chamber having a
membrane disposed therein, said membrane having a channel extending
between an upstream chamber region adapted to hold the electrolyte
solution of such different-length fragments, and a downstream
chamber region adapted to hold an electrolyte solution, where said
channel (i) has a selected minimum cross-sectional dimension in the
range between 2 and 100 nm, (ii) a net surface charge within a
given pH range that includes the selected solution pH, and (iii),
attached to a wall portion of the channel, a capture nucleic acid
having a sequence complementary to the target sequence, applying
across the channel, a voltage potential sufficient to move nucleic
acid sequences in said solution through said channel, where said
sequences can hybridize to complementary target sequences bound to
the channel wall portion, releasing captured target sequence from
said channel by applying a voltage potential across said channel
effect to dissociate hybridized nucleic acids, and detecting
released target sequences.
15. The method of claim 14, wherein said releasing includes
applying a voltage across said channel effective to selectively
release target sequences from the capture nucleic acid, based on
the degree of complementarity between the target sequence and the
capture nucleic acid.
16. The method of claim 14, wherein said membrane includes an array
of channels, each having attached to a wall portion thereof, a
capture nucleic acid with a selected sequence complementary to a
selected one of a plurality of different sequences.
17. The method of claim 16, wherein one of said electrodes includes
a plurality of electrode elements, each associated with one of said
membrane channels, said controller is operatively connected to each
of said electrode elements to apply and regulate the voltage
applied across each of the channels, and said releasing includes
applying a voltage across each of said channels, individually, a
voltage potential effective to selectively release target sequences
from the capture nucleic acid, based on the degree of
complementarity between the target sequence and the capture nucleic
acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application Ser. No. 60/298,733 entitled
"Nanopump Delivery Device and Method" filed on Jun. 15, 2001; and
U.S. Provisional Patent Application Ser. No. 60/298,450 entitled
"Nanopump Device for Detecting DNA Sequences" filed on Jun. 15,
2001, the disclosures of which are incorporated as if fully
rewritten herein.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of (i) nanopump
delivery devices and methods; and (ii) to the field of DNA sequence
determination.
SUMMARY OF THE INVENTION
[0003] A. Nanopump Delivery Device and Method
[0004] The invention includes a self-contained delivery device for
delivery a selected volume of stored electrolyte solution at
selected time intervals. The device includes a housing having a
delivery port, and contained within the housing, a chamber
containing an upstream supply reservoir for holding a quantity of
electrolyte solution, a downstream delivery reservoir for receiving
electrolyte solution from the supply reservoir and, disposed
between the two reservoirs, a nanopore channel membrane of the type
described above.
[0005] Also included are a pair of electrodes placed in the chamber
on either side of the membrane, a one-way valve connecting the
delivery reservoir to the delivery port within the housing,
allowing solution to flow out of the delivery port only, and a
controller contained within said housing. The controller provides
(i) a power source operatively connected to the electrodes for
applying across the electrodes, a voltage potential effective to
pump electrolyte solution from the supply to delivery reservoir, at
a selecting pumping rate, and (ii) a timer for controlling the
duration and timing of application of the voltage potential to the
electrodes, to pump a selected volume of solution through said
membrane, at selected time intervals.
[0006] The housing may be adapted for implantation at a body site,
and the solution may contain a therapeutic drug for delivery at
said site. The voltage applied to the electrodes may be, for
example, in the 1-5 volt range, and the channels may have a minimum
dimension in a selected range between about 2 and 30 nm. The timer
is preferably designed to apply across the electrodes, a pulsed
voltage whose duration is effective to pump a selected volume of
supply solution across said membrane.
[0007] B. Nanopump Device for Detecting DNA Sequences
[0008] The invention also includes a device for detecting a target
nucleic acid sequence contained in a solution of solution of
nucleic acid fragments and having a selected pH. The device
includes a chamber, and a membrane disposed in said chamber and
having a channel extending between an upstream chamber region
adapted to hold the electrolyte solution of such different-length
fragments, and a downstream chamber region adapted to hold an
electrolyte solution, where said channel has a selected minimum
cross-sectional dimension in the range between 2 and 100 nm and a
net surface charge within a given pH range that includes the
selected solution pH. Attached to a wall portion of the channel, is
a capture nucleic acid having a sequence complementary to the
target sequence. Upstream and downstream electrodes disposed in the
upstream and downstream chamber regions, respectively, are in
contact with electrolyte solution placed in the corresponding
chamber regions.
[0009] A controller in the device includes a power source
operatively connected to the electrodes for applying a selected
voltage potential across the channel, to move individual nucleic
acid sequences contained in the solution through the channel, where
the sequences can hybridize to complementary target sequences bound
to the channel wall portion.
[0010] The controller may further include a voltage regulator for
regulating the voltage applied across said channel, to effect
selective release of the target sequence from the capture nucleic
acid, based on the degree of complementarity between the target
sequences and the capture nucleic acid.
[0011] The membrane may include an array of channels, each having
attached to a wall portion thereof, a capture nucleic acid with a
selected sequence complementary to a selected one of a plurality of
different sequences. In this embodiment, one of the electrodes may
include a plurality of electrode elements, each associated with one
of said membrane channels, and the controller may be operatively
connected to each of the electrode elements to apply and regulate
the voltage applied across each of the channels. The controller may
be effective to place across the electrodes, a voltage potential
effective to move nucleic acid fragments electrophoretically
through said channel. The channel(s) in the membrane preferably
have a minimum dimension in the 2-25 nm range.
[0012] Also disclosed is a method for detecting a target nucleic
acid sequence contained in an electrolyte solution of nucleic acid
fragments and having a selected pH. The method includes placing the
solution in a chamber having a membrane disposed therein, the
membrane having a channel extending between an upstream chamber
region adapted to hold the electrolyte solution of such
different-length fragments, and a downstream chamber region adapted
to hold an electrolyte solution, where the channel (i) has a
selected minimum cross-sectional dimension in the range between 2
and 100 nm, (ii) a net surface charge within a given pH range that
includes the selected solution pH, and (iii), attached to a wall
portion of the channel, a capture nucleic acid having a sequence
complementary to the target sequence. There is applied a voltage
potential sufficient to move nucleic acid sequences in the solution
through the channel, where the sequences can hybridize to
complementary target sequences bound to the channel wall portion.
Captured target sequences are released from the channel by applying
a voltage potential across the channel effective to dissociate
hybridized nucleic acids, and released target sequences are
detected.
[0013] The releasing step may include applying a voltage across the
channel effective to selectively release target sequences from the
capture nucleic acid, based on the degree of complementarity
between the target sequence and the capture nucleic acid.
[0014] The membrane may include an array of channels, each having
attached to a wall portion thereof, a capture nucleic acid with a
selected sequence complementary to a selected one of a plurality of
different sequences. In this embodiment, one of the electrodes
includes a plurality of electrode elements, each associated with
one of the membrane channels. The controller in this embodiment is
operatively connected to each of the electrode elements to apply
and regulate the voltage applied across each of the channels. The
releasing step may include applying a voltage across each of said
channels, individually, a voltage potential effective to
selectively release target sequences from the capture nucleic acid,
based on the degree of complementarity between the target sequence
and the capture nucleic acid.
[0015] These and other objects and features of the invention will
be more fully appreciated when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates the pH dependence of charge on a silicon
surface;
[0017] FIG. 2 illustrates the pH dependence of charge on a silicon
surface derivatized with amine groups;
[0018] FIG. 3 illustrates the principle of electro-osmotic flow in
a channel having charged surface groups;
[0019] FIG. 4 shows, in simplified view, a nanopump constructed in
accordance with the invention;
[0020] FIG. 5 shows plots of flow rates in the nanopump of the
invention as a function of applied voltage, for various pore
sizes;
[0021] FIG. 6 illustrates electroosmotic flow principles in a
nanopore pump;
[0022] FIGS. 7A-7D illustrate steps in the production of a nanopore
filter used in the nanopump of the invention;
[0023] FIGS. 8A and 8B illustrate the operation of a nanopump
filtration device constructed in accordance with the invention;
[0024] FIGS. 9A and 9B illustrate the operation of a nanopump
drug-delivery device constructed in accordance with the
invention;
[0025] FIGS. 10A and 10B illustrate the construction of a nanopore
opening formed by two nanopore planar channels;
[0026] FIG. 10C shows key elements in a nanopore device constructed
in accordance with the invention for determining DNA fragment
lengths;
[0027] FIG. 11 illustrates elements of a DNA sequence detecting
device constructed in accordance with the invention; and
[0028] FIG. 12 illustrates electrokinetic forces acting on a DNA
molecules bound to a wall surface in the SNP-sequence detecting
device.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention, called a "nanopump", is a fluid pump
comprising a collection of parallel arrays of multiple channels
which, in their smallest dimensions are in the approximate range of
5 nm-100 nm. The channels, e.g., rectangular or U-shaped channels,
are fabricated from materials such as silicon and silicon oxides,
polymers, or metals, such as titanium, the surfaces of which
exhibit a net surface charge when exposed to electrolyte solutions
at appropriate pH levels. Under the influence of an electrical
field applied across the array, electroosmotic flow is induced,
pumping aqueous subject matter from one side of the array to the
other.
[0030] By controlling the geometry of the multiple-channel arrays,
the individual channel width, surface charge density and polarity,
together with controlling the array thickness and electrolyte
properties, the flow properties can be tailored for highly specific
applications.
[0031] A. Theory of Operation
[0032] The nanopump is based on electroosmotic flow. Electroosmotic
flow is a basic physical-chemical phenomenon, but has special
properties when it is applied to materials such as silicon or
silicon oxides. A further examination of silicon (Si) surface
chemistry is required to ensure that the basic process is well
described.
[0033] The chemical state of the silicon surface can be either in
an oxidized form or oxide-free, bare silicon, terminated by Si-H
groups. The silicon surface, after a wet cleaning step and a final
oxidative step, is hydrophilic. The thickness of this SiO.sub.2
layer is between 0.6 and 2.0 nm, its nominal value depending on
oxidation conditions and the measurement techniques used. This
chemical oxide, often referred to as "native" oxide, forms a
passivation layer with a dangling-bond defect density in the range
of 10.sup.12 cm.sup.-2 at the Si/SiO.sub.2 interface. The defect
densities reported are about two orders of magnitude higher than
for thermal oxides. Recent studies suggest that the quality of the
native oxides is strongly dependent on the applied chemistry in
which they are formed, and that higher-quality oxides can be
obtained with alternate chemistries such as D1 water/ozone.
[0034] Wet-chemically grown oxides are hydrophilic in contrast to
thermal oxides. This difference is caused by the way the oxygen
atom is bound to the silicon on the surface. Thermal oxides are
characterized through the formation of siloxane rings, which are
very stable against hydrolysis. Wet-chemically grown oxides are
generally covered with surface hydroxyl groups (Si--OH) called
silanol groups, and are very similar in their behavior with respect
to silica gels. Geometrical considerations and chemical
measurements indicate an average surface density of around five
hydroxyl groups per nm.sup.2, but this number can typically range
from 2-12 hydroxyl groups per nm.sup.2. It is important to note
that not all hydroxyl groups formed on a surface are chemically
equivalent owing to structural differences in their coordination,
but in general the surface hydroxyl groups on a hydrous oxide have
donor properties similar to those of their corresponding
counterparts in solution, the hydroxides.
[0035] FIG. 1 illustrates the dissociation of silanol(-) groups,
such as groups 16, at the surface 18 of a silicon substrate 20, at
low and high pH extremes. The adsorption of metal ions and protons
can be understood as competitive complex formation with
deprotonated surface groups (Si--O--) which behave like Lewis
bases. This means that the adsorption of species on a hydrous oxide
surface of Si can be compared with complex formation reactions in
solution. However, the extent of adsorption also depends strongly
on the surface charge of the oxide i.e., the number of hydroxyl
groups and the degree of dissociation and on the pH of the
solution. Silanol groups are completely ionized at pH levels above
9 (right in FIG. 1), creating a negative surface potential. Below
pH 4 (left in FIG. 1) the silanol groups are protonated and the
surface 20 is virtually neutral. Between these two pH extremes, the
surface becomes progressively more deprotonated, and negatively
charged, with increasing 5 pH.
[0036] FIG. 2 shows a silicon substrate having a portion of its
silyl groups are modified to amino groups, such as groups 24.
Methods for derivatizing a glass or silicon surface with amino
groups are well known. A hydrolytically stable amino-silica glass
coating material can be applied on the inner surfaces of nanopump
channels in order to create a surface which, depending on the pH of
the medium, can be either positively or negatively charged.
Aminopropyltriethoxysilane, or a similar silane reagent, is used to
introduce primary amino groups onto the silica surfaces.
[0037] Following such treatment, as illustrated in FIG. 2, the net
charge at the surface of the coating material depends on the degree
of protonation of the amino groups and the degree of ionization of
the silanol groups, thus enabling manipulation of the magnitude and
direction of the electroosmotic flow (EOF). At lower pH (at levels
somewhat below pH factor 6.0), the coating bears a net positive
charge, which results in an electroosmotic flow from the cathode
toward the anode and minimizes the wall-solute interactions of
basic species. At higher pH (at levels somewhat above pH factor
6.5), the coating surface bears a net negative charge and the
coated nanopore behaves like an uncoated one, having an EOF in the
cathodic direction. Such an amino-silica glass coating is extremely
stable under both acidic and basic conditions.
[0038] Electroosmotic flow is also influenced by addition of
certain organic bases to a running buffer. For example, addition of
N,N,N',N'-tetramethyl-1,3-butanediamine (TMBD) in the running
electrolyte effects electroosmotic flow and the migration behavior
of basic proteins in bare fused-silica capillaries. Depending on
the electrolyte pH (4.0, 5.5 and 6.5, respectively) and additive
concentration the electroosmotic flow can be either cathodic or
anodic. A similar Langmuirian-type dependence of the electroosmotic
flow on the concentration of TMBD in the running electrolyte was
found at the three experimented pH values, which may be indicative
of the specific adsorption of the additive in the immobilized
region of the electric double layer at the interface between the
capillary wall and the electrolyte solution.
[0039] B. Electroosmotic Flow
[0040] Most surfaces, including silicon as noted above, obtain a
surface electric charge when they are brought into contact with
electrolyte solutions. This surface charge influences the ion
distribution in the polar medium forming the electric double layer.
Gouy and Chapman modeled the region near the surface as a diffuse
electrical double layer (EDL), where they equated the non-uniform
ion distribution to the competing electrical and thermal diffusion
forces. Stern later presented the basis for the current model, in
which the Stern plane splits the EDL into an inner, compact layer
and an outer, diffuse layer.
[0041] In the inner layer, also known as the Stern layer, the
geometry of the ions and molecules strongly influences the charge
and potential distribution, with the Stern plane located near the
surface at roughly the radius of a hydrated ion. The inner layer
between the surface and the Stern plane is considered to be
immobile. When the ions are within the Stern plane, thermal
diffusion will not be strong enough to overcome electrostatic, or
Van der Waals forces and they will attach to the surface to become
specifically adsorbed.
[0042] In the outer diffuse layer, the ions are far enough away
from the surface that they are mobile. Electrokinetic transport
phenomena such as electroosmosis can be understood in terms of the
surface potential at the surface of the shear (approximately at the
Stern plane), known as the zeta potential (.xi.), because these
phenomena are only directly related to the mobile part of the
EDL.
[0043] Because of the EDL, the net charge density (.sigma..sub.e)
within the diffuse layer is not zero. If an electric field is
applied along the length of the channel, a body force is exerted on
the ions in the diffuse layer of the EDL. The ions will move under
the influence of the applied electrical field, pulling the liquid
with them and resulting in electroosmotic flow. The fluid movement
is carried through to the rest of the fluid in the channel by
viscous forces. This electrokinetic process is called
electroosmosis.
[0044] FIG. 3 represents anodal electroosmotic flow induced through
a channel 26 filled with an electrolyte solution 28. The channel is
lined with negatively charged ionized silanol groups, such as
groups 30. Potential difference is established by placing
electrodes 32, 34 at opposite ends of channel. Since fluid motion
is initiated by the electrical body force acting on the ions in the
diffuse layer of the EDL, electroosmotic flow depends not only one
the applied electrical field but also the net local charge density
in the liquid.
[0045] Prior studies of EDL and electroosmotic flows are limited to
systems with simple geometries such as cylindrical capillaries with
circular cross sections and slit-type channels formed by two
parallel plates. However, for the channels embodied in the present
invention, and in other fluidic devices produced by micro-machining
techniques, the cross-sectional shape is close to planar. In such a
situation, the EDL field is two-dimensional and will influence the
two-dimensional flow field in the rectangular microchannel.
[0046] B. Pump Structure
[0047] FIG. 4 is a pictorial representation of a nanopump 38
constructed according to the present invention. The pump includes a
nanopore filter or membrane 40 providing a plurality of
rectangular-shaped (substantially planar) parallel nanochannels,
such as microchannels 42, each channel connecting a donor or supply
reservoir 44 to receiver or recipient reservoir 46. The nanopump is
fabricated from silicon-based materials using the techniques
described herein. A plurality of ports 48 feed donor reservoir 44
and a second plurality of ports 50 draw excess from the receiver
reservoir 46. A pair of electrodes 52, 54 are used to apply a
potential difference across the membrane are suitably connected to
a power source.
[0048] FIG. 5 is a graph that depicts the relationship between
electrolyte flow rate and voltage applied to nanopumps with
different channel widths. In the preferred embodiment, a nanopore
membrane is placed between two chambers. This structure promotes
electroosmotic flow when a potential difference is applied across
the membrane. The resultant flow rate is related to the porosity of
the membranes and to the size of the channel. Unexpectedly, optimal
flow rates are observed at a channel dimension of about 20 nm.
Greater channel dimensions, e.g., 27 and 49 nm, exhibit lower flow
rates. The data in FIG. 5 were obtained using channels with
poly-silicon and crystalline silicon material sidewalls with a pore
geometry that is about 45 .mu.m in length, about 20 to 40 nm in
width, and about 5 .mu.m in depth.
[0049] FIG. 6 illustrates the relationship between channel width
and flow rate. This phenomenon may be partially related to the
increased proportion of the overall channel volume occupied by the
diffuse ionic double layer as the channel width is decreased. Flow
rate is also influenced by electrolyte properties including pH,
ionic strength and ionic species.
[0050] In operation, the reservoirs in the pump are filled with an
electrolyte solution whose pH is effective to impart a net charge,
e.g., net negative charge, to the walls of the nanopore membrane.
The power source is then activated to apply a selected voltage
potential across the electrodes, producing electro-osmotic pumping
across the membrane from the supply to the recipient reservoir.
[0051] C. Fabrication Process
[0052] The nanopump is created through a microfabrication process
using bulk and surface micromachining. In the preferred embodiment,
the microfabricated nanopump comprises a surface-micromachined
array of channels on top of an anisotropically etched silicon wafer
that provides mechanical support. The selection of channel pore
size (the minimum channel dimension) is in 5-100 nm range, e.g., 5,
10, 20, 30, 50 or 80 nm, preferably 10-30 nm pore size.
[0053] To reach a desired pore size in the tens of nanometers
range, strategies have been developed based on the use of a
sacrificial oxide layer sandwiched between two structural layers.
This process is discussed in the co-pending patent application
incorporated herein by reference. Because the flow rate of a
nanopump varies according to the material being pumped, it is
important to tailor new nanopumps for specific applications as
discussed above.
[0054] A nano-channel is formed by sandwiching a SiO.sub.2
sacrificial layer, the thickness of which determines the nominal
pore size, between a polysilicon structural layer and the silicon
wafer. For this design, increasing the number of entry holes
maximizes the flux. Phosphate buffered saline (PBS) fluxes as high
as 1.0 mL/cm2-hr have been attained for membrane filters with 30
nm-sized pores. Filtration tests showed greater than 99% of 100
manometer beads (actual log reduction of greater than 5) were
retained with 50 nanometer pores.
[0055] FIGS. 7A-7D are pictorial representations of the
cross-section of a wafer after successive stages in the
microfabrication process used to create nano-channel arrays. The
nano-channel arrays are microfabricated from silicon and silicon
nitride (Six,Ny) architectures. The critical dimension (i.e.,
width) of the nanopump channels will be defined by the thickness of
sacrificial silicon oxide films 60, a parameter that can be
controlled to sub-nanometer resolution. The fabrication process
allows for dense arrays of nano-channels, thus improving the
utility of these arrays for pumping applications. The fabrication
process is summarized here for review. A detailed and specific
discussion of this fabrication process is presented, for example,
in U.S. Pat. Nos. 6,044,981, 5,985,328, 5,985,164, 5,948,255,
5,928923, 5,798,042, 5,770,076, and 5,651,900, all of which are
incorporated herein by reference.
[0056] As a first step, and with reference to FIG. 7A, a structural
polysilicon layer 56 is deposited over a silicon nitride layer 58
(an etch-stop layer) and etched with the nanopore mask. Following
this, a sacrificial oxide layer 60 (FIG. 7B) is grown using thermal
oxidation to define the nanopump array thickness. Thermal oxidation
gives thickness control to sub-nm resolution across in entire 4"
silicon wafer.
[0057] A second polysilicon structural layer 62 is then deposited
over the pores and planarized to allow access to the nanopump
channels from the front face of the array. A silicon nitride
protective layer is then deposited and etch holes, such as hole 64
are opened on the backside of the wafer. The bulk silicon is
removed through these etch holes up to the etch stop layer of the
array, giving the structure shown in FIG. 7C.
[0058] The structure is then released by etching the protective
nitride layers and the sacrificial oxide layers in a concentrated
HF bath. In particular, sacrificial layer 60 is etched in the
region between entry holes and the lower opening 64, producing
defined-size nanopore channels 68 (FIG. 7D). Surface modification
of the polysilicon structure reverts it to a hydrophilic surface,
thus making it useful as a filter for bio-fluids.
[0059] D. Molecular Filtration and Separation
[0060] Many clinical and research applications involve separation
of specific biological molecules, such as nucleic acids and
proteins, for further analysis and characterization.
[0061] FIGS. 8A and 8B show the basic elements of a nanopump based
separator device 70 constructed in accordance with the invention.
The device generally includes a separation chamber 70 having
upstream and downstream ends 72, 74, respectively, and a plurality
of membranes 76, 78, 80 that partition the chamber into a plurality
of separation regions 82, 84, 86, and 88, including an upstream
chamber region 82 and a downstream chamber region 88. Each
membrane, such as membrane 76, is a nanopore filter of the type
described above, and has a selected channel size for filtering
macromolecules, e.g., proteins or nucleic acid fragments, of
selected sizes.
[0062] The device also includes electrodes 90, 92 disposed in the
upstream and downstream chamber regions, respectively, and a power
source 94 connecting the two electrodes. Each chamber region may
further include a side inlet and outlet (not shown) for removing
liquid contained in each chamber region.
[0063] In operation, a mixture of macromolecules or other solute
species and, optionally, small particles, such as virus particles
or colloids or liposomes, in an electrolyte are placed in the
downstream chamber regions, and the other chamber regions are
filled with a suitable electrolyte solution, in particular, one
whose pH supports a net charge on the membrane wall surfaces. By
applying a selected voltage across the electrodes, liquid in the
each chamber is pumped in a downstream direction across the
immediately downstream membrane. At each membrane, macromolecules
that are larger than the membrane pore size will be retained in the
upstream channel region, and those that are smaller will pass
through into the downstream channel region.
[0064] FIG. 8A shows the device with a sample 96 of different-sized
macromolecules in an electrolyte solution placed in upstream
channel region 82, and a suitable electrolyte solution placed in
each of the upstream channel regions. When a voltage of appropriate
polarity and field strength is applied across electrodes 90, 92,
sample 96 is drawn first through membrane 76, which discriminates
between the largest macromolecules (or particles) in the sample and
all other sample species. Similarly, as solution in the next
upstream channel region 84 migrates is pumped through membrane 78,
this membrane blocks passage of macromolecules of a certain size,
and passes others, producing a successively greater concentration
of smaller-sized macromolecules at each upstream channel region. At
the end of the filtration process, the macromolecules contained in
each channel region, including the upstream-most region, can be
removed, e.g., by flowing a lateral stream of electrolyte through
each channel region.
[0065] The system of stacked nanopumps described above may be
modified for performing the specific isolation and purification of
a particular protein or oligonucleotide from a complex mixture or
crude cell extract rapidly and precisely using electroosomotic
flow. For specific protein separation, the surfaces of the silicon
nanopump channels can be chemically modified to contain covalently
linked ligands with strong and specific affinity to their
conjugate. The chemistry of silanization is well established and
can be easily modified for specific ligand attachment for use to
capture a variety of enzyme families.
[0066] For specific oligonucleotide isolation and purification,
separate nucleic acid oligonucleotides complementary to the nucleic
acid sequence intended for capture can be synthesized relatively
inexpensively using traditional synthesis methods. These capture
oligonucleotides could then be covalently linked to the surfaces of
the nanopump channels using established methodology and can be used
to capture any specific oligonucleotide of interest.
[0067] Most importantly, this device allows for the simple and
rapid purification of multiple specific biomolecules at the same
time. Each nanopump level can be `programmed` to collect a specific
molecule in a complex mixture by simply by inserting the correct
nanopump `module` primed with the appropriate protein capture
ligand or nucleic acid oligonucleotide. In simple terms, this
device is capable of performing numerous molecular isolations from
one sample with one simple electroosmotic run that is both
user-customizable and scalable.
[0068] In summary, the proposed device has several advantages over
current laboratory methods for rapid and precise separation,
purification and isolation. With the use of nanopump array modules,
exact size-exclusion fractionation can be performed on multiple
complex samples in less time and with precise resolution. Also,
specific purification and isolation for a variety of molecules can
be recovery efficiency. Finally, this device can perform the
separation, filtration, and affinity purification of biomolecules
by simply switching user-customizable modules, eliminating the need
for multiple expensive electrophoretic and liquid chromatographic
equipment.
[0069] E. Implantable Drug-Delivery Device
[0070] FIGS. 9A and 9B illustrate an implantable drug delivery
device 100 constructed according to the present invention, and
shown at initial and half-spent stages of operation, respectively.
The device generally includes a housing 102 having a delivery port
104. The housing is formed of a suitable biocompatable material
that allows its placement at an implantation site in a body.
[0071] Contained in the housing is a supply reservoir 108, a
nanopump 110, constructed in accordance with the invention, and a
one-way check valve 112 that allows one-way flow of solution
contained in reservoir 108 from the pump to the exit port, via a
channel 103. The reservoir, pump and valve are all part of a
continuous flow system, as shown. Also shown is a vent structure
114 that includes a vent 116 and a plunger 118 disposed within
reservoir 108.
[0072] As solution is pumped out of the reservoir through exit port
104, the reservoir space is progressively filled by movement of
plunger 118 toward the pump, as indicated in FIG. 9B, with the
reservoir volume upstream of the plunger being filled progressively
by fluid available at the implantation site.
[0073] The nanopump is activated by means of a pair of electrodes
120, 122 disposed on either side of a nanopore membrane 125 in the
pump, and constructed in accordance with the invention. More
particularly, electrode 122 is contained within an upstream supply
reservoir 124 in the pump, and electrode 120, within a downstream
recipient reservoir 126 in the pump. Application of a voltage
potential across the two electrodes is effective to pump
electrolyte drug solution in a downstream direction, in accordance
with the invention.
[0074] The power source in the device, for applying a potential
across the pump electrodes, is a battery pack indicated at 130. The
battery pack has a voltage of preferably 1-5 volts. Activation of
voltage from the battery pack to the electrodes is through a timing
control unit 132 which is a small processor designed to produce an
activation signal for a selected signal duration, e.g., 1-5
seconds, at selected time intervals, e.g., every 4 or 8 hours.
[0075] In operation, the device, including the supply reservoir and
the nanopump, is filled with an electrolyte solution, typically a
drug solution having a selected drug concentration. Before
implantation, the control unit is adjusted to produce desired
metered amounts of drug solution, e.g., p1 to n1 amounts, at
selected time intervals, e.g., every eight hours. The loaded,
programmed device is then implanted at an internal body site, e.g.,
at a subcutaneous site, to deliver a metered amount of drug
solution at selected time intervals.
[0076] Power requirements to induce electroosmotic flow are to be
low due to the relatively porosity provided by the multiple
parallel channel arrays. Moreover, the same geometry favors fluid
flow. Exquisite on-off control, and thus control of drug input rate
and pattern, is provided by the selected switching circuitry. For
example, pulse, square wave and continuous patterns are achievable
simply by controlling the length of time the switch 127 is closed.
As described, having a miniaturized timer in the circuitry provides
a means of providing "on-board" intelligence. Alternatively, the
switch may be closed magnetically. Thus external control is
possible by simply placing a magnet close to the skin surface,
providing a drug input rate and pattern controlled by the patient
himself, or his caregiver.
[0077] F. Sizing Nucleic Acid Fragments
[0078] The nanopump described herein can be adapted, in accordance
with another aspect of the invention, to perform oligonucleotide
length analysis using a rapid, one-step process that potentially
limits complex sample preparation. For this application, a silicon
micromachined membrane that contains an array of nano-sized pores
with dimensions of approximately 2 nm is fabricated. An electrical
potential across the pore is used to assist in moving
oligonucleotides from the sample through the pores. The pore
geometry is selected to allow single oligonucleotides to proceed
through the pore in a linear fashion (i.e. end-to-end).
[0079] A separate sensing array allows for monitoring the pore
blockage by nucleic acid molecules as a function of time. For
example, examining the change in electrical potential across the
pore as the oligonucleotide moves through it can provide a measure
of fragment length (i.e. number of base pairs). It is common in the
study of neurobiology and biophysics to form biological membranes
or planar lipid membranes (such as lipid bilayer films) and to
introduce nano-sized channels. These channels can be made to stay
open for extended periods of time. Kasianowicz et. Al, Proc Nat
Acad Sci (USA), 93(24):13770 (1996) reasoned that applying a
transmembrane voltage could cause polyanionic oligonucleotides to
flow through a membrane channel as an extended linear chain. When
these molecules were present in the channels, they could be
detected as a reduced or blocked level of normal ionic flow
measured as reduced ionic current. Measurement of the time and
magnitude of such blockages was demonstrated as a method of
recording the molecular length, and other characters, of the
particular molecule.
[0080] In the case of the nanopump envisioned here, the biological
membrane is replaced with a membrane containing nanopores that are
fabricated using silicon micromachining, as described above, but
where two "planar" channels placed end to end and at right angles
with respect to each other, to form a one-dimensional (ID), pore
(i.e. channel) shaped like a rectangular parallelepiped. The pore
geometry can be designed with precise and selectable nano-sized
channel widths in the 2-50 nm size range. The pore height and pore
length can also be designed with microsized dimensions in the 1-50
nm size range.
[0081] A diagram of a typical 2D nanopore channel 128 is shown in
FIG. 10A. A selected number of pores, with the typical shape shown
in, are normally fabricated into a planar, membrane-like structure,
as described above.
[0082] In order to determine strand length the DNA strand must
traverse a man-made nano-sized pore described above in a linear
fashion. This is accomplished, in accordance with one embodiment of
the invention, by using a second 2D pore 130 located adjacent and
perpendicular to the first 2D pore as seen in FIG. 10B. Using this
design, the intersection 132 between the microfabricated pores can
have nano-size dimensions in both planes and a nano-sized
pore-length so it more closely approximates a 1D (that is, linear
as opposed to planar) biological pore.
[0083] Native DNA consists of one long molecule that makes up a
chromosome. It is a two-stranded spiral (double-helix) that
includes about 3 billion nucleotides arranged in subunits called
base pairs. A segment of DNA carrying genetic instructions, or
gene, is approximately 100,000 base pairs long. To perform many DNA
analytical experimentation (e.g. sequencing, Southern
hybridization) the long DNA molecule is usually digested into
smaller, single-stranded fragments that typically contain <500
base pairs (FIG. 12). The diameter, D, of a single-stranded, 100
base pair oligonucleotide is approximately 2 nm and the length, L,
is approximately 200 nm. In the present invention, by measuring the
exact time necessary for the passage of one molecule through the
channel, it is possible to correlating this time with the length of
the molecule. In this manner, exact oligonucleotide length can be
established.
[0084] The construction of an electronic device 131 for measuring
fragment length, based on the above principles, is shown FIG. 10C.
In this case, a silicon micromachined, 1D nano-sized pore is
created as discussed above, at the intersection of two
channel-forming substrates 128, 130. The size of this pore, shown
at 132, is S.sub.p.times.S.sub.p, measuring 2 nm.times.2 nm. The
pore's channel length, L.sub.p, is selectable within a range of 5
to 50 nm.
[0085] Electrodes 134 (E1), 136 (E2) located in buffer solutions
can be placed on both sides of the nanopore membranes or attached
to the surfaces of the silicon membrane at a point near the pore
location, as shown in the figure. When the device includes an array
of such pores, an array of surface-attached electrodes can used to
monitor the ion current passing through more than one pore. Surface
metalization of silicon doping methods, common to the
microelectronic industry, can be used to form single electrodes or
electrode arrays on the surface of the silicon. That is, multiple
pores can be created and each pore can be monitored by a separate
set of E1 and E2 electrodes. Another option to multiple-pore
monitoring is to have a common E1 electrode and an array of E2
electrodes. Using multiple pores will allow for a more rapid data
acquisition rate and a more extensive analysis.
[0086] Another advantage to a microfabricated nano-pore membrane
channel is that a third electrode 138 (E3) can be added to the
channel architecture, providing the advantage of using a fixed
voltage of common E1 and E2 electrodes for pumping through the
channel, while monitoring ion current using an array of E3
electrodes. The E3 electrode must be on the order of 5 nm
thickness, and may be created by doping the silicon itself to
increase its conductivity using methods common in the
microelectronic industry.
[0087] G. DNA Sequence Analysis
[0088] A basic design of a nanopump array intended for DNA sequence
analysis, in accordance with another aspect of the invention, is
given in FIG. 11. The central element 140 includes a series of
nanopump channel arrays, such as arrays 142, 144, micromachined out
of a single silicon wafer.
[0089] The nanopump array layer is sandwiched between upper and
lower microfabricated layers 146, 148, respectively, upper layer
146 is fitted with discrete "donor" reservoirs, such as reservoirs
150, 152, which align above and are continuous with one side of the
individual nanopumps. Each of the upper donor reservoirs is fitted
with an electrode, such as electrodes 154, 156 in reservoirs 150,
152, respectively. A similar reservoir layer 148 is aligned below
the nanopump layer, but in the case of this lower layer, the
individual receiving reservoirs, such as reservoirs 158, 160, are
also connected via channels, such as channel 162, 164, to a
"common" receiving reservoir 166.
[0090] An optical flow-cell 168 is included in the channel leading
to the receiving reservoir and a fluorescence optical detection
system is positioned to measure the fluorescence signal of the
fluid flowing through this common channel. Each of the three layers
is aligned and sealed so that each nanopump is continuous with the
corresponding donor and receiving reservoirs. The reservoirs and
connecting channels are filed (primed) with electrolyte solution
prior to use.
[0091] After fabrication and assembly, the surfaces of the channels
are chemically grafted with reactive chemical groups (such as
primary amino or thiol groups) using standard silane reagents.
Alternatively the channels may be linked with a ligand such as
avidin (or its binding partner/receptor, biotin). Capture DNA
sequences are subsequently grafted to the surface directly or
through avidin/biotin interactions. For attachment, the capture
sequences are added to the common receiving reservoir and a voltage
difference is applied between the common reservoir (anode) 167 and
any or all of the individual donor reservoirs (cathodes). The DNA
sequences migrate under the influence of the electric field from
the common reservoir through the chemically modified (or ligand
modified) nanopump channels, into the donor reservoirs. The
polarity may be periodically reversed (cycled) to optimize the
chemical grafting of the capture DNA to the nanopump channel
surfaces as they pass. After capture DNA sequence grafting, the
reservoirs and channels are washed by passage of buffer throughout
the system.
[0092] Samples of fluorescent-labeled DNA probes of known sequence
are added to the individual donor (upper) reservoirs. Current is
passed through all the nanopumps in the array (anode in donor
reservoirs, cathode in receiving reservoirs). As the probe DNA
sequences pass through the nanopump channels, hybridization occurs.
Again, cycling of the electrode polarity between the donor and
receiving reservoir, and thus reversing the electrophoretic
movement of the DNA probes, may be used as a strategy to promote
optimal hybridization to capture DNA sequences bound to the
nanopump channel surfaces.
[0093] Dehybridization of probe and capture DNA sequences is
controlled by a combination of applied current (electrophoretic
force, EF) and electroosmotic flow (tangential fluid flow force,
TFFF). Importantly, these two forces may be in the same or opposite
directions and may be applied as a gradient or pulsed. For example
in the case of negatively charged DNA molecules (DNA is highly
negatively charged by virtue of charged phosphate groups in each
residue of the backbone structure), EF will always be in the
direction of the cathode. Electroosmotic flow, and hence the TFFF,
may be either cathodal or anodal, depending on the net surface
potential at the plane of fixed charges on the nanopore surfaces.
By adjusting and fine tuning EF and TFFF, the selectivity of
denaturation of hybridized DNA will be improved, thus allowing for
the discrimination of dehybridization of probe and capture DNA with
fewer mismatched base pairs. SNP may be detected in this fashion
with greater precision relative to EF alone. In the nanopump array
configuration illustrated in FIG. 11, dehybridization would be
expected to occur as the circuit is closed between the individual
donor reservoirs and the common receiving reservoir. Electronic
circuitry would be designed so that a spectrum of voltages, voltage
pulses or perhaps cycles of polarity reversals, would be applied in
sequence between individual donor reservoirs and the common
receiving reservoir. When dehybridization occurs, the passage of
the fluorescent-labeled probe DNA is detected by the optical
system. Probe sequence identity is assured by energizing one donor
reservoir at a time. Conditions under which the probe and capture
DNA sequences dehybridize (voltage, and perhaps temperature and
ionic strength) would be recorded, correlated with the probe
sequence identity and related to the degree of mismatches base
pairs. The flow-through configuration inherent in the nanopump
design may also permit temperature adjustments and ionic gradients
to control de-hybridization.
[0094] The combined effect of electrophoretic force and
electroosmotic fluid flow provided by the nanopump design may
improves the resolution of detachment of target probe DNA sequences
from capture sequences (FIG. 12). Such a system may resolve either
detachment (dehybridization) of probes containing mismatched base
pairs (i.e., base-pair mismatch analysis) and/or different DNA
chain lengths with same degree of hybridization.
[0095] The flow through feature of the nanopump array also obviates
the need for reversing polarity to drive away non-specific analytes
or nonreacted molecules. Buffer is simply flowed across bound probe
layer to wash.
[0096] The flow through feature also permits changing the
temperature and ionic strength of the running buffer up-stream of
the nanopump array. This approach may permit the use of temperature
gradients (in range of melting temperature, T.sub.m of duplex DNA)
and/or ionic gradients in addition to electrophoretic forces and
electroosmotic flow to refine dehydridization conditions and thus
improve resolution of mismatched base pairs.
[0097] Although the invention has been described with respect to
particular embodiments and examples, it will be appreciated that a
variety of modifications and changes may be made without departing
from the claimed invention.
* * * * *