Molecular sieve abstract
A device for utilizing a non-gel self-assembled nano-feature array
molecular sieve for analyzing molecules is provided. The molecular
sieve device comprises an ordered array of self-assembled nano-features
which function as a molecular sieve to separate molecules based
on a suitable characteristic. A system for integrating the non-gel
ordered self-assembled nano-feature array molecular sieve of this
invention into a device for separating molecules based on a characteristic
and a method for separating a wide range of molecules using the
non-gel ordered self-assembled nano-feature array molecular sieve
of the invention are also provided.
Molecular sieve claims
What is claimed is:
1. An electrophoretic device for producing a mobility distribution
in a sample containing a plurality of molecules comprising: a non-gel
self-assembled nanofeature array sieve having proximal and distal
ends, the sieve comprising a substrate made of a first material
and a plurality of self-assembled nanofeatures made of a second
material wherein each nanofeature has a center point, a cross-sectional
dimension and an outer surface and wherein the nanofeatures are
fixedly attached to the substrate in a an ordered periodic array
such that the distance between the center points of two adjacent
nanofeatures defines a regular lattice spacing and the distance
between the outer surfaces of two adjacent nanofeatures defines
a pore size for the sieve and wherein the pore size of the sieve
is chosen such that a molecule of the sample can be transported
through the sieve at a characteristic velocity, the ordered periodic
array of nanofeatures being defined by a plurality of catalytic
germination spots deposited in a corresponding ordered periodic
array on the substrate, each of the nanofeatures being anchored
to and self-assembled from a separate one of the plurality of catalytic
germination spots; a molecular reservoir in fluid communication
with the proximal end of the sieve for introducing the molecules
into the sieve; a detector arranged at the distal end of the sieve
such that the molecules passing out of the sieve are detected and
a signal generated; an electrical field generator for producing
an electric field in field communication with the sieve, the reservoir
and the detector such that the electrical field induces the molecules
in the reservoir to move through the sieve to the detector; and
a monitor in signal communication with the detector to communicate
the signal to a user.
2. The electrophoretic device according to claim 1 further comprising
a sieve body having proximal and distal ends and defining an internal
volume and wherein the sieve is confined within the internal volume.
3. The electrophoretic device according to claim 1 one of the
substrate or sieve body is transparent.
4. The electrophoretic device according to claim 1 wherein the
sieve body is made of a material selected from the group consisting
of silicon, alumina, glass and plastic.
5. The electrophoretic device according to claim 1 wherein the
substrate is made of a material selected from the group consisting
of silicon, alumina, glass and plastic.
6. The electrophoretic device according to claim 5 wherein the
substrate is further coated with a metal selected from the group
consisting of gold, aluminum and titanium.
7. The electrophoretic device according to claim 5 wherein the
metal is anodized Al.
8. The electrophoretic device according to claim 1 wherein the
catalytic germination spots are made from a material selected from
the group consisting of Ni, Co or a Ni/Co alloy.
9. The electrophoretic device according to claim 1 wherein the
nanofeatures have a uniform size.
10. The electrophoretic device according to claim 1 wherein at
least two differently dimensioned nanofeatures are disposed on the
substrate surface.
11. The electrophoretic device according to claim 1 wherein the
nanofeatures have a cross-sectional dimension of about 10 to 100
nm.
12. The electrophoretic device according to claim 1 wherein the
array has a lattice spacing of about 10 to 100 nm.
13. The electrophoretic device according to claim 1 wherein the
array has a pore size of about 10 to 100 nm.
14. The electrophoretic device according to claim 1 wherein the
substrate has an area of about 1 mm.sup.2 to 1 cm.sup.2.
15. The electrophoretic device according to claim 1 wherein the
periodic array is a uniform periodic array having a uniform lattice
spacing between nanofeatures.
16. The electrophoretic device according to claim 1 wherein the
periodic array has at least two different lattice spacings.
17. The electrophoretic device according to claim 1 wherein the
periodic array is a staggered periodic array.
18. The electrophoretic device according to claim 1 wherein the
nanofeatures are made of carbon.
19. The electrophoretic device according to claim 1 wherein the
sieve body forms at least one channel.
20. The electrophoretic device according to claim 1 wherein the
sieve body forms a plurality of parallel channels.
21. The electrophoretic device according to claim 1 wherein the
detector is a laser induced fluorescence spectrometer.
22. The electrophoretic device according to claim 1 wherein the
detector comprises a binary supergrating optic.
23. The electrophoretic device according to claim 22 wherein the
detector comprises a photodiode array comprising a plurality of
spatially separated photodiodes.
24. The electrophoretic device according to claim 1 wherein the
detector comprises a CCD camera.
25. The electrophoretic device according to claim 1 wherein the
electrical field generator is a pair of electrodes.
26. The electrophoretic device according to claim 1 wherein the
electric field is a constant DC field having a DC field vector and
a field strength.
27. The electrophoretic device according to claim 26 wherein the
electric field further comprises a pulsed field having a pulsed
field vector angled transverse to the DC field vector.
28. The electrophoretic device according to claim 26 wherein the
field strength is about 10 to 10000 V/cm.
29. The electrophoretic device according to claim 1 wherein the
molecules have a size of about 3 bp to 100 Mbp.
30. The electrophoretic device according to claim 1 wherein the
molecules are DNA or RNA.
31. The electrophoretic device according to claim 1 wherein the
molecules are labeled with a dye.
32. The electrophoretic device according to claim 1 wherein the
reservoir comprises one device selected from the group consisting
of an agarose gel, an electrokinetic injector and a positively charged
gold wire.
33. The electrophoretic device according to claim 1 wherein the
nanofeatures are chemically functionalized.
34. The array sieve according to claim 1 wherein the nanofeatures
are grown by self-assembly on the substrate.
35. The array sieve according to claim 1 wherein the nanofeatures
are nanotubes.
36. The array sieve according to claim 1 wherein the characteristic
velocity of the molecules is size dependent.
37. A method of separating molecules in a sample by size dependent
mobility comprising utilizing an electrophoretic device according
to claim 1.
38. A method of separating a plurality of molecules in a sample
comprising the steps of: providing a non-gel self-assembled nanofeature
array sieve having proximal and distal ends, the sieve comprising
a substrate made of a first material and a plurality of self-assembled
nanofeatures made of a second material wherein each nanofeature
has a center point, a cross-sectional dimension and an outer surface
and wherein the nanofeatures are fixedly attached to the substrate
in an ordered periodic array such that the distance between the
center points of two adjacent nanofeatures defines a regular lattice
spacing and the distance between the outer surfaces of two adjacent
nanofeatures defines a pore size for the sieve and wherein the pore
size of the sieve is chosen such that a molecule of the sample can
be transported through the sieve at a characteristic velocity, the
ordered periodic array of nanofeatures being defined by a plurality
of catalytic germination spots deposited in a corresponding ordered
periodic array on the substrate, each of the nanofeatures being
anchored to and self-assembled from a separate one of the plurality
of catalytic germination spots; introducing the sample into the
proximal end of the sieve; applying power to an electrical field
generator in field communication with the sieve such that an electric
field having a field vector and a field strength is projected from
the proximal to the distal end of the sieve such that the sample
is induced to move from the proximal to the distal end of the sieve;
and detecting the distribution of the sample at the distal end of
the sieve and communicating the distribution to a user.
39. The method according to claim 38 wherein the characteristic
velocity of the molecules is size dependent.
40. The method according to claim 38 wherein the nanofeatures are
nanotubes.
Molecular sieve description
FIELD OF THE INVENTION
The present invention is directed to a self-assembled nano-array
molecular sieve for the separation of molecules.
BACKGROUND OF THE INVENTION
This invention relates in general to self-assembled nanometer-scale
arrays used as molecular sieves in the separation of molecules by
differential transport through the array, and in particular to self-assembled
carbon nanotube arrays used as electrophoretic sieves for DNA sequencing
and separation of biological molecules comprising a self-assembled
carbon nanotube array arranged on a substrate and an electromagnetic
field generator for applying a potential across the array sieve
producing a characteristic mobility in the molecules.
Electrophoresis is the predominant technique for separating DNA
fragments obtained from restriction maps of complete genomes (millions
of base pairs long) and for large-scale sequencing projects, like
the Human Genome Initiative. Electrophoresis has also become an
essential tool for clinical chemistry applications. In conventional
electrophoretic sieves, electrophoretic separation occurs by differential
transport of polyelectrolytes, such as, DNA molecules and proteins,
through a medium or device in the presence of an electric field.
The medium acts as a sieve, producing a size-dependent mobility
in the molecules. In slab gel electrophoresis, the sieving medium
is provided by a slab gel of agarose or polyacrylamide polymers,
which contain nanometer-size pores. For example, an agarose gel
is made by placing agarose into solution with a suitable solvent
and the pore size depends on the concentration of agarose in solution
according to the equation:
where a is the pore size and A is the concentration of the agarose
in (g/mL). Pore size for an agarose gel with A.about.0.1 to 1.0
g/mL is in the range of 100 to 500 nm.
Polyacrylamide polymers are produced through a polymerization reaction
of acrylamide and methylenebisacrylamide. By controlling the conditions
of the reaction, such as acrylamide concentration and the degree
of cross-linking, the pore structures thus formed can be reproducibly
controlled and can have pore sizes as small as a few nanometers.
Because of the small pore size, polyacrylamide gel electrophoresis
(PAGE) is the method of choice for separating small DNA strands
(<1000 bases) for DNA sequencing or genetic mutation studies,
such as cancer detection or toxicology.
Despite the wide-range of pore sizes available, and the well-documented
reproducibility of the conventional gel electrophoresis techniques,
severe throughput limitations, size limitations and the need for
cleaner, hardier and more user-friendly technologies have led a
number of researchers to look for ways to improve the automation
and rapidity of electrophoretic techniques. For example, sequencing
speed is limited in gel electrophoresis because the electric fields
used to push the molecules through the sieve must be kept low to
avoid Joule heating of the gel, which could cause degradation of
the gel material. In addition, gels are not very durable, requiring
constant replacement which results in the further requirement for
extra-plumbing and reservoirs to allow facile replacement of the
gel adding to both the size and complexity of the gel-electrophoresis
equipment.
One recent advance has been capillary electrophoresis (CE). In
capillary electrophoresis, a small diameter capillary acts as the
sieve for the molecules. CE has gained widespread popularity because
the small-scale CE sieve structures allow for the facile dissipation
of excess heat, which in turn allows for the use of higher electric
fields resulting in a reduction in sequencing time. However, CE
systems still contain gels and would thus not be appropriate for
extended periods of use, because of the added complexity of storing
replaceable gels and injecting them periodically in the micro-channel
structure.
Hybridization techniques have offered some promise for rapid separation.
However, technical issues in data collection, such as low signal-to-noise
ratios and analysis, such as computationally intensive combinatorial
analysis have prevented hybridization techniques from becoming the
standard in DNA analysis.
More recently, researchers at Princeton have introduced micro-fabricated
arrays as artificial sieving structures to replace polymer-based
sieves. These artificially fabricated arrays have several advantages
compared to both the polymer gels and the capillaries including:
1) the possibility of using ultra-high fields enabling higher speed
separation and real-time monitoring of DNA samples; 2) the use of
a non-viscous medium leading to higher durability and which in turn
could possibly lead to the development of permanent sieves; 3) the
flexibility and controllability of the configuration of the sieving
structure would allow for analysis over a broad range of molecular
sizes, from simple DNA fragments to full chromosomes, and the production
of devices on a very large scale for parallel processing; 4) because
these structures can also be fabricated with extremely regular sized
and spaced sieve features, the separation resolution should be improved
leading to further miniaturization of the electrophoresis device,
including the possibility of an electrophoresis analyzer on a chip;
and 5) because the sieves can be built from inert substances analysis
can be made of a variety of biomolecules.
One example of a micro-fabricated array formed using ion-beam lithography
was disclosed by Duke et al., in Electrophoresis, vol. 18 pages
17-22 (1997), incorporated herein by reference. Duke et al. produced
a periodic array of pillars 100 nm in diameter and 100 nm apart
and demonstrated the ability to differentiate the electrophoretic
mobility of DNA molecules between 7.2 and 43 kilobases (kb). Other
disclosures of lithographically produced arrays for the use in electrophoresis
devices are described in U.S. Pat. Nos. 5110339 and 5837115
both of which are incorporated herein by reference.
While this method clearly shows promise for providing nano-scale
array sieves for separating DNA molecules having several thousand
base pairs, the lower limit for array features made using such lithographic
techniques is about 100 nm, indicating that smaller DNA molecules
cannot be separated. Moreover, due to the time-intensive nature
of e-beam lithography, it is not suitable for the economical fabrication
of large dense arrays of with pore sizes approximately below 50
nm. This in turn limits the application of such sieves to DNA separation
but not sequencing, which requires the separation of DNA molecules
as small as a few base pairs long. In order to provide separation
of molecules of up to 600 bases, inter-post separations of approximately
15 to 30 nm will be needed. Moreover, despite the progress made
in recent years in developing new techniques to produce smaller
and smaller features via lithographic techniques such as ion beam
or electron beam lithography, it is clear that increasingly costly
efforts are being required to sustain the progress. While deep UV
and X-ray lithography offer hope of incremental improvements in
resolution, lithographic and patterning techniques for reproducibly
producing features at the 10 to 30 nm level are essentially nonexistent
at the present time. The resolution limit of currently available
patterning technologies make it apparent that entirely new approaches
will be needed to sustain the rapid progress that has characterized
the last few decades of semiconductor technology development.
One novel approach to making nanometer-scale structures utilizes
self-assembly of atoms and molecules to build up functional structures.
In self-assembled processing, atom positions are determined by fundamental
physical constraints such as bond lengths and angles, as well as
atom-to-atom interactions with other atoms in the vicinity of the
site being occupied. Essentially, self-assembly uses the principles
of synthetic chemistry and biology to "grow" complex structures
from a set of basic feedstocks. Utilizing such techniques molecular
motors have been synthetically produced containing fewer than 80
atoms. Two relatively simple examples of self-assembled structures
formed using chemical vapor deposition (a process commonly used
in thin film deposition, including crystal-growth) include carbon
fullerenes ("buckyballs") and carbon nanotubes. In addition,
dense arrays of carbon nanotubes have been grown on surfaces and
such arrays have been utilized as nano field emitter arrays, atomic
force microscopy probes, nanoscale transistors, actuators, high-Q
mechanical resonators and a variety of sensors.
While fabrication processes based on self-assembly at the nanometer
scale have a number of advantages over the conventional lithography
techniques, including avoiding the blanket film depositions, lithography,
and subtractive processing characteristic of conventional nano-scale
manufacturing, until recently there has been no method based on
self-assembly for fabricating uniform nano-arrays of the type necessary
to build an instrument for electrophoretic separation. Instead,
the nano-arrays formed have been disordered on a fine scale with
uncontrolled spacing between individual nanotubes and a significant
spread in nanotube diameters.
Recently, a technique has been developed that relies on self-assembly
to produce geometrically regular nanotube arrays with excellent
uniformity. This process is based upon the self-organizing formation
of highly uniform pore arrays in anodized aluminum films. First,
a nanochannel alumina structure is formed by anodizing an aluminum
film under conditions that lead to hexagonally-ordered arrays of
narrow channels with very high aspect ratios. The nanochannel alumina
structure is then used as a template for the growth of nanotube
arrays of carbon and other materials, including metals and some
semiconductors. The full process technique is disclosed in Appl.
Phys. Lett., vol. 75 pg 367 (1999), and is incorporated herein
by reference. Utilizing this technique the authors were able to
grow nanotube arrays comprising uniform carbon nanotubes with a
diameter of 32 nm. Despite the promise of this new technique, there
have been no attempts to adapt the technology to grow array structure
capable of being used as an electrophoretic sieve for analyzing
and sequencing DNA and RNA.
Accordingly, a need exists for a durable non-gel sieving device
capable of analyzing a wide range of molecular sizes under the influence
of ultra-high electric fields and capable of being produced on a
large scale for parallel processing.
SUMMARY OF THE INVENTION
The present invention is directed to a device and system for utilizing
a non-gel self-assembled nano-array molecular sieve for analyzing
biomolecules. In one particular embodiment this invention utilizes
a non-gel ordered self-assembled array of nano-features that functions
as an electrophoretic sieve to separate biomolecules. This invention
is also directed to systems for integrating the non-gel ordered
self-assembled nano-array sieve of this invention into a device
for separating biomolecules. This invention is also directed to
novel methods for separating a wide range of biomolecules using
the non-gel ordered self-assembled nano-array sieve of the invention.
In one embodiment, the self-assembled nano-array of the present
invention is incorporated into an electrophoretic micro-device comprising
a self-assembled nano-array according to the present invention,
a field generator for applying an electric field across the sieve,
and a detector. The self-assembled nano-array sieve is designed
to differentially transport polyelectrolytes, such as DNA molecules,
proteins, etc., through the sieve in the presence of an electric
field to produce a characteristic distribution of the introduced
molecules. The nano-array is placed in line-of-sight with the detector
such that as the molecules exit the sieve, they flow through the
optical detection region of the detector. The detector then analyzes
the exiting molecules such that the identity of the exiting molecule
can be determined.
In one embodiment, the self-assembled nano-array sieve comprises
a substrate having a periodic array of features such that in the
presence of an appropriate feedstock the atoms of the feedstock
self-assemble on the ordered features of the substrate to produce
an ordered array of nano-features having non-random alignment and
size distribution. In one embodiment the size, shape and pattern
of the self-assembled nano-array features grown on the substrate
are adapted such that molecules within a specified size range can
be separated. The substrate wafer is preferably made of a substance
that reacts with the feedstock to produce the self-assembled structures.
In another embodiment the nano-array features self-assemble into
nanotubes having a specified diameter and height suitable for use
in the sieve of the current invention.
In an alternative embodiment, the substrate is made of a semiconductor
such as, for example, oxidized silicon or aluminum oxide, coated
with a metal catalyst film such as, for example, Ni or Co. In this
embodiment, the silicon can be further doped to adjust the electronic
properties of the substrate surface.
In another alternative embodiment, the self-assembled nano-array
sieve is confined in a channel and the outer surface of the array
optionally covered with a cap layer so as to enclose the nano-array
sieve such that the DNA molecules are confined within the sieve
during separation. In such an embodiment, either the cap layer or
the substrate must be transparent to light such that optical detection
schemes can be utilized to analyze the molecular distribution. In
this embodiment, reservoirs can be integrated into the channel and
cap to provide an entrance point for the molecules to be tested.
In another alternative embodiment, the nano-array features are
self-assembled from an inert material such as, for example, carbon
utilizing a carbon feedstock gas such as, for example, acetylene.
In yet another alternative embodiment, the detector comprises a
laser induced fluorescence system such as, for example, a laser
diode emitter in conjunction with a binary superimposed grating,
or conventional optics and a photomultiplier tube.
In still another alternative embodiment, the electrophoresis system
of the present invention is utilized in combination with a DNA sequencer.
In such an embodiment, the DNA strands would be labeled with a specific
dye for each of the chain-terminating dideoxynucleotides and a binary
superimposed grating in conjunction with four spatially separate
optical detectors would be utilized to determine the identity of
the tested molecules. In this embodiment, preferably the carbon
nanotube array would have a pore size of about 15 nm and the molecules
would have a size up to about 1000 bases long.
In still yet another alternative embodiment, the invention is directed
to a system for the detection of substances comprising multiple
sieve paths and detectors as described above, such that parallel
processing of molecules can be carried out.
In still yet another additional embodiment, the invention is directed
to a method for separating molecules based on molecular size. The
method comprises analyzing molecules introduced into the self-assembled
nano-array sieve as described above.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features and advantages of the present invention
will be better understood by reference to the following detailed
description when considered in conjunction with the accompanying
drawings wherein:
FIG. 1 is a schematic view of an embodiment of a nano-array sieve
according to the invention.
FIG. 2 is a schematic view of an embodiment of a nano-array sieve
in operation according to the invention.
FIG. 3a is a schematic view of an embodiment of a process for forming
a nano-array sieve according to the invention.
FIG. 3b is a schematic view of an embodiment of a process for forming
a nano-array sieve according to the invention.
FIG. 3c is a schematic view of an embodiment of a process for forming
a nano-array sieve according to the invention.
FIG. 3d is a schematic view of an embodiment of a process for forming
a nano-array sieve according to the invention.
FIG. 4 is an SEM image of an embodiment of a nano-array sieve according
to the invention.
FIG. 5 is a schematic view of an embodiment of a molecular sieve
device according to the invention.
FIG. 6 is a schematic view of an embodiment of a molecular sieve
device according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a molecular sieve instrument
comprising a non-gel self-assembled nano-scale periodic array sieve
for discriminating molecules based on a characteristic mobility.
As shown in FIG. 1 the molecular sieve 10 comprises a substrate
12 having proximal 14 and distal ends 16 and an array 18 of self-assembled
nano-features comprising an ordered repeating pattern of nano-features
20 arranged on the substrate 12 of the molecular sieve 10. In operation,
an electric field 22 is applied across the length of the array 18
from the proximal 14 to the distal 16 end such that molecules 24
introduced onto the array 18 surface move through the array 18 to
a detector (not shown) in a time dependent on a characteristic of
the molecule 24 such as, for example size, charge, chemical inertness,
etc.
Although in the embodiment shown in FIG. 1 the self-assembled
array 18 of nano-features comprises periodic rows of uniform nano-features
20 attached to the substrate 12 any other arrangement of nano-features
20 suitable to function as a molecular sieve 10 can be utilized
such as, for example, staggered arrays of uniform nano-features
or uniform rows of nano-features having alternating sizes. In another
alternative embodiment the spacing of the nano-features 20 is varied
to produce a gradient gel for which sieving properties vary spatially.
Additionally, while FIG. 1 only shows a molecular sieve 10 deposited
on a flat substrate surface 12 any geometry of substrate 12 suitable
for use as a molecular sieve 10 could be utilized, such as, for
example, a curved, corrugated or tubular substrate. It will be understood
that the design of the molecular sieve 10 according to the present
invention is necessarily driven by the molecules 24 to be separated
as the sieving ability of the molecular sieve 10 is sensitively
dependent on both the geometrical size, shape and spacing of the
nano-features 20 as well as the size and properties of the molecules
24. The molecular sieve 10 itself has two characteristic sizes:
(1) the nano-feature size and (2) the nano-feature spacing, or pore
size. Using an iterative process, the nano-features 20 can be optimized
to separate molecules 24 of a specified size or other physical property,
then other parameters can be selected to optimize operation, such
as, for example, electric field 22 strength, detector and the mechanical
properties of the sieve body. Accordingly, although the self-assembled
molecular sieve 10 of the embodiment shown in FIG. 1 comprises a
uniform array 18 of uniform cylindrical nano-features 20 it should
be understood that any shape, size or spacing of nano-features 20
as described above, can be utilized in the molecular sieve 10 of
the current invention such that the pore size and sieving properties
of the array 10 are suitable for use as a separation device for
the molecules 24 of interest.
For example, a sieve must possess specific properties to separate
double strand DNA (dsDNA) and single strand DNA (ssDNA) molecules.
The DNA molecule generally appears in its dsDNA configuration. The
dsDNA molecule can span a wide variety of sizes, ranging from a
few kilo-base-pairs (kbp) to many Mbp, such as, for example, chromosomes.
To provide sequencing capabilities, on the other hand, the molecular
sieve 10 must be able to process ssDNA molecules which are produced
by Maxam-Gilbert or Sanger sequencing reactions. In contrast to
the dsDNA molecule, the ssDNA molecule used for sequencing is typically
no more than 1000 bases long. Accordingly, the design of a molecular
sieve 10 intended to separate either form of DNA requires that the
conformational behavior of ssDNA and dsDNA under electrophoresis
be known. This behavior, in turn, strongly relies on the physical
properties of the array 18 and sieve 10. For example, if the gaps
or pores between the nano-features 20 in the arrays 18 are smaller
than 30 nm, pulsed electrophoresis utilizing a pulsed electric field
22 which requires the bending of DNA, may not be feasible because
of the minimum bending diameter of dsDNA (D.sub.b.about.30 nm).
Also, the value of the electric field 22 E compared to the electric
field E.sub.0 required to balance the thermal motion of Kuhn segment
will influence the stretching configuration of the molecules 24.
The relaxation time, or Rouse time T.sub.R, associated with the
molecules may also impose a limit on the design dimensions of a
molecular sieve 10 for sequencing. Table 1 below, compares the
physical properties of the ssDNA and dsDNA molecules.
TABLE 1 Properties of dsDNA and ssDNA Property ssDNA dsDNA Nucleotide
length (n) n bases n base pairs Persistence length (p) .about.0.75
nm .about.50 nm Kuhn length (b) 1.5 nm (.about.3 bases) 100 nm (.about.300
base pairs) # Kuhn segments (N) .about.n/3 .about.n/300 DNA contour
N .multidot. b N .multidot. b length (L) .about.500 nm (n.about.1
kb) .about.30 .mu.m (n.about.100 kbp) Random coil gyration .about.N.sup.3/5
.multidot. b .about.{1/3 .multidot. p .multidot. L .multidot. radius
(R.sub.g) [1 - p/L + p/L .multidot. e.sup.-L/p ]}.sup.1/2 .about.50
nm (n.about.1 kb) .about.700 nm (n.about.100 kbp) Minimum bending
.about.0.50 nm .about.30 nm diameter (D.sub.b) Charge/Kuhn -1 e
-50 e length (q) Friction 8 .multidot. 10.sup.-12 kg/s 4 .multidot.
10.sup.-10 kg/s coefficient/Kuhn segment (.zeta.) Electrophoretic
2 .multidot. 10.sup.-8 m.sup.2 /s .multidot. V 2 .multidot. 10.sup.-8
m.sup.2 /s .multidot. V mobility (.mu..sub.0 = q/.zeta.) Electric
field 10.sup.5 V/cm 50 V/cm strength to balance thermal motion at
T.about.25.degree. C. (E.sub.0 = k .multidot. T/q .multidot. b)
Time associated 10.sup.-8 s 10.sup.-3 s with Brownian motion of
Kuhn segment at T.about.25.degree. C. (.tau. = .zeta. .multidot.
b.sup.2 /k .multidot. T) Rouse time 10.sup.-5 s 1 s (T.sub.R = .tau.
.multidot. N.sup.2 /(3 .multidot. .pi..sup.2)) (n.about.1 kb) (n.about.100
kbp)
A schematic diagram of the mechanism of separation in a molecular
sieve 10 is shown in FIG. 2. Three DNA molecules 24a, 24b, and 24c,
are pictured at even time intervals (t1 to t4), as they migrate
through a row of nano-features 20 in a molecular sieve 10. The longest
molecule 24c is most hindered by the interaction with the nano-features
20 and is consequently the last to arrive at the next row. The reason
for this demonstrated behavior is that molecules 24 having different
sizes relative to the pores between the nano-features 20 in the
array sieve 10 interact with the nano-features 20 differently. For
randomly coiled molecules whose gyration radius is smaller than
the pore size 26 of the self-assembled molecular sieve 10 such
as for molecule 24a, sieving, also called Ogston-sieving, occurs
and the migration speed is dependent on the gyration radius of the
molecule 24a. For larger strands (such as the DNA molecules 24b
and 24c), the molecules need to uncoil to fit through the pores,
as shown at t2. To overcome this limitation, the electric field
22 can be increased to allow stretching of the molecules 24 to occur.
Under the influence of the electric field 22 the molecule 24b then
reptates though the pores with snake-like movement and the migration
speed is dependent on molecular size up to a given size. However,
there is an upper limit to the effectiveness of uniform array sieves.
For example, at higher electric fields or for longer strands, molecules
wrap themselves around the nano-features 20 as shown for molecule
24c at t2 with both sides of the molecule 24c hanging on either
side of the nano-feature 20 under the tension of the electric field
22. In molecular sieve 10 such as that shown in FIG. 1 molecules
24 slide or hook around the nano-features 20 as in a pulley system,
thus freeing the molecules 24. This episodic "hooking",
unlike reptation or Ogston sieving, does not segregate between molecular
sizes in uniform arrays, and does not appear to be an effective
sieving mechanism. Likewise, at very high fields, DNA molecules
24 are fully-stretched and slide through the nano-features 20 unperturbed
because of the regularity of the sieve 10 (a phenomenon absent in
gels because of the randomness of the sieving structure). Accordingly,
if a sieve 10 is made having gaps, or pores, 26 between nano-features
20 of 15 nm, the maximum length of the molecule 24 that would obey
the Ogsten sieving regime would be n.about.50 bases, while for molecules
24 longer than 50 bases, reptation or even hooking might occur.
Such physical limitations can be overcome by using staggered arrays,
as shown in FIG. 2 or by pulsing a second electric field 28 at a
field vector angled in relation to the vector of the driving electric
field 22. Accordingly, the design of the molecular sieve 10 as well
as the operating conditions of the electric field 22 will depend
on the size and size range of the molecules 24 to be separated.
While the self-assembled nano-arrays 18 contemplated in many of
the embodiments discussed in the present application are constructed
of carbon nanotube nano-features 20 made from pyrolizing an acetylene
feedstock over a substrate having an ordered array of germination
points, the nano-features 20 can be of any shape and made by any
process and from any material suitable for making self-assembled
nano-features, such as, for example, spheres or pyramids made of
other atomic materials or even biomolecules, such as, for example,
proteins. In another embodiment, the nano-features 20 are further
functionalized for a variety of applications, such as, for example,
being made hydrophilic or hydrophobic, being charged either negatively
or positively, or being derivatized with specific chemical groups,
etc.
The substrate 12 can be made of any material which can withstand
the temperatures required for growth of the nano-features 20 and
which can be modified to provide a suitable ordered array of germination
points for growing the nano-features 20 of the array 18 such as,
for example, metallized Si oxide wafers, anodized alumina, glass,
or even polymeric plastics. In turn, any suitable catalyzing metal
can be used to activate the germination points on the surface of
the substrate 12 such as, for example, nickel or cobalt. Alternatively,
the catalyzing metal could be an alloy of two or more metals such
as a Co/Ni alloy. The metal catalysts could also be produced by
pyrolysis of inorganic or organic metal-containing compounds, such
as, for example, Ferric Nitrate or Cobalt Chloride.
One possible process for forming the molecular sieve 10 of the
present invention comprising nanotube nano-features 20 utilizing
a chemical vapor deposition (CVD) technique is shown schematically
in FIGS. 3a to 3d. In a first step, as shown in FIG. 3a, a substrate
12 such as, for example, silicon is coated with a high purity metal
film 30 such as, for example, Al. The metal is then anodized in
a multistep process to produce germination points or pores 32 in
the highly ordered sieve array 18 in the metal oxide surface 30
as shown in FIG. 3b. Next, a small amount of catalytic material
34 such as, for example, Co or Ni is electrochemically deposited
in the bottom of the pores 32 to serve as a catalyst for carbon
nanotube nano-feature 20 growth, as shown in FIG. 3c. Finally, as
shown in FIG. 3d, the nanotube nano-features 20 are self-assembled
by pyrolysis of a suitable feedstock, such as, for example, acetylene
gas diluted in nitrogen and/or ammonia at about 650.degree. C. and
the nanotube nano-features 20 then partially exposed by etching
the metal oxide film 50 in a mixture of phosphoric and chromic acids.
The anodized surface 30 and the catalyzed pores 32 serve as a template
such that the nanotube nano-features 20 self-assembled in an ordered
array 18 corresponding to the pores 32 in the anodized metal substrate
30. A detailed description of this method for the self-assembled
growth mechanism of a well-ordered self-assembled carbon nanotube
array is found in, Appl. Phys. Let. vol. 73 page 3845 (1998), which
is incorporated herein by reference.
FIG. 4 is an SEM micrograph of a self-assembled nano-array 18 comprising
nanotube nano-features 20 produced by this process. As shown, utilizing
this technique, nanotube nano-features 20 of uniform length and
cross section and with only a 5% spread in diameter can be grown
in very regular hexagonal arrays of parallel nanotubes. Moreover,
the relatively low vapor deposition growth temperature makes the
technique compatible with basic Si processes. Furthermore, because
the pore diameter is proportional to the anodization voltage, the
pore size can be controlled from 10 nm up to several hundred nm
with nano-feature areal densities as high as 10.sup.11 cm.sup.-2.
Voltage control of the nanochannel diameter also offers the possibility
of optimizing the nano-feature diameter for sieving molecules with
different baselengths.
Determination of an appropriate shape, size and spacing of self-assembled
nano-features 20 for a specific molecular sieve 10 can be determined
via any traditional theoretical electrodynamic calculation method.
The description and modeling of polyelectrolytes (e.g., DNA, RNA
and almost every protein) is difficult due to the competing interaction
of short and long range interactions, which introduce new length
scales and new degrees of freedom to the well understood concepts
of neutral polymer physics. Techniques must take into account both
the interplay of various length scales inherent to systems of polyelectrolytes
and also in the different regimes of concentrations of chains, but
also the crucial role of screening interactions of polyelectrolytes
by the counter-ions and salt ions in solution.
Suitable simulations may employ potential functions for the short-range
interactions, such as, for example, Lennard-Jones field, multibody
interactions (Morse-type potentials), bonded interactions and for
the long-range electrostatic interactions. Such molecular dynamics
simulations may use any combination of conventional algorithms,
such as, for example, (a) the Nose-Hoover algorithm for constant
pressure and constant temperature systems; (b) optimized or non-optimized
interpolation Particle-Particle-Particle-Mesh and multiple algorithms
with tree data structures for the treatment of the long range electrostatic
potentials in periodic and non-periodic systems; and (c) efficient
domain decomposition techniques that allow for efficient parallelization
for short-range forces. Multiple steps for the integrations can
also be utilized to model separate time scales. Simple multibead-spring
models, which can be mapped onto realistic polymer and/or polyelectrolytes
can be utilized as approximations to investigate chain conformations
of polymer blends and polyelectrolyte solutions in various concentration
regimes and in solvents of different quality such that the counter-ions
and salt ions can be taken into account. Some examples of conventional
modeling techniques that may be utilized include: the non-equilibrium
molecular dynamics (NEMD) technique and the non-equilibrium Brownian
dynamics (NEBD) technique. NEMD is a convenient and precise method
to investigate the response of a complex fluid to an external, e.g.
flow field. The NEMD technique is based on the numerical solution
of Newton's equations of motion for many particle systems whose
intermolecular potentials are given. NEBD and the "equivalent"
Fokker-Planck equations treat the dissipative part of the NEMD motions
in a less precise, but very efficient way.
In one embodiment, as shown in FIG. 5 the self-assembled molecular
sieve 10 is incorporated in an electrophoretic instrument 40 which
comprises a substrate 42 a sieve body 44 having proximal 46 and
distal 48 ends and sieve walls 50 enclosing an internal volume 52
a non-gel self-assembled periodic nano-array sieve 54 arranged within
the internal volume 52 an electric field generator 56 in field
communication with the array sieve 54 a molecular reservoir 58
in fluid communication with the proximal end 46 of the sieve body
44 and a detector 60 in signal communication with the distal end
48 of the sieve body 44.
In operation, molecules 62 are placed into the molecular reservoir
58 and power is supplied to the electric field generator 56. The
application of power to the electric field generator 56 produces
an electric field 64 having a specified charge vector and strength.
This electric field 64 is applied across the sieve 54 from the proximal
46 to the distal 48 end of the sieve body 44 such that the polyelectrolyte
molecules 62 stored in the molecular reservoir 58 at the proximal
end 46 of the sieve body 44 are induced to move through the sieve
54 from the proximal 46 to the distal end 48 of the sieve body 44
to the detector 60 under the influence of the electric field 64
at a rate dependent on a characteristic of the molecule 62 such
as, for example, size. The detector 60 is arranged with the end
of the sieve 54 such that molecules 62 passing out of the sieve
54 are analyzed by the detector 60. Utilizing this technique, sequencing
with a 0.1% length resolution can be performed with self-assembled
nano-array sieves 54 in a time of 100 seconds over a 10 cm sieve
length. For separation based on conformation, full separation can
be performed in seconds over a 1 cm array sieve 54.
The sieve body 44 itself can be made of any material suitable for
micromachining utilizing standard lithographic or MEMS techniques
to enclose the array sieve 54 such as, for example, aluminum oxide
or silicon. In a preferred embodiment, the sieve body 44 further
comprises a cap layer 66 which can be of any design such that the
cap layer 66 makes intimate contact with the array sieve 54 while
maintaining a uniform separation from the existing substrate 42.
Such a cap layer 66 could be made of any suitable material, such
as, for example, aluminum oxide or silicon. Such a cap layer 66
could be formed by any conventional MEMS process, such as growth
or deposition over a sacrificial layer (not shown) deposited to
encapsulate the self-assembled nano-array sieve 54 wherein the sacrificial
layer is subsequently removed to expose the self-assembled nano-array
sieve 54 itself. Alternatively, the cap layer 66 and channel 68
could be formed in a single deposition step with the self-assembled
nano-array sieve 54 being embedded into the boundaries of the channel
68 such that alternative diffusion paths are avoided during electrophoresis.
In a more preferred embodiment, one of the substrate 42 the cap
layer 66 or sieve walls 50 is transparent such that an optical
detector 60 can be used to interrogate the array sieve 54.
Although the sieve body 44 shown in FIG. 5 comprises a single sieve
54 confined within a narrow channel 68 any geometry of sieve channels
68 can be utilized. One alternative embodiment is shown in FIG.
6 which shows a multiple channel sieve array 54 in which the array
channels 68 are aligned in parallel on a single substrate 42 such
that multiple or parallel processing of molecular samples can be
carried out at one time. In this embodiment, multiple molecular
reservoirs 58 are utilized for introduction of the molecules 62
to the sieve body 44. A single electric field generator 56 is utilized
to apply a uniform across the entire array sieve 54. It should be
understood that while parallel and single array channels 68 are
shown in FIGS. 5 and 6 any suitable alternative geometry of channels
68 may be utilized such as, for example, folded channels may be
used to increase the length of the sieve path 54 without increasing
the size of the sieve body 44. Although the embodiment shown in
FIG. 6 discloses a multi-channel array in which the sieve channels
68 are separated by sieve walls 60 the multi-channel array could
alternatively comprise a single "sheet" of nano-array
without walls between the array channels 68. In such an embodiment,
the array channels 68 would be determined by the way the loading
of the molecules 62 is performed because under the influence of
the electric field 64 no significant lateral diffusion or intermixing
of the samples should occur. For example, if loading can be achieved
over a width of 0.5 mm, a substrate 12 1 cm on a side could hold
10 array channels 68 while if the array channel 68 width is only
50 .mu.m, up to 96 array channels 68 could be included on a single
substrate 12. Such an embodiment could be used to develop an electrophoresis
device on a chip for mobile detection and analysis of samples. In
such an embodiment a portable power source (not shown) would also
be integrated into the device 40.
Although the above embodiments comprise open reservoirs 58 into
which molecules 62 are injected, the molecules 62 can be introduced
into the nano-array sieve 54 by any conventional technique dependent
only on the narrowness of the array channel 68. For example, the
molecules 62 could be stored in a thin slab of agarose gel which
could then be positioned next to one of the edges of the array sieve
54. In such an embodiment, the molecules 62 would be released by
application of the electric field 64. Alternatively, the apparatus
could include means to inject molecular samples in narrow bands
directly into the nano-array sieve 54. One potential technique is
the gold wire technique in which a narrow strip of gold is used
as a "starting line". Molecules 62 are bound to the gold
by biasing the strip with a positive voltage. When the bias is switched
off, the molecules instantaneously migrate away from the strip in
a thin band. Another potential technique is electrokinetic injection,
which allows for the introduction of single molecules into a sieve.
This technique is disclosed in a number of published articles, such
as, for example, J. Chromatog. A., vol. 894 pg. 191-201 (2000),
which is incorporated herein by reference.
The electrical field generator 56 can comprise any suitable generator
such that an electromotive force is applied to the molecules 62
across the length of the sieve 54 via an electric field 64 such
as, for example, positive and negative electrodes 70 in electrical
contact with the proximal 46 and distal 48 ends of the sieve body
44. In such an embodiment, the electrodes could be made of any suitable
conducting material, such as, for example, Al, Au or Ag. While a
steady, uniform electric field 64 is contemplated in the embodiment
described above, any suitable electric field 64 can be utilized,
such as, for example, DC fields and pulsed fields. Additionally,
any suitable electric field strength can be utilized such that sufficient
separation of molecules can be obtained within a suitable timeframe.
In a preferred embodiment, the field strength of the electric field
64 is between about 10 and 10000 V/cm.
The detector 60 can comprise any suitable detection means such
that the molecules 62 exiting the sieve can be monitored, such as,
for example, a laser fluorescence spectrometer comprising a standard
488-nm laser source arranged such that the beam from the laser interrogates
the distal end 48 of the array sieve 54 and the resulting fluorescence
is collected by standard optics and photomultiplier tubes. Although
the standard laser-induced fluorescence detector described above
can be utilized for general separation and mobility analysis, in
an alternative embodiment, a full DNA sequencing detector can be
integrated into the device, utilizing four-color coding of the Sanger
products. In such an embodiment, as shown schematically in FIGS.
5 and 6 the detector would comprise a light source 72 a binary
superimposed grating 74 for multi-wavelength diffraction and a four
element photodetector array 76. Alternatively, a single molecule
detection technique for the imaging of individual molecules 62 could
be utilized. In this embodiment a standard epi-fluorescence microscope
with mercury lamp excitation and an inexpensive charge-coupled device
(CCD) camera or alternatively an image intensified camera can be
utilized to obtain time-resolved images. Suitable cameras are sold
commercially by a number of manufacturers, such as, for example,
the I-PentaMAX Gen IV Intensified CCD Camera sold by Roper Scientific.
In a preferred embodiment of the sequencing detector 60 described
above, the light source 72 is a blue semiconductor light source
emitting at a wavelength of 488 nm, the binary grating 74 is a binary
superimposed grating composed of an array of equal sized pixels,
each having two values in the index of refraction, spatially patterned
according to a preprogrammed specific algorithm such that fluorescence
emissions from the molecules 62 excited by the light from the light
source 72 are separated based on the wavelength of the emission.
In operation, during a Sanger reaction, the DNA molecules 62 are
labeled with a specific dye for each of the four reactions in the
presence of a chain-terminating dideoxynucleotides. In this manner,
a particular dye is associated with a particular base (Adenine,
Thymine, Cytosine or Guanine). Accordingly, when light from the
light source 72 impinges on the molecules 62 an emission with a
wavelength based on the specific chemistry of the dye will be emitted.
The emission from the molecules 62 is then focused on the binary
grating 74 which separates the four different wavelengths of the
emission light and focuses the emission onto one of four spatially
separated detectors 76. The output of each detector 76 is then monitored
as a simple binary data stream to determine whether there is an
input signal or not. A further discussion of the above technique
is disclosed in IEEE Photonic Tech. L., vol 10., pp. 839-841 (1998),
which is incorporated herein by reference.
The signal monitor system for any of the above detection schemes
can comprise any suitable digital signal processor capable of measuring
the signal change from the detector 72 and transmitting that information
to the user, such as, for example, a printed circuit board having
a pre-amplifier, an AD converter and driver circuit, and a programmable
chip for instrumentation specific software; or a multichip module
comprising those elements.
A particular embodiment of the electrophoretic molecular sieve
40 shown in FIG. 5 and described above the self-assembled nano-array
sieve 54 is fabricated according to the design shown in FIGS. 1
and 4 via the process outlined in FIGS. 3a to 3d, comprising a uniform
array 54 of uniform self-assembled carbon nanotube nano-features
having a mean diameter of .about.15 nm and a pore size of about
.about.30 nm on a 10 mm substrate. A combination DC and pulsed electric
field generator 56 is utilized to supply the electric field 64 to
the sieve 54.
A comparison of the design parameters of the electrophoretic molecular
sieve device 40 and the prior art electrophoretic devices is shown
in Table 2 below.
TABLE 2 Device Design Parameters of Prior Art Devices and Prototype
Device Device Volkmuth Duke Turner Proposed Prototype Technique
E-beam E-beam E-beam Nanotube Nanotube Lithography Lithography Lithography
Arrays Arrays Post 1 mm 1-2 mm 100 nm 10-100 nm .about.15 nm diameter
Post Spacing 2 mm 2-4 mm 200 nm 30-100 nm .about.30 nm PoreSize
1 mm 1-2 mm 100 nm 10-100 nm .about.15 nm Device Size 15 mm <10
mm <10 mm Field 1 V/cm .about.3 V/cm 1.3-13 V/cm 10-1000 10-1000
Strength V/cm V/cm Field Type DC DC, Pulsed DC DC, Pulsed DC, Pulsed
DNA dsDNA dsDNA dsDNA ssDNA, ssDNA dsDNA Process Fractionation Fractionation
Fractionation Sequencing Sequencing Fractionation DNA Size 3-100
kbp 10-200 kbp 7.2-43 kbp .about.1 bp-Mbp <1 kb Dynamics Hooking
Hooking Linear Migration
The device 40 can be optimized for mutation detection using multiple
DNA fragments generated by dideoxy fingerprinting reactions through
a nano-DOVAM-S (Detection Of Virtually All Mutations-SSCP) data
analysis technique. This type of technique utilizes conventional
single-strand conformation polymorphism (SSCP), but is much more
efficient at mutation detection because it provides the redundancy
of electrophoresis under five sets of sensitive and complementary
conditions. By varying the operating conditions of the instrument
the effects of electrophoresis with different array matrices, buffers,
temperatures, and additives on mutation detection can be determined.
DOVAM-S is a robust method for scanning genomic DNA, its advantages
include the parallel analysis of 45 to 50 samples using multiple
amplified products per lane on each gel and the increase in sensitivity
of mutation detection to.about.100%. Utilizing the nano-DOVAM-S
method, mutations can be detected even if only one of the ten single-stranded
segments displays an altered electrophoretic migration. A full discussion
of the DOVAM-S technique is disclosed in, BioTechniques, vol. 26
pp. 932-942 (1999), which is incorporated herein by reference.
While the generic nature of the conditions for DOVAM-S and the
marked effect of buffers, additives and temperature make it suitable
for nano-array molecular sieve devices, alternative methods could
be utilized, including simple SSCP, as well as SSCP in combination
with another method in order to generate redundancy of mutation-containing
segments, such as, for example, dideoxy fingerprinting (ddF) and
bidirectional dideoxy fingerprinting (Bi-ddF) in which SSCP is combined
with a Sanger dideoxy termination reaction using only one dideoxy
terminator. Another alternative method is restriction endonuclease
fingerprinting (REF) which combines SSCP with restriction endonuclease
digestion to generate multiple mutation containing segments.
In the DOVAM-S process, the genomic DNA is first amplified by PCR
to produce segments to be analyzed by nano-DOVAM-S. The use of an
electrophoresis sieve device in the method nano-DOVAM-S should permit
the use of extremely small volume preparations facilitating rapid
PCR performance at much reduced cost. Several micro and nano-scale
PCR methods are available including capillary microfabricated silicon
microreactors coupled to micro-separation platforms for the preparation,
performance and delivery of PCR, such as, for example, J. Chromatogr.
A., vol. 853 pp. 107-120 (1999). Micro/nano-PCR methodologies are
currently commercially available or in development by Caliper and
Nanogen. PCR primers are labeled with one of four fluorescent dyes
used in the BigDye.TM. terminators manufactured by PE Applied Biosystems
for ABI sequencing (R6G (green), ROX (red), R110 (blue), and TAMRA
(black)). Several (up to 18) PCR products from one sample are typically
pooled for the nano-DOVAM-S assay.
The samples would then be loaded into the apparatus and the device
run such that the region of interest (150-300 nucleotides) migrated
past the detector in approximately a 20 minute interval. Potentially
a CCD detector could be utilized that would make 15 measurements
per second over multiple array channels such that 18000 measurements
could be taken and analyzed in a single data run. Using 10 lanes
and three samples with 20 segments each per lane, and detecting
all lanes in parallel, the present technique could potentially scan
600 DNA segments per 20 minutes or 1-2 Mb of DNA per day, this is
compared to the 1-2 Mb of DNA per month rate of analysis of conventional
gel-electrophoresis techniques.
Utilizing the above technique, it is anticipated that mutations
of various types, including missense, nonsense, splice mutations,
insertions and deletions can be detected. Included among the point
mutations are transitions (G:C>A:T at and not at CpG sites; A:T>G:C)
and transversions (G:C>T:A; C>C:G; A:T>T:A; A:T>C:G).
Although the above discussion has focused on utilizing the molecular
sieve of the current invention for detecting mutations, such a device
could be utilized for any purpose for which conventional gel-based
and micro-machined molecular sieves can be used, such as, for example,
DNA fingerprinting, sequencing, etc.
Although specific embodiments are disclosed herein, it is expected
that persons skilled in the art can and will design alternative
molecular sieves, methods to produce the molecular sieves and/or
molecular sieve instruments that are within the scope of the following
claims either literally or under the Doctrine of Equivalents. |