U.S. patent application number 12/256409 was filed with the patent office on 2010-06-17 for microfluidic apparatus, systems, and methods for performing molecular reactions.
Invention is credited to Andrew A. Berlin, Selena Chan, Tae-Woong Koo, Xing Su, Lei Sun, Narayanan Sundararajan, Yuegang Zhang.
Application Number | 20100151454 12/256409 |
Document ID | / |
Family ID | 35054803 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100151454 |
Kind Code |
A1 |
Sundararajan; Narayanan ; et
al. |
June 17, 2010 |
MICROFLUIDIC APPARATUS, SYSTEMS, AND METHODS FOR PERFORMING
MOLECULAR REACTIONS
Abstract
Disclosed herein are methods, apparatuses, and systems for
performing nucleic acid sequencing reactions and molecular binding
reactions in a microfluidic channel. The methods, apparatuses, and
systems can include a restriction barrier to restrict movement of a
particle to which a nucleic acid is attached. Furthermore, the
methods, apparatuses, and systems can include hydrodynamic focusing
of a delivery flow. In addition, the methods, apparatuses, and
systems can reduce non-specific interaction with a surface of the
microfluidic channel by providing a protective flow between the
surface and a delivery flow.
Inventors: |
Sundararajan; Narayanan;
(Santa Clara, CA) ; Sun; Lei; (Santa Clara,
CA) ; Zhang; Yuegang; (Cupertino, CA) ; Su;
Xing; (Cupertino, CA) ; Chan; Selena; (San
Jose, CA) ; Koo; Tae-Woong; (Cupertino, CA) ;
Berlin; Andrew A.; (San Jose, CA) |
Correspondence
Address: |
Pillsbury Winthrop Shaw Pittman LLP;(INTEL)
P.O. Box 10500
McLean
VA
22102
US
|
Family ID: |
35054803 |
Appl. No.: |
12/256409 |
Filed: |
October 22, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10815264 |
Mar 31, 2004 |
7442339 |
|
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12256409 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
Y10T 436/117497
20150115; Y10T 436/143333 20150115; G01N 21/658 20130101; C12Q
2521/319 20130101; C12Q 2565/629 20130101; C12Q 2565/619 20130101;
C12Q 2565/632 20130101; C12Q 1/6869 20130101; C12Q 2565/629
20130101; C12Q 1/6827 20130101; C12Q 1/6869 20130101; C12Q 2521/319
20130101; C12Q 1/6827 20130101; Y10T 436/11 20150115 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method to detect a nucleotide, comprising: a) restraining
movement of a nucleic acid molecule attached to a single particle
using a restriction barrier located within a first channel; b)
contacting the nucleic acid molecule with an exonuclease to release
the nucleotide, wherein the nucleotide is a terminal nucleotide;
and c) identifying the released nucleotide by associating the
released nucleotide with a surface enhanced Raman
spectroscopy-active surface, irradiating the released nucleotide
with a detection laser beam and measuring Raman emission from the
irradiated nucleotide, thereby detecting the nucleotide.
2. The method of claim 1, wherein the restriction barrier comprises
a plurality of walls.
3. The method of claim 2, wherein the restriction barrier comprises
a first angled wall and a second angled wall positioned relative to
the first angled wall to capture the single particle having the
surface with the attached nucleic acid molecule.
4. The method of claim 1, wherein a gradient force optical trap
captures the single particle downstream of the restriction barrier,
transports the single particle upstream of the restriction barrier,
and release the single particle.
5. The method of claim 1, wherein a gradient force optical trap
captures the single particle downstream of the detection laser
beam, the detection laser beam and the restriction barrier are
moved downstream of the captured single particle, and the single
particle is released.
6. A method to determine a nucleotide sequence of a nucleic acid
molecule, comprising: a) restraining movement of a single particle
using a restriction barrier located within a first channel, wherein
the nucleic acid molecule is attached to the single particle; b)
contacting the nucleic acid molecule with an exonuclease to release
a terminal nucleotide; and c) identifying a first released
nucleotide and a second released nucleotide by irradiating the
first released nucleotide and then the second released nucleotide
with light from a detection light source, by associating the first
released nucleotide and the second released nucleotide with a
surface enhanced Raman spectroscopy-active surface, and measuring
Raman emission from the irradiated first released nucleotide and
then from the second released nucleotide, thereby determining a
nucleotide sequence of the nucleic acid.
7. The method of claim 6, wherein the restriction barrier comprises
a plurality of walls.
8. The method of claim 7, wherein the restriction barrier comprises
a first angled wall and a second angled wall positioned relative to
the first angled wall to capture the single particle having the
surface with the attached nucleic acid molecule.
9. The method of claim 6, wherein a gradient force optical trap
captures the single particle downstream of the light from the
detection light source, transports the single particle upstream of
the restriction barrier, and releases the single particle.
10-45. (canceled)
46. The method of claim 3, wherein the first angled wall and the
second angled wall form a first opening at least 1 micron in width
or diameter and a second opening less than 10 microns in width or
diameter, wherein the first opening has a greater width or diameter
than the second opening.
47. The method of claim 4, wherein the gradient force optical trap
is operable as optical tweezers.
48. The method of claim 1, wherein the particle has a diameter
between 0.1 and 20 microns.
49. The method of claim 6, wherein there is a gap between a wall of
the channel and the restriction barrier.
50. The method of claim 6, wherein a portion of a flow path in
optical communication with the detection light source is coated
with silver, gold, platinum, copper or aluminum.
51. The method of claim 1, wherein a single nucleotide is attached
to a single particle.
52. The method of claim 1, further comprising sequentially
detecting a plurality of nucleotides.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to devices, systems,
and methods for performing molecular reactions, and more
specifically to microfluidic devices, systems, and methods for
performing molecular reactions.
[0003] 2. Background Information
[0004] Determination of the entire sequence of the human genome has
provided a foundation for identifying the genetic basis of diseases
such as cancer, cystic fibrosis, sickle cell anemia and muscular
dystrophy. However, a great deal of work remains to be done to
identify the genetic variations associated with each disease and to
develop more sensitive and accurate tests for these diseases. This
development would be accelerated greatly if efficiency and product
yields of present methods were improved.
[0005] Current methods for determining nucleic acid sequence
information are laborious, expensive, and inefficient. This is
indicative of the shortcomings of many current molecular reactions
used in biotechnology, such as DNA synthesis or carbon nanotube
production, which provide a low reaction yield of the desired
product. In addition, many side reactions occur in macromolecule
reactions that yield undesirable products. Finally, lack of
synchronization in multi-molecule polymer reactions (e.g.
exonuclease DNA sequencing) results in inaccurate results.
[0006] To attempt to overcome their shortcomings, current methods
usually require a large excess of reagent, which results in more
side reaction and higher cost. Furthermore, in many cases reactions
must be performed in multiple steps and require nucleic acid
manipulation. For example, a desired "block" of a macromolecule is
synthesized and then linked (i.e. ligated) to form a final product
(e.g. insert a modified gene into a plasmid). This results in
inefficient methods with relatively low yield and low product
purity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 illustrates confinement in a restriction barrier 140
of a bead 150 with an attached nucleic acid molecule 160 in a
microfluidic channel 100.
[0008] FIGS. 2A and 2B illustrate an exemplary system and method
provided herein, wherein a bead 150 with an attached nucleic acid
molecule 160 assembled downstream of a detection unit, is captured
by optical tweezers 210, transported and released upstream of a
restriction barrier 140.
[0009] FIGS. 3A-3C illustrates an embodiment wherein fluidic
alignment is used in a nucleic acid molecule synthesis reaction.
FIG. 3A illustrates fluidic alignment. FIG. 3B illustrates exposure
of an end (i.e. terminus) of the nucleic acid molecules 310. FIG.
3C illustrates addition of nucleotide subunits 320 to the nucleic
acid molecule 310.
[0010] FIGS. 4A-4H provides a series of schematic diagrams
illustrating exonuclease nucleic acid sequencing with fluidic
alignment and fluidic focusing.
[0011] FIGS. 5A and 5B illustrate a microfluidic system that
includes protective flows 350 and a delivery flow 360. A target
molecule 310 is aligned using a magnetic force (FIG. 5A) or an
electric field (FIG. 5B), before a second molecule is delivered to
a target region of the target molecule 310 by a delivery flow
360.
[0012] FIG. 6 illustrates a microfluidic system that includes a
single protective flow 350 and a delivery flow 360 that is located
in the top portion of a microfluidic channel 410, above the
protective flow 350.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The invention generally provides improved apparatuses,
systems and methods for performing biomolecular reactions in a
microfluidic device. In one group of related embodiments, referred
to herein as "the nucleic acid capture" embodiments, the disclosed
systems, apparatus, and methods provide for the capture of a single
nucleic acid molecule in a microfluidic channel upstream from an
optical detector. These embodiments allow sequential detection of
one or more nucleotides released from a nucleic acid molecule.
Accordingly, the disclosed methods, systems and apparatus are of
use for the rapid, automated sequencing of at least a portion of a
nucleic acid molecule.
[0014] Methods for isolating single nucleic acid molecules on a
solid support and manipulation of these molecules with optical
tweezers have been developed. However, the lasers used by optical
tweezers could potentially affect optical detection methods such as
Raman spectroscopy. Furthermore, the integration of optical
tweezers and a Raman detector is limited by the field of view when
using the same microscope objective (-100 microns for a 20.times.
objective). The use of two microscope objectives directly adjacent
to each other further separates the optical tweezers component from
the Raman detection on a scale of several centimeters. However, the
use of two microscope objectives placed on top and below the
substrate requires that the material of the microfluidic channel is
optically transparent and has the same transmission properties from
a top and bottom layer.
[0015] The methods provided herein overcome these problems by
facilitating the isolation and manipulation of single nucleic acid
molecules without interfering with optical detection of the nucleic
acid molecule or a single nucleotide. Methods can be performed in
the microfluidic channel to functionalize a solid support, e.g., a
particle such as a bead, with the single nucleic acid molecule. A
single bead with a single nucleic acid molecule attached, can be
transported and released upstream of a detector using optical
tweezers, for example. The optical tweezers are typically a
gradient force optical trap, such as a single-beam gradient force
optical trap, that captures the single particle downstream from the
laser beam.
[0016] The released bead can then flow downstream and either become
trapped in a restriction barrier or attached to a surface. Once the
bead is confined, the optical tweezers can be removed so that they
do not interfere with an optical detector downstream. Single
nucleotides can be cleaved from the bead using an exonuclease, for
example. The single nucleotides are then detected using
spectroscopic methods such as surface enhanced Raman spectroscopy
(SERS). The inclusion of a restriction barrier in a microfluidic
channel and the immobilization of an optically transported bead,
allows removal of the optical tweezers from the optical path of a
detection device, thereby preventing interference from the
additional light source of the optical tweezers close to the
collection volume of the detector.
[0017] Accordingly, presented herein is a system that includes a
surface enhanced Raman spectroscopy (SERS) detection unit and a
first channel in optical communication with the SERS detection
unit, wherein the first channel contains a restriction barrier to
restrain movement of a single particle with an attached nucleic
acid molecule. The SERS detection unit typically includes a
detection light source, typically a laser light source, for
irradiating a molecule, and a detection unit for detecting Raman
emission from the irradiated molecule.
[0018] These systems are used to analyze the nucleic acid molecule
attached to the surface of the particle. For example, the system
can be used to perform a method to determine a nucleotide sequence
of a nucleic acid molecule. The method includes restraining
movement of a single nucleic acid molecule attached to a single
particle in a first channel that includes a restriction barrier, by
capturing the single particle in the restriction barrier. The
captured nucleic acid molecule is then contacted with an agent that
removes nucleotides, e.g., an exonuclease, to release a terminal
nucleotide which is then detected using surface enhanced Raman
spectroscopy (SERS). Typically, a first released nucleotide and a
second released nucleotide are identified after associating the
released nucleotides with a SERS-active surface such as a SERS
substrate or a metal nanoparticle in solution, irradiating the
released nucleotides with a detection laser beam and measuring
Raman emission from the irradiated nucleotides. Additional released
nucleotides can be detected. By detecting the released nucleotides
a nucleotide sequence of the nucleic acid molecule is
determined.
[0019] In other aspects, a nucleic acid molecule restrained in the
restriction barrier can be analyzed using methods other than by
exonuclease treatment in a sequencing reaction. For example, the
system can be used to amplify a nucleic acid molecule attached to
the surface of the particle using known amplification methods, such
as the polymerase chain reaction. Furthermore, the trapped nucleic
acid molecule can be analyzed by hybridization using probes, such
as fluorescently labeled and/or Raman-labeled probes. In certain
aspects, multiple smaller nucleic acid probes with different labels
can be contacted with the restrained nucleic acid molecule. Each
nucleic acid probe identifies a nucleotide sequence to which it
binds. Therefore, binding of the probes to the restrained nucleic
acid molecule identifies the presence of a nucleotide sequence in
the restrained nucleic acid molecule and provides and provides a
type of barcode.
[0020] In another embodiment, an apparatus is provided for
performing sequencing methods. The apparatus includes a surface
enhanced Raman spectroscopy (SERS) detection unit; a first channel
in optical communication with the detection unit, and a restriction
barrier upstream of the detection unit to restrain movement of the
particle. The apparatus can optionally include a second channel in
fluid communication with the first channel, forming a junction with
the first channel downstream from the SERS detection unit.
Furthermore, the apparatus optionally includes optical tweezers
having a laser capable of moving from a starting position
downstream from the SERS detection unit, and typically downstream
from the first channel and the second channel to a position
upstream from the SERS detection unit. Alternatively, the SERS
detection unit can have a moveable stage to effect the same
transport of a nucleic acid molecule from downstream of the
detection unit to upstream of the detection unit. The apparatus can
further include a third channel, wherein the third channel is in
fluid communication with the second channel and wherein the third
channel and the first channel form a junction downstream of the
detection unit. The optical tweezers are typically a gradient force
optical trap that captures a single particle.
[0021] In another embodiment, provided herein is an apparatus
including, a first channel including a restriction barrier
comprising a first angled wall and a second angled wall positioned
relative to the first angled wall to form a first opening at least
1 micron in width or diameter and a second opening less than 10
microns in width or diameter, wherein the first opening has a
greater width or diameter than the second opening. In certain
aspects, the second opening is less than 1 micron in width or
diameter. The apparatus can further include a light source and a
detector to detect a surface enhanced Raman spectroscopy emission
of a molecule irradiated by the light source. In these aspects the
first channel is in optical communication with the light source and
the detector.
[0022] In another embodiment, provided herein is a system
including: [0023] a) a light source [0024] b) a detector to detect
a surface enhanced Raman spectroscopy emission of a molecule
irradiated by the light source; and [0025] c) a first channel in
optical communication with the light source and the detector,
wherein the first channel includes a restriction barrier comprising
a plurality of angled walls to restrain movement of a single
particle upstream of light emitted by the light source.
[0026] In yet another embodiment, a method is provided to detect a
nucleotide, that includes restraining movement of a particle that
includes a nucleic acid molecule attached to its surface, using a
restriction barrier located within a first channel; contacting the
nucleic acid molecule with an exonuclease to release the nucleotide
from the nucleic acid molecule; and identifying the released
nucleotide using surface enhanced Raman spectroscopy (SERS). SERS
is typically performed by irradiating the released nucleotide with
a detection laser beam and measuring Raman emission from the
irradiated nucleotide.
[0027] Also provided is a method to restrain movement of a single
nucleic acid molecule immobilized on a single particle in a first
channel by capturing the single particle in a restriction barrier.
In one aspect, the restriction barrier includes a first angled wall
and a second angled wall positioned relative to the first angled
wall to capture the single particle having the surface with the
attached nucleic acid molecule. As discussed, in one aspect the
first angled wall and the second angled are spaced apart to allow a
nucleotide, the nucleic acid molecule, and/or a protein to pass
through the restriction barrier. Since the nucleic acid molecule
for example, is attached to the particle, it is held within the
restraint by the particle. However, when a biomolecule such as a
nucleotide, is released from the particle, it is released from the
restraint. Since the captured single nucleic acid molecule is
accessible to other reactants, it can be used in biochemical
reactions, such as exonuclease reactions, to detect a terminal
nucleotide of the captured nucleic acid molecule, which is useful
for determining at least partial nucleic acid sequence information
regarding the captured nucleic acid molecule.
[0028] As used herein, "a" or "an" may mean one or more than one of
an item.
[0029] "Nucleic acid" encompasses DNA, RNA, single-stranded,
double-stranded or triple stranded and any chemical modifications
thereof, although single-stranded nucleic acids are preferred.
Virtually any modification of the nucleic acid is contemplated. As
used herein, a single stranded nucleic acid may be denoted by the
prefix "ss", a double stranded nucleic acid by the prefix "ds", and
a triple stranded nucleic acid by the prefix "ts."
[0030] A "nucleic acid" may be of almost any length, from 10, 20,
30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,
400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500,
4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000,
30,000, 40,000, 50,000, 75,000, 100,000, 150,000, 200,000, 500,000,
1,000,000, 1,500,000, 2,000,000, 5,000,000 or even more bases in
length, up to a full-length chromosomal DNA molecule.
[0031] A "nucleoside" is a molecule comprising a base (A, C, G, T
or U) covalently attached to a pentose sugar such as deoxyribose,
ribose or derivatives or analogs of pentose sugars.
[0032] A "nucleotide" refers to a nucleoside further comprising at
least one phosphate group covalently attached to the pentose sugar.
In some embodiments, the nucleotides are ribonucleoside
monophosphates or deoxyribonucleoside monophosphates although in
certain embodiments it is anticipated that nucleoside diphosphates
or triphosphates could be produced. In other embodiments,
nucleosides may be released from the nucleic acid molecule and
detected as discussed below. It is contemplated that various
substitutions or modifications may be made in the structure of the
nucleotides, so long as they are still capable of being released
from the nucleic acid by a deconstruction reagent. For example, in
certain embodiments the ribose or deoxyribose moiety may be
substituted with another pentose sugar or a pentose sugar analog.
In other embodiments, the phosphate groups may be substituted by
various analogs such as fluorescent labels.
[0033] Nucleic acid molecules to be sequenced can be prepared by
any technique known in the art. In certain embodiments, the nucleic
acids are naturally occurring DNA or RNA molecules. Virtually any
naturally occurring nucleic acid may be prepared and sequenced by
the disclosed methods including, without limit, chromosomal,
mitochondrial and chloroplast DNA and ribosomal, transfer,
heterogeneous nuclear and messenger RNA.
[0034] As used herein, the term "specific binding pair member"
refers to a molecule that specifically binds or selectively
hybridizes to, or interacts with, another member of a specific
binding pair. Specific binding pair members include, for example a
receptor and a ligand, or an antigen and an antibody. For example,
the first specific binding pair member can be a protein, such as an
antibody molecule, or fragment thereof, and the second specific
binding pair member can be a biomolecule, such as a protein, that
includes an epitope recognized by the antibody. In one example, the
first specific binding pair member is a receptor and the second
specific binding pair member is a ligand.
[0035] As used herein, the terms "analyte" refer to any atom,
chemical, molecule, compound, composition or aggregate of interest
for detection and/or identification. Non-limiting examples of
analytes include an amino acid, peptide, polypeptide, protein,
glycoprotein, lipoprotein, nucleoside, nucleotide, oligonucleotide,
nucleic acid, sugar, carbohydrate, oligosaccharide, polysaccharide,
fatty acid, lipid, hormone, metabolite, cytokine, chemokine,
receptor, neurotransmitter, antigen, allergen, antibody, substrate,
metabolite, cofactor, inhibitor, drug, pharmaceutical, nutrient,
prion, toxin, poison, explosive, pesticide, chemical warfare agent,
biohazardous agent, radioisotope, vitamin, heterocyclic aromatic
compound, carcinogen, mutagen, narcotic, amphetamine, barbiturate,
hallucinogen, waste product and/or contaminant.
[0036] A "biological sample" includes, for example, urine, blood,
plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid,
tears, mucus, and the like. In certain aspects, the biological
sample is from a mammalian subject, for example a human subject.
The biological sample can be virtually any biological sample, as
long as the sample contains or may contain a second specific
binding pair member. For example, the sample can be suspected of
containing a protein that has an epitope recognized by an antibody
included as the first specific binding pair member. The biological
sample can be a tissue sample which contains, for example, 1 to
10,000,000; 1000 to 10,000,000; or 1,000,000 to 10,000,000 somatic
cells. The sample need not contain intact cells, as long as it
contains sufficient quantity of a specific binding pair member for
the methods provided. According to aspects of the methods provided
herein, wherein the biological sample is from a mammalian subject,
the biological or tissue sample can be from any tissue. For
example, the tissue can be obtained by surgery, biopsy, swab,
stool, or other collection method. In other aspects, the biological
sample contains, or is suspected to contain, or at risk for
containing, a pathogen, for example a virus or a bacterial
pathogen.
[0037] As used herein, the term "nanocrystalline silicon" refers to
silicon that comprises nanometer-scale silicon crystals, typically
in the size range from 1 to 100 nanometers (nm). "Porous silicon,"
which contains nanosized silicon crystals, refers to silicon that
has been etched or otherwise treated to form a porous
structure.
[0038] As used herein, "operably coupled" means that there is a
functional interaction between two or more units of an apparatus
and/or system. For example, a Raman detector may be "operably
coupled" to a computer if the computer can obtain, process, store
and/or transmit data on Raman signals detected by the detector.
[0039] The term "binds specifically" or "specific binding
activity," when used in reference to an antibody means that an
interaction of the antibody and a particular epitope has a
dissociation constant of at least about 1.times.10.sup.-6,
generally at least about 1.times.10.sup.-7, usually at least about
1.times.10.sup.-8, and particularly at least about
1.times.10.sup.-9 or 1.times.10.sup.-10 or less. As such, Fab,
F(ab').sub.2, Fd and Fv fragments of an antibody that retain
specific binding activity, are included within the definition of an
antibody.
[0040] As used herein, the term "antibody" is used in its broadest
sense to include polyclonal and monoclonal antibodies. The term
antibody as used herein is meant to include intact molecules as
well as fragments thereof, such as Fab and F(ab').sub.2, Fv and SCA
fragments which are capable of binding an epitopic determinant.
[0041] (1) An Fab fragment consists of a monovalent antigen-binding
fragment of an antibody molecule, and can be produced by digestion
of a whole antibody molecule with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain. [0042] (2) An Fab' fragment of an antibody molecule
can be obtained by treating a whole antibody molecule with pepsin,
followed by reduction, to yield a molecule consisting of an intact
light chain and a portion of a heavy chain. Two Fab' fragments are
obtained per antibody molecule treated in this manner. [0043] (3)
An (Fab').sub.2 fragment of an antibody can be obtained by treating
a whole antibody molecule with the enzyme pepsin, without
subsequent reduction. A (Fab').sub.2 fragment is a dimer of two
Fab' fragments, held together by two disulfide bonds. [0044] (4) An
Fv fragment is defined as a genetically engineered fragment
containing the variable region of a light chain and the variable
region of a heavy chain expressed as two chains. [0045] (5) A
single chain antibody ("SCA") is a genetically engineered single
chain molecule containing the variable region of a light chain and
the variable region of a heavy chain, linked by a suitable,
flexible polypeptide linker.
[0046] The term "antibody" as used herein includes naturally
occurring antibodies as well as non-naturally occurring antibodies,
including, for example, single chain antibodies, chimeric,
bifunctional and humanized antibodies, as well as antigen-binding
fragments thereof. Such non-naturally occurring antibodies can be
constructed using solid phase peptide synthesis, can be produced
recombinantly or can be obtained, for example, by screening
combinatorial libraries consisting of variable heavy chains and
variable light chains (see Huse et al., Science 246:1275-1281
(1989). These and other methods of making, for example, chimeric,
humanized, CDR-grafted, single chain, and bifunctional antibodies
are well known to those skilled in the art (Winter and Harris,
Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546,
1989; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring
Harbor Laboratory Press, 1988); Hilyard et al., Protein
Engineering: A practical approach (IRL Press 1992); Borrabeck,
Antibody Engineering, 2d ed. (Oxford University Press 1995.
[0047] Methods for raising polyclonal antibodies, for example, in a
rabbit, goat, mouse or other mammal, are well known in the art
(see, for example, Green et al., "Production of Polyclonal
Antisera," in Immunochemical Protocols (Manson, ed., Humana Press
1992), pages 1-5; Coligan et al., "Production of Polyclonal
Antisera in Rabbits, Rats, Mice and Hamsters," in Curr. Protocols
Immunol. (1992), section 2.4.1). In addition, monoclonal antibodies
can be obtained using methods that are well known and routine in
the art (Harlow and Lane, supra, 1988).
[0048] As used in this invention, the term "epitope" refers to an
antigenic determinant on an antigen, to which the paratope of an
antibody binds. Antigenic determinants usually consist of
chemically active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics.
[0049] Examples of types of immunoassays of the invention include
competitive and non-competitive immunoassays in either a direct or
indirect format. Those of skill in the art will know, or can
readily discern, other immunoassay formats without undue
experimentation.
[0050] In performing a method of the present invention, "blocking
agents" can be included in the incubation medium. "Blocking agents"
are added to minimize non-specific binding to a surface and between
molecules.
[0051] The term "receptor" is used to mean a protein, or fragment
thereof, or group of associated proteins that selectively bind a
specific substance called a ligand. Upon binding its ligand, the
receptor triggers a specific response in a cell.
[0052] The term "polypeptide" is used broadly herein to mean two or
more amino acids linked by a peptide bond. The term "fragment" or
"proteolytic fragment" also is used herein to refer to a product
that can be produced by a proteolytic reaction on a polypeptide,
i.e., a peptide produced upon cleavage of a peptide bond in the
polypeptide. A polypeptide of the invention contains at least about
six amino acids, usually contains about ten amino acids, and can
contain fifteen or more amino acids, particularly twenty or more
amino acids. It should be recognized that the term "polypeptide" is
not used herein to suggest a particular size or number of amino
acids comprising the molecule, and that a peptide of the invention
can contain up to several amino acid residues or more. A protein is
a polypeptide that includes other chemical moieties in addition to
amino acids, such as phosphate groups or carbohydrate moieties.
[0053] FIG. 1 illustrates an exemplary microfluidic channel 100 for
performing methods provided herein that involves trapping a bead
150 with an attached single nucleic acid molecule 160 in a
restriction barrier 140. The microfluidic channel 100 includes an
immobilization structure, which in certain aspects of the invention
is a restriction barrier 140. The restriction barrier 140 in this
example includes a first angled wall 120 and a second angled wall
130 between which a single particle is captured. The first angled
wall 120 and second angled wall 130 are spaced apart to allow a
molecule such as a nucleotide molecule, a nucleic acid molecule
and/or a protein such as an exonuclease, to flow between them.
However, the first angled wall 120 and second angled wall 130 are
spaced close enough to each other to retain the particle 150.
[0054] Accordingly, the first angled wall 120 and second angled
wall 130 form a first opening 190 that is large enough to allow a
single particle 150 to be captured by the restriction barrier 140,
and a second opening 195 through which the particle 150 cannot
pass. It will be understood that the height of the restriction
barrier 140 is typically determined by the channel depth and that
the dimensions of the restriction barrier 140 and its openings 190,
195 need to be commensurate with and are a function of the particle
150 dimensions. In certain example, the first opening 190 is
between about 0.1 micron and about 100 microns in width and height,
for example between about 1 micron and about 10 microns, or greater
than 0.1, 0.25, 0.5, 1 microns, 2.5 microns, or 5 microns, and less
than 1 millimeter, 500 microns, 250 microns, 100 microns, 50
microns, 25 microns, 10 microns. Opposite from the first opening
190 is a second opening 195 formed by the first angled wall 120 and
the second angled wall 130 that is smaller than the diameter of the
particle 105 so that the particle 150 is retained in the
restriction barrier 140, yet large enough to allow molecules, such
as nucleotides, nucleic acid molecules, or proteins to pass
through. The second opening 195 is typically less than 10, 5, 4, 3,
2, or 1 micron, or less than 100, 10, 5, 4, 3, 2, or 1 nanometer,
and typically at least 1, 2, 3, 4, 5, or 10 nanometers. The
restriction barrier 140 is positioned within the microfluidic
channel 100, such that when a particle 150 enters the microfluidic
channel 100 it is captured in the restriction barrier 140 because
fluid flow in the channel 100 moves the particle 150 from the large
opening 190 towards the small opening 195. Typically, a particle
150 cannot exit a restriction barrier 140 along the direction of
fluid flow after it enters the restriction barrier 140 provided
that flow in the channel 100 continues to be directed into the
restriction barrier 140.
[0055] The restriction barrier can include a plurality of walls,
such as a plurality of angled walls. It will be understood that
various configurations of walls can be used in the restriction
barrier to restrain a particle. The restriction barrier can be
configured in any way that provided that it restrains a particle
that has an attached nucleic acid molecule that can be contacted
with another molecule, such as an exonuclease. Furthermore, the
restriction barrier is configured so that a nucleotide cleaved from
a nucleic acid molecule attached to a captured particle, can is not
trapped in the restriction barrier. Therefore, the restriction
barrier typically has a first opening on the upstream portion of
the barrier that is large enough for a nucleic acid molecule
attached to a particle to enter the barrier, and a second opening
on the downstream end of the barrier that is small enough to trap
the nucleic acid molecule attached to the particle, inside the
barrier. For example, the restriction barrier can be L-shaped using
rectangular brackets, or funnel shaped, allowing a nucleic acid
molecule and/or a protein to flow through, while still retaining
the particle. Other possibilities for the restriction barriers
include using polymer filters or porous membranes such as cellulose
acetate and alumina membranes with pore sizes smaller than the
particle diameter.
[0056] Where the restriction barrier includes a first angled wall
and a second angled wall, many different angles can be used for the
walls provided that the restraining barrier can restrain a particle
yet provide access to a nucleic acid molecule attached to the
particle. For example, the first angled wall and the second angled
wall can form an angle of between 5.degree. and 90.degree., or
between about 10.degree. and 75.degree.. The first and second
angled walls can be identical lengths or they can be different
lengths. In one aspect, one angled wall is longer and functions as
a ramp to guide a particle into the restriction barrier. The first
and second walls are typically between 1 micron and 10 millimeters
in length and between 10 nanometers and 1 micron in width.
[0057] Molecular interactions or attractive forces between the
angled walls and the bead or nucleic acid can be used to restrain
and/or position the bead, or to assist in this restraint or
positioning. In other words, the restraint of the particle need not
be physical but also can include chemical attachment or other
forces. For example, magnetic forces can be employed.
Alternatively, the angled walls can include a first specific
binding pair member and the beads or nucleic acid molecule can
include a second specific binding pair member. For example, an
angled wall can include one or more avidin moieties attached to its
surface and a particle can include one or more biotin moieties. By
the binding of a first specific binding pair member to a second
specific binding pair member, the particle is further restrained
within the restriction barrier. Furthermore, functionalizing the
material of the walls can be used to reduce adhesion of particle to
the wall.
[0058] The restriction barrier walls can be made from a variety of
materials. For example, the walls can be molded in the microchannel
and made of an identical composition as the microchannels. Methods
for micromolding microfluidic devices are provided herein Any
crosslinkable or polymerizable fluid could be used to construct the
angled walls of the restraining barrier. For example, hydrogel can
be used to construct the barrier. Fabrication of the barriers is
done by essentially filling the channels first with the
polymerizable fluid and then exposing it to a polymerizing agent
such as UV radiation, through a mask and then flushing the
unpolymerized fluid leaving the polymerized restriction barrier
within the microchannel. The mask determines the shape and
dimensions of the barrier.
[0059] A particle used in the present invention methods can be a
wide range of sizes, shapes, and materials, provided that a nucleic
acid can be attached to the surface of the particle, the particle
can be captured, transported, and released by optical tweezers, and
movement of the particle can be restrained in a restriction
barrier. For example, the particles can be surface-functionalized
microsphere beads. Suitable beads of varying sizes are commercially
available (Bangs Laboratories, Inc., Fishers, Ind.). The
microsphere beads typically have a diameter of between about 0.1
microns (.mu.) and about 20.mu., for example between about 0.5.mu.
and about 10.mu., and as a more specific example between about
1.mu. and about 5.mu.. The microsphere beads can be made of
materials such as polystyrene, glass, polysaccharides such as
agarose, and latexes such as styrene butadiene. In certain aspects,
the particle can be in the form of magnetic or non-magnetic beads
or other discrete structural units.
[0060] Various methods can be used to attach a single nucleic acid
molecule to a particle. As discussed in more detail below, methods
are known for immobilizing a nucleic acid to a solid support, such
as a particle. To attach a single nucleic acid molecule to a
particle using virtually any known method for immobilization, a
sample containing the nucleic acid molecule can be diluted prior to
coupling it to a particle. At an appropriate dilution, each
particle will have a statistical probability of binding zero or one
nucleic acid molecule. Particles with one attached nucleic acid
molecule can be identified using, for example, fluorescent dyes and
flow cytometer sorting or magnetic sorting.
[0061] In another embodiment, a single molecule is attached to a
particle by contacting nucleic acid molecules that are immobilized
in a channel surface with particles. In illustrative examples, the
nucleic acid molecules are spaced far enough apart on the channel
surface so that a particle can only bind to one nucleic acid
molecule (See U.S. patent application Ser. No. 10/748,802, filed
Dec. 30, 2003, entitled "METHOD AND DEVICE FOR ISOLATING SINGLE
POLYMERIC MOLECULES," inventors Narayanan Sundararajan and Xing Su,
and U.S. patent application Ser. No. 10/781,238, filed Feb. 18,
2004, entitled "METHOD AND DEVICE FOR ISOLATING AND POSITIONING
SINGLE MOLECULES," inventors Narayanan Sundararajan, Xing Su, and
Tae-Woong Koo). Typically, the particle is attached to the nucleic
acid molecule as the result of binding of a first specific binding
partner on the particle to a second specific binding partner on the
nucleic acid molecule. For example, the particle can include avidin
moieties on its surface and the nucleic acid molecule can include
biotin moieties.
[0062] More specifically, in certain examples nucleic acid
molecules can be modified/labeled on one end and immobilized to
specific binding positions on a substrate surface of a channel,
such that the shortest distance between two adjacent binding
positions is at least about two times the length of particles to
which the nucleic acids are to be attached. The specific binding
positions, which typically are formed by immobilizing a first
specific binding pair member on the channel surface, can be
downstream from the detection unit and downstream from a flow of
nucleic acid molecules. Nucleic acid molecules include a second
specific binding pair member that binds to the first specific bind
pair members on the channel surface.
[0063] A nucleic acid labeled with a specific binding pair member
preferably is bound to the substrate surface via the non-labeled
end only. Unbound nucleic acid molecules (i.e., nucleic acid
molecules that have not attached to a binding position) are
typically washed off of the substrate surface after the
immobilization of the nucleic acid molecules. Immobilized nucleic
acid molecules are subsequently contacted with particles that have
specific binding members on their surface that bind to a specific
binding pair member attached to the nucleic acid molecule. Free
particles can be carried away from the channel surface by the flow
within the channel. After the particles bind to the nucleic acid
molecules they are released from the substrate by breaking the
association of the nucleic acid molecule with the specific binding
position on the substrate surface. Optical tweezers are then used
to capture a single particle with an attached single nucleic acid
molecule and transport the single particle upstream of a
restriction barrier. As indicated above, the nucleic acid molecules
are spaced far enough apart on the surface to assure that a
particle cannot bind to more than a single nucleic acid
molecule.
[0064] According to another aspect of the invention, a single
nucleic acid molecule associated with a particle can be isolated by
introducing particles with varying numbers of attached nucleic acid
molecules into an applied electric field, and isolating particles
having only one attached nucleic acid molecule from the remainder
of particles. Separation of the particles based on the number of
attached nucleic acid molecules occurs because the nucleic acid
molecule changes the charge of the particle, therefore also
affecting its mobility in an applied electrical field.
[0065] In certain aspects, the immobilization structure for
restraining movement of the particle is a surface of a flow
channel, or a substrate attached to a surface of the flow channel,
upstream from the SERS detection unit. Various molecular
interactions or forces can be used to restrain a particle on a
surface provided that attachment of the particle to the
immobilization structure is strong enough to withstand the flow of
liquid in the channel. For example, magnetic forces can be used.
Furthermore, the immobilization structure can include a
cross-linking agent, for immobilizing the particle. Optical
tweezers are used to transport and release the particle within the
channel so that it can be carried by the flow of the channel into
contact with the immobilization structure.
[0066] When the methods provided are used to determine a nucleotide
sequence of a nucleic acid molecule, the identity and sequence of
between 1 nucleotide and all of the nucleotides of the nucleic acid
molecule can be determined. For example, a nucleotide sequence of
less than 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500,
1000, 1500, or 2000 nucleotides can be determined.
[0067] FIG. 2 illustrates a specific example of a system for
performing a method provided that involves optical tweezers 210 and
a restriction barrier 140. The microfluidic system 200 includes a
first channel 100; a second channel 220 in fluid communication with
the first channel 100, wherein the first channel 100 delivers
particles 150 into the first channel 100; a third channel 230 in
fluid communication with the first channel 100, wherein the third
channel 230 delivers nucleic acid molecules 160 into the first
channel 100; optical tweezers 210 to capture, transport, and
release a single particle 150; and a SERS detection unit in optical
communication with the first channel at a detection area 240
downstream of the restriction barrier.
[0068] Using this system, which is typically a MEMS system,
particles 150 are transported into the first channel 100 by the
flow of the second channel 220 where they contact nucleic acid
molecules 160 delivered from the third channel 230, downstream of
the junction of the second channel 220 and third channel 230 with
the first channel 100. A single nucleic acid molecule 160 is
attached to the surface of the particle 150. The optical tweezers
210 then capture a single particle 150 with an attached nucleic
acid molecule 160 downstream form the restriction barrier 140 and
downstream of a junction of the first channel 100 and the second
channel 220 and/or third channel 230, transport the particle 150
upstream of a restriction barrier 140, and release the particle
150. The particle 150 is then moved by the flow of the first
channel 100 into the restriction barrier 140 inside which its
movement is restrained. Next, an exonuclease or other molecular
destruction reagent in the first channel 100 contacts the nucleic
acid molecule 160 and cleaves a terminal nucleotide. The cleaved
nucleotide travels downstream in the first channel 100 and is
detected by the SERS detection unit in a detection area 240. The
detection area 240 is an area in the first channel 100, or a
channel connected thereto, downstream of the restriction barrier
140. The SERS detection unit is in optical communication with the
detection area 240.
[0069] A reaction area is an area within the first channel where a
nucleic acid molecule is made available to an exonuclease or other
destruction agent. The reaction area is located upstream of the
SERS detection unit. Typically, the reaction area contacts and/or
surrounds the immobilization substrate, for example the restriction
barrier. In certain aspects, the reaction area is located upstream
of the junction of a first channel, which carries particles, and a
second channel, which carries nucleic acid molecules.
[0070] In methods, systems, and apparatus provided, optical
tweezers are positioned in a first channel to capture a single
particle downstream from a restriction barrier in the channel,
transport the particle upstream of the restriction area, and
release the particle so that it can be captured in the restriction
barrier.
[0071] "Optical tweezers" are tightly focused beams of laser light
that can be used to trap and remotely manipulate polarizable
objects (See e.g., U.S. Pat. Nos. 5,620,857; 5,100,627; and
4,893,886). Originally proposed for the trapping of atoms, such
devices are also capable of trapping macroscopic, polarizable
objects such as latex and glass spheres in the micron size range as
well as biological material such as viruses, bacteria, yeast and
protozoa, ranging in size from 20 nm to 100 microns. Not wanting to
be limited by a particular theory, the basic principle behind
optical tweezers is the gradient force of light which manifests
itself when a transparent material with a refractive index greater
than the surrounding medium is placed in a light intensity
gradient. As light passes through the polarizable object, it
induces fluctuating dipoles in the material. These dipoles interact
with the electromagnetic field gradient, resulting in a force
directed towards the brighter region of the light. Hence the object
is pulled into the focus of the laser beam which is the local
maximum of the light rigid.
[0072] Single-beam gradient force traps have been demonstrated for
neutral atoms and dielectric particles. Generally, the single-beam
gradient force trap includes a strongly focused laser beam having
an approximately Gaussian transverse intensity profile. In these
traps, radiation pressure scattering and gradient force components
are combined to give a point of stable equilibrium located close to
the focus of the laser beam. Scattering force is proportional to
optical intensity and acts in the direction of the incident laser
light. Gradient force is proportional to the optical intensity and
points in the direction of the intensity gradient.
[0073] Particles in a single-beam gradient force trap are confined
transverse to the laser beam axis by a radial component of the
gradient force. Stabilizing the particle along the axis direction
of the trap is achieved by strongly focusing the laser beam to have
the axial component of gradient force dominate the scattering force
in the trap region.
[0074] The wavelengths of the laser light source of the optical
trap can be in the visible range, but are typically in the infrared
frequencies. One example of an optical trap laser is a standard
laser emitting a coherent light beam substantially in the infrared
range of wavelengths, for example, 0.8 .mu.m to 1.8 .mu.m. Optical
trapping can also be conducted using visible wavelengths for
example at 532 nm and 1064 nm, or using an argon ion laser (488 nm,
514 nm) or a HeNe laser (632 nm). The light beam from the laser
impinges upon a combination of optics elements for focusing the
light beam with a sufficient degree of convergence to form a
single-beam gradient force optical trap for confining particles at
a desired position. The combination of optics elements includes an
adjustably mounted diverging lens and a high convergence lens.
[0075] In another series of related embodiments, referred to herein
as "the hydrodynamically focusing methods," methods are provided to
control position-specific reactions using microfluidic alignment
and hydrodynamic focusing. These methods overcome the current need
for methods that are simpler, more efficient, and less costly.
"Hydrodynamic focusing" is a reduction in cross-sectional dimension
or area of a first flow along a flow axis by contacting the first
flow with two side flows within a channel, wherein the direction of
the two side flows is the same as the direction of the first flow.
The side flows are termed sheath flows and they "squeeze" the first
flow due to conservation of mass. The "side" flows need not
necessarily be in the lateral direction but can also be in the
vertical direction.
[0076] Suitable sources for introducing fluids into microfluidic
channels include, but are not limited to, pumps, such as syringes
or micro-fluidic pumps, and channels or chambers having sufficient
pressure to promote flow. Electrokinetic and electroosmotic flows
driven by voltages can also be used. Furthermore, electrodes can be
fabricated into the outlets to focus the stream as well. The fluids
may be introduced as either steady flows or intermittent flows (for
example discrete pulses). The flow rates of the fluids can be the
same or different.
[0077] A hydrodynamic focusing system can receive fluids from the
sources. The hydrodynamic focusing system includes a first
micro-fluidic inlet channel coupled with a first fluid source at a
first end thereof, and typically a second micro-fluidic inlet
channel coupled with a second fluid source at a first end thereof,
and a third micro-fluidic inlet channel coupled with a third fluid
source at a first end thereof. For example, the first fluid can be
a hydrodynamically-focused delivery flow, wherein the second and
third fluids can be protective flows. Other configurations of flows
is possible such as the 3-D focusing or using chimney structures
where the sample stream enters the bottom through the hole to the
main microfluidic channel.
[0078] The channels represent micro-sized fluid passages that can
have a cross-sectional dimension, such as a channel width, height,
or diameter, of less than approximately 1000 .mu.m, 500 .mu.m, or
100 .mu.m. These minute dimensions promote laminar flow, which is
conducive to hydrodynamic focusing. There is no known minimum or
maximum length for the channels, although often the lengths are in
a range between at least several times the channels widths and
several centimeters. The first inlet channel approaches the second
inlet channel from a first side thereof, at an angle, and the third
inlet channel, when present, typically approaches the second inlet
channel from a second, opposite, side thereof, at an angle.
[0079] A wide variety of focusing fluids can be employed. Examples
include but are not limited to water, aqueous solutions, organic
solvents, organic solutions, and mixtures thereof. The use of a
relatively viscous fluid may be appropriate to promote laminar flow
and reduce diffusion. Generally the fluids in the channels should
be compatible, relatively inert, and should not solidify.
[0080] The focusing fluids are received from the inlet channels
into a focusing manifold of the hydrodynamic focusing system. The
focusing manifold is coupled with second ends of each of the inlet
channels. The focusing manifold represents a junction where the
second ends of the inlet channels come together and join. The
fluids may be discharged from the channels into the focusing
manifold where they contact each other and hydrodynamic focusing is
initiated. Hydrodynamic focusing generally involves contacting a
plurality of flows in the focusing manifold and focusing or
otherwise reducing a cross-sectional dimension or area of one or
more flows along a flow axis in the confines of the hydrodynamic
focusing system. In a representative example of hydrodynamic
focusing, a delivery fluid and a protective fluid are contacted in
the focusing manifold. In the laminar flow regime, which generally
occurs in micro-fluidic channels and chambers, the fluids do not
mix significantly, but tend to come into alignment as side-by-side
co-axial flows. The laminar flow regime may be characterized by a
Reynolds number that is less than approximately 2300, 1000, 100,
10, 1, 0.1, or 0.01. A small amount of diffusion at interfaces
between the fluids may be tolerated. At contact, the fluids may
exert hydrodynamic forces or pressures on one another. Within the
confines of the hydrodynamic focusing system, for example at the
entrance to the outlet channel, the forces or pressures may focus
or otherwise reduce cross-sectional dimensions or areas of the
flows along a flow axis.
[0081] When fluids delivered through the inlet channels have
similar hydrodynamic forces, for example similar flow rates and
pressures, the regions occupied by the fluids in the outlet channel
tend to be similar. As shown in FIG. 5, each of the fluid flows
occupy approximately one-third proportion of the flow cross section
of the outlet channel, although this is not required. Optionally,
different hydrodynamic forces may be employed to allow one fluid to
occupy a larger or smaller region than another fluid. Increasing
the flow rate or pressure of one or more fluids generally increases
the amount of focusing and decreasing the flow rate or pressure of
one or more fluids generally decreases the amount of focusing.
[0082] The amount of focusing also depends upon, and tends to vary
inversely with, the cross-sectional area of the outlet channel
available for flow. The outlet channel can be dimensioned about the
same as any one of the inlet channels, although this is not
required. Increasing the cross-sectional area can decrease the
amount of focusing and decreasing the cross-sectional area can
increase the amount of focusing. By adjusting the dimensions of the
outlet channel, and the flow rates and pressures of the fluids, the
amount of focusing may be varied from a small amount to a large
amount.
[0083] Existing technologies attempt to direct a reactant to a
desired position by specific binding, such as by DNA hybridization
which requires specific sequences and special targets such as
single-stranded DNA. Furthermore, methods have been developed for
directing reactants to a subcellular position with laminar flow of
a width of about 10 .mu.m (Takayama et al, Nature, 2001, 411,
p1016). This technology uses hydrodynamic focusing to deliver a
reagent to one side of a cell that includes about a 10 .mu.m
section of the cell. However, this technology does not allow the
precision of focusing necessary for directing a reactant to a
specific region of another molecule.
[0084] Synchronization of multiple-molecule polymer reactions is
another unsolved problem. For example in exonuclease DNA
sequencing, also called direction DNA sequencing, individual DNA
molecules bind to an exonuclease with different rates due to their
un-controlled conformation. The difference in initial binding rate
is amplified during step-wise nucleotides removal, as the
nucleotide removal is faster than the initial binding (Wuite et al,
Nature, 2000, 404, p103-106). This rate difference makes it very
difficult to detect the removed nucleotides. For example, among 100
copies of a nucleic acid molecule having a sequence
5'-ATCGATACGATCG, at a particular timepoint during an exonuclease
reaction, some copies of the nucleic acid molecule may be releasing
the 3' terminal guanidine residue, while others may be releasing
the penultimate 3' terminal cytidine residue.
[0085] To synchronize nucleic acid sequencing reactions, the use of
thermostable and photoactivating enzymes has been disclosed (See
e.g., U.S. Pat. No. 5,674,743). However, thermostable or
photoactivating enzymes with exonuclease activity do not exist now.
Furthermore, even if such an enzyme is engineered, it will be very
difficult to limit the enzyme's catalytic activity to a certain
temperature range, or to a photoactivated state.
[0086] Another presently unsolved problem is that site-specific
reactions on molecules often give lower yield due to side
reactions. For example, in solid-phase oligo-nucleotide synthesis,
the per-step yield for short products (i.e. <50 nucleotides) can
be as high as 99%. However, the yield drops dramatically as the
length increases, for example to below 90% at lengths of over 100
nucleotides. Furthermore, side-products accumulate fast from
reactions in regions within the oligonucleotide chain, rather than
at the ends. When the length of a synthesized nucleic acid molecule
reaches 100 nucleotides, over 80% of the reaction products are
found to be side products due at least in part to the fact that
some ends of the nucleic acid molecule are buried in the polymer
chain. This limits the synthesis of long oligonucleotides and hence
their availability for various applications.
[0087] Provided herein are methods that overcome these shortcomings
of current methods, by using fluidic alignment to lock the
conformation and orientation of a reactant before confining a
reaction to a very small area (e.g., linear scale .about.0.1 .mu.m
for sequencing) by hydrodynamic focusing. This results in simpler,
more synchronized methods that provide higher yield and higher
purity products. Better synchronization is achieved because a
molecule can be delivered precisely to a desired reactive region
(i.e. a target region) of another molecule, and this target region
can be moved over time as a reaction proceeds. Higher yield is the
result of the fact that microfluidic alignment exposes an end of a
reactant when an end reaction is desired (e.g., exonuclease
reaction, carbon nanotube end modification), thereby enabling the
reagent to find the end easily. Higher purity is the result of the
fact that by focusing the reactant flow, side reactions on other
parts of a molecule can be minimized. Furthermore, the invention
methods are simpler than existing methods because fluidic control
allows continuous synthesis, rather than the multiple steps
required in most current methods.
[0088] Methods provided herein control position-specific reactions
using microfluidic alignment and hydrodynamic focusing. In order to
confine reactions to the desired position of molecules such as DNA,
proteins, and carbon nanotubes, the entire molecule or one end of
the molecule is immobilized on a surface. Microfluidic alignment
followed by drying is then used to "lock" the molecules in a
specific conformation. Once the molecules have been immobilized on
the surface in the desired location and conformation, microfluidic
hydrodynamic focusing can be utilized to direct a reagent
specifically to a desired position of the molecule. Furthermore,
the hydrodynamically focusing methods are useful to synchronize
nucleic acid sequencing reactions.
[0089] Not wanting to be limited by a particular theory, it is
believed that the invention method is based in part on the
following facts: (a) fluidic alignments of nucleic acids and
semiconductor nanowires have been reported in the literature; (b)
both double-stranded and single-stranded nucleic acids have been
aligned; (c) two and three-dimensional microfluidic focusing have
been developed (See e.g., U.S. patent application Ser. No.
10/609,227, filed Jun. 26, 2003, inventors Narayan Sundararajan and
Andrew Berlin); (d) Sub-micron fluidic focusing on a silicon chip
has been reported (Knight et al, "Hydrodynamic Focusing on silicon
chip", Phy. Rev. lett., 1998, 80, p3863-3866.); (e)
fluidic-controlled reaction in select regions of a cell has been
demonstrated (i.e. "Laminar flows--sub cellular positioning of
small molecules", Takayama et al, Nature, 2001, 411, p1016); and
(f) fluidic alignment of DNA on orthogonal directions has been
demonstrated (Wooley et al, Nano Lett., 2001, 1, p345-348;
illustrating that once the DNA is aligned and dried on surface, its
conformation is locked even if it is exposed to a 2.sup.nd fluid
flow that is to another direction).
[0090] In addition to the above facts, the methods are based, in
part, on the reactivity of surface-bonded DNA and protein molecules
has been demonstrated by various reactions that are performed on
DNA and protein chips (e.g. hybridizations and polymerizations).
Furthermore, exonuclease reactions on oligonucleotides that are
immobilized on surface have been successfully performed. These
studies indicate that enzymes (or reactants) react with immobilized
nucleic acids, proteins, or other molecules such as carbon
nanotubes, even when their conformations are locked.
[0091] Accordingly, provided herein is a method for contacting a
first molecule with a second molecule within a microfluidic device,
that includes delivering at least one hydrodynamically focused flow
through the microfluidic device, wherein the hydrodynamically
focused flow brings the second molecule into contact with the first
molecule only at a target region of the first molecule.
[0092] The methods provided are useful, for example, for improving
the yield of an oligonucleotide synthesis reaction, as illustrated
in FIG. 3. Accordingly, molecules contacted in methods disclosed
herein include reactants typically used in nucleic acid synthesis,
such as the well known phosphoramidite method. Typically, the first
molecule is a nucleotide or a growing nascent nucleic acid molecule
310 to which monomers 320, phosphoramidite nucleotides, (i.e. the
second molecule) are incorporated. A nascent nucleic acid molecule
310 that is attached to a solid support 330 can be aligned using
fluidic alignment (FIG. 3A). Fluidic alignment exposes the ends of
the nucleic acid molecules 310 (FIG. 3B). As a result of the
fluidic alignment, monomers (i.e. phosphoramidite nucleotides 320)
have better access to the end of the nucleic acid 310 (FIG. 3C),
which is expected to result in better yield.
[0093] As is well known, in the phosphoramidite method, a series of
deprotection, coupling, capping, and oxidation steps are repeated
until a single stranded nucleic acid molecule of interest is
synthesized. The nucleic acid molecule is formed 3' to 5', the
direction opposite that of nucleic acid synthesis within cells. The
first step typically utilizes a substrate that has a protected form
of the terminal (3') monomer chemically bound to its matrix. The
nascent oligonucleotide chain will stay attached to this substrate
as each activated monomer, one by one, is linked to its 3'
neighbor. The substrate is contained in a reaction vessel, such as
a microchannel, where it reacts with reagents released into the
reaction chamber at specific times throughout a synthesis cycle.
Protected monomers are typically deoxyribonucleoside
3-phosphoramidites containing dimethoxytrityl (DMT) blocking groups
on the 5'-oxygen atoms. These monomers are activated by treatment
with a weak acid prior to chain elongation. In methods of the
present invention, either the entire nucleic acid molecule
synthesis reaction, or parts of this reaction, are performed while
a fluid flow aligns a nascent nucleic acid molecule 310.
[0094] Referring to FIG. 4, hydrodynamic focusing methods can be
used in nucleic acid exonuclease sequencing reactions. The method
can be performed, for example, in a microfluidic device, such as a
MEMS device, that includes valves 420 for controlling the flow
between microchannels 410. Typically, a nucleic acid molecule 310
(i.e. a first molecule) is immobilized on a solid support 330.
Furthermore, the nucleic acid molecule 310 is aligned before it is
contacted with an exonuclease (i.e. a second molecule) (not shown).
Valves 1 and 3 are opened to allow for in situ nucleic acid
synthesis, such as disclosed in FIG. 3, or immobilization of a
nucleic acid molecule 310 on a solid support 330. For example, as
shown in FIGS. 4 B, nucleic acid molecules can be captured on the
solid support by flowing a solution that contains the nucleic acid
molecules through microchannels so that the nucleic acid molecules
contact the solid support 330. Various methods for immobilizing a
nucleic acid molecule 310 to a solid support 330 are known.
[0095] The captured nucleic acid molecules 310 are then aligned
using fluid flow (represented by arrows) and surface tension (FIG.
4C). If a nucleic acid molecule 310 is synthesized in situ within
the microfluidic channel, the nucleic acid molecule 310 is aligned
during synthesis, as disclosed with respect to FIG. 3. Alignment of
the nucleic acid molecule 310, in addition to facilitating nucleic
acid synthesis, facilitates accurate positioning of at least one
hydrodynamically focused flow at a target region of the nucleic
acid molecule 310. The target region, for example, can be the end
of the nucleic acid molecule 310. The microchannel is then dried to
"freeze" or "lock" the nucleic acid molecule 310 in the desired
aligned confirmation as illustrated in FIG. 4D. Valves 1 and 3 are
closed, and valves 2 and 4 are opened to dry the microchannel.
Next, exonuclease molecules, or other deconstruction reagents, in a
buffer are delivered to a 3' terminus of the nucleic acid molecule
310 in a delivery flow using two-dimensional hydrodynamic fluidic
focusing (FIG. 4E). A delivery flow is a flow that transports a
suspended molecule in a microchannel to a site of a reaction. In
this example, the delivery flow transports the deconstruction
reagent to the target region of the nucleic acid molecule. As
disclosed herein, for reactions involving terminal nucleotides of a
nucleic acid molecule 310, the hydronyamic flow can be focused, for
example, to a width of about 0.05 to about 0.15 microns (linear
scale), to include a 3' terminus of the nucleic acid molecule. Upon
exposure of a terminus of the nucleic acid molecule 310 to the
exonuclease molecules, terminal nucleotides 430 are cleaved by the
exonuclease molecules and carried downstream for analysis (FIG.
4F). Analysis is typically SERS analysis performed using a SERS
detection unit (not shown). Typically, only the target region of
the nucleic acid molecules 310 are digested, although it is
possible that a much lower rate of digestion can occur at
non-target regions of the nucleic acid molecule 310 if a small
number of exonuclease molecules escape from the hydrodynamically
focused flow.
[0096] Immobilized molecules such as nucleic acids, nanotubes, or
nucleic-acid wrapped nanotubes can be aligned using any of a number
of known techniques. An exemplary method for aligning nucleic acids
on a substrate is known as molecular combing. (See, e.g., Bensimon
et al., Phys. Rev. Lett. 74:4754-57, 1995; Michalet et al., Science
277:1518-23, 1997; U.S. Pat. Nos. 5,840,862; 6,054,327; 6,225,055;
6,248,537; 6,265,153; 6,303,296 and 6,344,319.) In this technique,
nucleic acids or other hydrophilic polymers are attached at one or
both ends to a substrate, such as a silicon chip. The substrate and
attached nucleic acids are immersed in a solution, such as an
aqueous buffer, and slowly withdrawn from the solution. The
movement of the air-water-substrate interface serves to align the
attached nucleic acids, parallel to the direction of movement of
the meniscus.
[0097] The method of polymer alignment used is not limiting and any
known method, including but not limited to use of optical tweezers,
DC and/or AC electrical fields, microfluidic flow, and/or magnetic
fields is contemplated. In another non-limiting example, nucleic
acids or other charged polymers can be aligned on a substrate by
free flow electrophoresis (e.g., Adjari and Prost, Proc. Natl.
Acad. Sci. U.S.A. 88:4468-71, 1991). The surface can comprise
alternating bands of conductive and non-conductive materials that
function as electrodes, or other types of microelectrodes can be
used. In the presence of an alternating current electrical field,
polymers including charged residues, such as the phosphate groups
on nucleic acids, will align with the field (Adjari and Prost,
1991). The method is not limited to nucleic acids and can be
applied to proteins or other polymers containing charged groups.
Where the charge on the polymer is not fixed, the net charge can be
manipulated, for example by changing the pH of the solution.
Fluidic alignment of various types of polymer molecules (i.e.
molecular wires or concatenated molecular chains), has been
demonstrated (Bensimon et al., Science, 265: 1096-98 (1994) (double
stranded DNA); Lieber et al., Science, 291:630 (2001)(semiconductor
nanowires); Lienemann et al., Nanoletters, 1:345 (2001)
(single-stranded DNA)).
[0098] In certain specific examples, the dynamic focusing
conditions are changed over time to move the target region to focus
on the retracting terminus of the nucleic acid molecule 310 as
terminal nucleotides 430 are cleaved by the exonuclease. This is
expected to result in improved synchronization of the cleavage
reaction because the exonuclease is delivered precisely to a
desired reactive region (i.e. target region) of the nucleic acid
molecule 310 as the terminus of the nucleic acid molecule 310
shortens. For example, the reaction can be performed in phases with
a different target region for each phase.
[0099] The target region of a dynamically focused flow can be
changed by changing the reaction conditions. Reaction conditions
can be changed, for example, by changing the pressure of the side
streams. Accordingly, side stream pressure can be reduced in a
second phase, to enlarge the width of the hydrodynamically focused
flow (FIG. 4G). This results in cleavage of nucleotides in a second
target region. For example, if the fluid flow is widened by
approximately 0.05 microns, during the second phase an additional
150 bases will be sequentially cleaved from the 3' terminus of the
nucleic acid molecule 310. After the second phase reaction stops,
additional phases can be performed using increasing
hydrodynamically focused flow widths (FIG. 4 H), to cleave
additional nucleotides. In order to minimize binding of reactants
to walls of the microfluidic channels, the side streams can be
protective streams.
[0100] As indicated above, the width and/or diameter of the
hydrodynamic flow can be controlled by changing pressure of side
streams that are used to focus the hydrodynamically focused
delivery stream. Furthermore, the target area can be changed, for
example, by increasing the pressure of one side flow, and reducing
the pressure of another side flow, by moving the delivery of the
flow within the channel, or by changing the viscosity of one or
more of the flows.
[0101] In another aspect, electrodes are used to change the
focusing characteristics and position as well (Wang et al.,
"Electrical Molecular Focusing For Laser Induced Fluorescence Based
Single DNA Detection", Technical Digest of the 15th IEEE
International Conference on MEMS (ISBN-0-7803-7187-9) (MEMS 2002)).
The electrodes can be arranged to provide 3-D electric molecular
focusing. A middle electrode can be applied with positive potential
and two side electrodes can be grounded. Negatively charged
biomolecules, such as DNA molecules and most proteins, are
concentrated to the middle electrode. One advantage of electrode
focusing is that it does not require continuous flow.
[0102] The width or diameter of the hydrodynamic focused flow in
the methods provided is typically less than 10 microns, and in
certain aspects is less than 5, 4, 3, 2, 1, 0.5, 0.4, 0.3, and 0.2
microns (linear scale), but at least 0.05 microns (linear scale).
For certain aspects of the invention for focusing a flow on a
nucleic acid molecule, such as nucleic acid synthesis reactions and
exonuclease sequencing reactions, a flow is focused to between
about 0.5 and about 0.05 microns (linear scale), between about 0.2
and about 0.1 microns (linear scale), or to about 0.1 microns
(linear scale). Finer focusing can likely be obtained using
electrode focusing.
[0103] In another aspect, methods provided herein are useful for
labeling nanotubes. Accordingly, in these aspects the first
molecule is a nanotube and the second molecule is a label. For
these aspects, the hydrodynamically focused flow is targeted at an
end of the nanotube. Therefore, the methods are useful for
directing labels to specific target regions of nanotubes. The width
of the focused flow is typically between about 0.05 and 0.2
microns. The labels can include virtually any label known to be
associated with nanotubes, including the disclosed Raman labels.
These aspects are useful for example, to label the ends of
nanotubes or to functionalize nanotubes with DNA. Furthermore, both
a functionalization reaction and attachment can be performed using
the same apparatus. These aspects can also be used for cutting
nanotubes at particular lengths after alignment and
immobilization.
[0104] Nanotubes can be made in a variety of shapes and sizes.
(See, e.g., Freitag et al., Phys. Rev. B 62:R2307-R2310, 2000;
Clauss et al., Europhys. Lett. 47:601-607, 1999; Clauss et al.,
Phys. Rev. B. 58:R4266-4269, 1998; Odom et al., Ann. N.Y. Acad.
Sci. 960:203-215, 2002). Nanotubes can have tube lengths of about
10 to 200 nm and a diameter of about 1.2 to 1.4 nm. The length or
diameter of the nanotubes to be used in methods of the present
invention is not limited and nanotubes of virtually any length or
diameter are contemplated. However, in a labeling method wherein
nanotubes are aligned and then labeled, nanotubes that are labeled
typically have a similar length (e.g. within 25 nm of each other)
so that a hydrodynamically focusing flow can contact the ends of
all the nanotubes.
[0105] Nanotubes can be prepared by known methods or obtained from
commercial sources, for example, CarboLex (Lexington, Ky.), NanoLab
(Watertown, Mass.), Materials and Electrochemical Research (Tucson,
Ariz.) or Carbon Nano Technologies Inc. (Houston, Tex.). Some
processing of either synthesized or purchased nanotubes may be
appropriate before use. Processing may include purification of
nanotubes from other contaminants, separation of nanotubes of mixed
diameter and/or length into nanotubes of discrete diameter and
length, and removal of nanotube end caps.
[0106] Carbon nanotubes can be produced by a variety of techniques
known in the art, including but not limited to carbon-arc
discharge, chemical vapor deposition via catalytic pyrolysis of
hydrocarbons, plasma assisted chemical vapor deposition, laser
ablation of a catalytic metal-containing graphite target, or
condensed-phase electrolysis. (See, e.g., U.S. Pat. Nos. 6,258,401,
6,283,812 and 6,297,592.) In some embodiments, nanotubes may be
size sorted by mass spectrometry (See, Parker et al., J. Am. Chem.
Soc. 113:7499-7503, 1991) before they are attached to a surface,
aligned, and end labeled. Alternatively, nanotubes may be sorted
using an AFM (atomic force microscope) or STM (scanning tunneling
microscope) to precisely measure the geometry of individual
nanotubes before labeling them. Other methods of size fractionation
known in the art, such as gas chromatography, time of flight mass
spectrometry, ultrafiltration or equivalent techniques are
contemplated. Once sorted, the carbon nanotubes can be attached to
a label.
[0107] In another embodiment, provided herein are methods to
prevent non-specific binding of proteins and other biomolecules or
particles on surfaces of microfluidic channels. The methods involve
using one or more laminar flows (protective flows) in a
microfluidic channel to protect one or more surfaces of the channel
from non-specific binding of biomolecules or particles. The
protective flows are adjacent to the surfaces where non-specific
binding is not desired, such as a surface of a microfluidic
channel, and have thicknesses that can be controlled. Typically,
the first molecule is immobilized on a solid support before it is
The protective flow can contain reduced levels of, or be free of
active biomolecules or particles that could bind to the specific
surfaces. Furthermore, the protective flow can include non-specific
blocking agents, such as bovine serum albumin or salmon sperm DNA,
as are known in the art.
[0108] Accordingly, in another embodiment, provided herein is a
method for contacting a first molecule with a second molecule
within a microfluidic device, including delivering through the
microfluidic device, at least one hydrodynamically focused delivery
flow having the first molecule and/or the second molecule suspended
therein, and at least one protective flow that at least partially
inhibits the second molecule from contacting a surface of the
microfluidic device. The protective flow(s) typically run parallel
and restrict the flow of the delivery flow, as shown in FIGS. 5A,
5B, and 6. The first molecule can be immobilized on the surface of
the microfluidic device and stretched, before and while it is
contacted with the second molecule in the delivery flow.
[0109] The protective flow can block at least some or all
detectable non-specific binding of the second molecule to a surface
of the microfluidic device. Furthermore, the protective flow
typically inhibits the delivery flow from contacting at least one
surface of the microfluidic device. In addition, the protective
flow typically includes an undetectable amount of the first
molecule or the second molecule. A second protective flow that at
least partially inhibits the second molecule from contacting a
second surface of the microfluidic device can also be provided.
[0110] In one specific example, a method is provided to inhibit
non-specific binding in a microfluidic device, including
[0111] a) immobilizing a first molecule on a surface of the
microfluidic device;
[0112] b) stretching the first molecule;
[0113] c) delivering at least one protective flow through the
microfluidic device; and
[0114] d) delivering through the microfluidic device, at least one
hydrodynamically-focused delivery flow having a second molecule
suspended therein, wherein the protective flow inhibits the
delivery flow from contacting the surface of the microfluidic
device as the first molecule binds to the second molecule.
[0115] Biomolecules or particles for bioreactions or specific
bindings are delivered by one or more separate laminar flows
(delivery flows) into a microfluidic channel. Delivery flows, such
as hydrodynamically focused delivery flows as disclosed herein, are
separated from the specific surfaces of the channel by protective
flows. It has been demonstrated that two adjacent laminar flows
containing different molecules, will not mix. For these aspects,
target biomolecules are typically immobilized in a channel through
various linkers as is known in the art, (e.g. DNA with one end
immobilized on a surface and the other linked with the target
biomolecules).
[0116] A first molecule, such as a target biomolecule, is held in
position in the delivery flow by stretching the target biomolecule.
This stretching can be the result of forces known in the art, such
as electrical fields, magnetic fields (with magnetic nanoparticles
attached to the molecules) or optical manipulation (with
polystyrene beads attached to the molecules). For example, methods
disclosed herein for aligning molecules can be used to stretch
molecules.
[0117] The protective flow can surround a delivery flow to focus
the delivery flow, thereby providing for more efficient molecule
delivery of a second molecule to the target molecule (i.e. first
molecule), as disclosed above related to hydrodynamic focusing
methods. Furthermore, the protective flow at least partially
inhibits, and in some aspects completely blocks, the delivery flow,
and molecules suspended in the delivery flow, from contacting a
surface of the microfluidic device. In addition, methods herein can
include a second protective flow, wherein the first and second
protective flows are adjacent to opposite surfaces of a delivery
flow. It has been demonstrated that two adjacent laminate flows
containing different molecules will not mix.
[0118] A wide variety of solutions can be delivered in the
protective flows, provided that the solutions do not increase
background values. For example, a protective flow can contain a
buffer solution, and optionally can include blocking agents, for
example BSA. It will be understood that a specific formulation of a
protective flow depends on the specific reaction being performed.
For example, standard ELISA concentrations of BSA blocking can be
used for methods involving antibody-antigen binding.
[0119] With reference to FIGS. 5-7, target molecules (e.g. nucleic
acid molecules 310) are immobilized at one end on an inner surface
650 of a microfluidic channel 410 by, for example, to a gold coated
region 540 of the surface 650 of the channel 410 by thiol-gold
interaction. The other end of the nucleic acid molecules 310 is
associated with a magnetic molecule 510, such as a magnetic
nanoparticle (FIG. 5A) or a charged moiety (FIG. 5B). The magnetic
nanoparticle or the charged moiety can be used to stretch the
target nucleic acid molecule 310 strands under a magnetic field or
an electrical field, respectfully. The magnetic field is generated
by a magnet 520 which is held near one side of the microfluidic
channel 410. As shown in FIG. 5B, an electrical field is generated
by a power source 560, 570 that is used to provide a current across
the microfluidic channel 410. A delivery flow 360 is confined to
the center of the channel by the protective flows 350. Therefore,
the active biomolecules or particles in the delivery flow 360 can
only reach the center part of the target molecule 310 strand that
is suspended in the center region of the channel 410. This helps to
reduce contact, and therefore non-specific binding, to the surfaces
of the channel 410 by biomolecules or particles in the delivery
flow 360.
[0120] FIG. 6 illustrates a microfluidic system for performing an
illustrative method provided herein that includes delivery of a
protective flow to reduce non-specific binding to a surface in a
labeling reaction. As discussed herein, these methods typically
involve an immobilizing a molecule, such as a specific binding pair
member, and delivery of a second specific binding pair member in a
delivery flow, to the immobilized molecule. In the example provided
in FIG. 6, a buffer solution 620 flows in a microfluidic channel
410 formed from a flat substrate 610 and a molded piece 630 made
for example of PDMS. A nucleic acid molecule 310 is immobilized at
one end on a surface 650, such as a gold surface, in the
microfluidic channel 410. The other end of the nucleic acid
molecule 310 is attached to a particle 670, such as a poly-styrene
bead, which can be manipulated by optical tweezers 640. Several
biotin moieties 680 are attached to the middle section of the
nucleic acid molecule 310. Under the manipulation of optical
tweezers 640, the nucleic acid molecule 310 is stretched, thereby
causing the biotin moieties 680 to be suspended near the center of
the channel. A delivery flow 360 that contains avidin-ferritin
conjugates 660 (i.e. second molecules) is then introduced into the
channel, but is confined to the upper (or center, if the geometry
in FIGS. 5A and 5B is used) part of the channel. The bottom portion
(i.e. substrate surface) of the channel is protected by the
protective flow of a buffer solution 350. Accordingly, the
avidin-ferritin conjugates 660 bind to the biotin-molecules 680 on
the center segment of the suspended nucleic acid molecule.
[0121] To confirm that the avidin-ferritin conjugate 660 binding
occurred, the substrate 610 can be rinsed and the channel 410
dried. After peeling off the PDMS piece 630, ferritin molecules 660
that are bound to the biotin moieties 680 on the nucleic acid
molecule 310 are detected on a clean substrate surface 650.
[0122] The width and/or diameter of the protective flow can be
controlled by changing the force used to produce the protective
flow, by changing the pressure of an adjacent flow, or by changing
the viscosity of the protective flow, as discussed above for
delivery flows. The width or diameter of the one or more protective
flows in the methods provided herein is typically less than 10
microns, and in certain examples is less than 5, 4, 3, 2, 1, and
0.5, microns (linear scale), but at least 0.2 microns (linear
scale).
[0123] As indicated, the protective flows inhibit the delivery flow
from contacting the surface of the microfluidic device as a first
molecule binds to a second molecule. Typically, this inhibition is
the result of the protective flows flowing between the delivery
flow and the surface of a microfluidic channel. Therefore, the
protective flow continues to flow as the first molecule binds the
second molecule. The protective flow typically continues to flow
through the binding reaction of the first molecule and the second
molecule.
[0124] Protective flows can be used to inhibit non-specific binding
to microchannel surfaces in virtually any binding reaction where it
is desirable to block non-specific binding to a surface of a
reaction microchannel. For example, protective flows can be used in
labeling reactions, immunoassays and receptor/ligand binding
assays. The use of protective flows in labeling reactions is
illustrated in FIGS. 5A-5B and 6, as discussed above. Regarding the
use of protective flows in immunoassays, an antibody can be
immobilized on the surface of the microchannel and stretched. Then,
a sample suspected of containing an antigen, such as a biological
sample, can be delivered in a delivery flow to the antibody binding
region of the antibody, wherein a protective flow separates the
delivery flow from the surface. Regarding the use of protective
flows in receptor/ligand binding reactions, a receptor can be
immobilized on the surface of a microchannel and stretched. Then a
sample suspected of containing a ligand, such as a biological
sample, can be delivered in a delivery flow to the ligand binding
region of the receptor, wherein a protective flow separates the
delivery flow from the surface. As another example, a nucleic acid
strand with binding agents at specific locations along the nucleic
acid, can be reacted with gold nanoparticles such that the gold
nanoparticles are focused away from the surface for further sample
interrogation using scanning probe microscopy techniques.
[0125] The following general teachings provided further details
regarding the methods, systems, apparatuses disclosed herein. One
or more molecules analyzed in the methods provided herein can be
labeled with a Raman label. With respect to nucleic acids,
typically the nucleic acid molecules are labeled before they are
attached to a particle in embodiments that utilize a restriction
barrier. Some or all of the nucleotides of a nucleic acid molecule
can be labeled. For example only purine residues of a nucleic acid
molecule can be labeled with a Raman label. Methods for determining
a nucleotide sequence can be repeated several times with different
labeled copies of a nucleic acid molecule or with different strands
of the nucleic acid molecule, to obtain addition and possibly
complete sequence information.
[0126] A Raman label can be any organic or inorganic molecule,
atom, complex or structure capable of producing a detectable Raman
signal, including but not limited to synthetic molecules, dyes,
naturally occurring pigments such as phycoerythrin, organic
nanostructures such as C.sub.60, buckyballs and carbon nanotubes or
nanoprisms and nano-scale semiconductors such as quantum dots.
Numerous examples of Raman labels are disclosed below. The skilled
artisan will realize that such examples are not limiting, and that
a Raman label can encompasses any organic or inorganic atom,
molecule, compound or structure known in the art that can be
detected by Raman spectroscopy.
[0127] Non-limiting examples of labels that can be used for Raman
spectroscopy include TRIT (tetramethyl rhodamine isothiol), NBD
(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,
terephthalic acid, isophthalic acid, cresyl fast violet, cresyl
blue violet, brilliant cresyl blue, para-aminobenzoic acid,
erythrosine, biotin, digoxigenin,
5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein,
5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein,
5-carboxy rhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino
phthalocyanines, azomethines, cyanines, xanthines,
succinylfluoresceins and aminoacridine. Polycyclic aromatic
compounds in general can function as Raman labels, as is known in
the art. These and other Raman labels can be obtained from
commercial sources (e.g., Molecular Probes, Eugene, Oreg.).
[0128] Other labels that can be of use include cyanide, thiol,
chlorine, bromine, methyl, phosphorus and sulfur. Carbon nanotubes
can also be of use as Raman labels. The use of labels in Raman
spectroscopy is known (e.g., U.S. Pat. Nos. 5,306,403 and
6,174,677). The skilled artisan will realize that Raman labels
should generate distinguishable Raman spectra when bound to
different types of nucleotide.
[0129] Labels can be attached directly to a nucleotide or other
biomolecule, or can be attached via various linker compounds. Raman
labels that contain reactive groups designed to covalently react
with other molecules, are commercially available (e.g., Molecular
Probes, Eugene, Oreg.).
[0130] In many of the methods provided herein, a reaction chamber
or reaction area of a channel contains an immobilized nucleic acid
molecule and a deconstruction reagent, such as an exonuclease. The
exonuclease catalyzes the sequential release of individual
nucleotides from the free end of the nucleic acid molecule. As the
individual nucleotides are released by the deconstruction reaction
and enter solution, they move down the flow path past a SERS
detection unit. The detection unit includes an excitation source,
such as a laser, that emits an excitatory beam. The excitatory beam
interacts with the released nucleotides, and/or labels attached to
the released nucleotides, so that electrons are excited to a higher
energy state. The Raman emission spectrum that results from the
return of the electrons to a lower energy state is detected by a
Raman spectroscopic detector, such as a spectrometer or a
monochromator.
[0131] The released nucleotides are spatially separated from the
nucleic acid molecule before detection by the detection unit.
Spatial separation acts to increase the signal-to-noise ratio of
the Raman detector by isolating the individual nucleotides.
[0132] In embodiments herein, the nucleic acid molecule is fixed in
place, as by attachment to an immobilization structure, such as by
being captured in a restriction barrier, and immersed in a
microfluidic flow down a flow path that transports the released
nucleotides away from the nucleic acid molecule and past a
detection unit. In non-limiting examples, the microfluidic flow may
result from a bulk flow of solvent past the nucleic acid molecule
and down a flow path, for example, a microcapillary tube or an
etched channel in a silicon, glass or other chip. In alternative
embodiments, the bulk medium moves only slowly or not at all, but
charged species within the solution (such as negatively charged
nucleotides) move down a flow path comprising a channel or tube in
response to an externally applied electrical field.
[0133] In the embodiments discussed above, the detection unit must
be capable of distinguishing between the common nucleotides
released from the nucleic acid molecule. At a minimum, the
detection unit must be able to distinguish between nucleotides
containing adenosine (A), guanosine (G), cytosine (C) and thymidine
(T) for sequencing DNA molecules. If RNA is being sequenced, the
detection unit must be able to distinguish between nucleotides
containing A, G, C and uridine (U). With a single nucleic acid
molecule per reaction chamber, it is not necessary that the
detection unit be capable of quantifying the amounts of each
nucleotide in solution, since the nucleotides move past the
detection unit 18 one at a time.
[0134] The skilled artisan will realize that analysis of DNA, will
result in the release of deoxyribonucleosides or
deoxyribonucleotides, (including thymidine), while analysis of RNA
will result in the release of ribonucleosides or ribonucleotides
(including uridine). Although nucleoside monophosphates will
generally be the form released by exonuclease activity, the
embodiments are not limited to detection of any particular form of
free nucleotide or nucleoside but encompass any monomer that may be
released from a nucleic acid by the activity of a deconstruction
reagent.
[0135] In some embodiments of the invention, a method disclosed
herein can be performed in a micro-electro-mechanical system
(MEMS). MEMS are integrated systems that include mechanical
elements, sensors, actuators, and electronics. All of those
components can be manufactured by known microfabrication techniques
on a common chip, including a silicon-based or equivalent substrate
(e.g., Voldman et al., Ann. Rev. Biomed. Eng. 1:401-425, 1999). The
sensor components of MEMS can be used to measure mechanical,
thermal, biological, chemical, optical and/or magnetic phenomena.
The electronics can process the information from the sensors and
control actuator components such pumps, valves, heaters, coolers,
filters, etc. thereby controlling the function of the MEMS.
[0136] The electronic components of MEMS can be fabricated using
integrated circuit (IC) processes (e.g., CMOS, Bipolar, or BICMOS
processes). They can be patterned using photolithographic and
etching methods known for computer chip manufacture. The
micromechanical components can be fabricated using compatible
"micromachining" processes that selectively etch away parts of the
silicon wafer or add new structural layers to form the mechanical
and/or electromechanical components.
[0137] Basic techniques in MEMS manufacture include depositing thin
films of material on a substrate, applying a patterned mask on top
of the films by photolithographic imaging or other known
lithographic methods, and selectively etching the films. A thin
film can have a thickness in the range of a few nanometers to 100
micrometers. Deposition techniques of use may include chemical
procedures such as chemical vapor deposition (CVD),
electrodeposition, epitaxy and thermal oxidation and physical
procedures like physical vapor deposition (PVD) and casting.
[0138] In some embodiments of the invention, MEMS devices include
various fluid filled compartments, such as microfluidic channels,
nanochannels and/or microchannels. These and other components of
the apparatus can formed as a single unit, for example in the form
of a chip as known in semiconductor chips and/or microcapillary or
microfluidic chips. Alternatively, an immobilization substrate,
such as a metal coated porous silicon substrate, can be removed
from a silicon wafer and attached to other components of an
apparatus. Any materials known for use in such chips may be used in
the disclosed apparatus, including silicon, silicon dioxide,
silicon nitride, polydimethyl siloxane (PDMS),
polymethylmethacrylate (PMMA), plastic, glass, quartz.
[0139] Techniques for batch fabrication of chips are well known in
the fields of computer chip manufacture and/or microcapillary chip
manufacture. Such chips may be manufactured by any method known in
the art, such as by photolithography and etching, laser ablation,
injection molding, casting, molecular beam epitaxy, dip-pen
nanolithography, chemical vapor deposition (CVD) fabrication,
electron beam or focused ion beam technology or imprinting
techniques. Non-limiting examples include conventional molding with
a flowable, optically clear material such as plastic or glass;
photolithography and dry etching of silicon dioxide; electron beam
lithography using polymethylmethacrylate resist to pattern an
aluminum mask on a silicon dioxide substrate, followed by reactive
ion etching. Known methods for manufacture of nanoelectromechanical
systems may be used for certain embodiments of the invention. (See,
e.g., Craighead, Science 290: 1532-36, 2000.) Various forms of
microfabricated chips are commercially available from, e.g.,
Caliper Technologies Inc. (Mountain View, Calif.) and ACLARA
BioSciences Inc. (Mountain View, Calif.).
[0140] In certain embodiments of the invention, part or all of the
apparatus can be selected to be transparent to electromagnetic
radiation at the excitation and emission frequencies used for Raman
spectroscopy, such as glass, silicon, quartz or any other optically
clear material. For fluid-filled compartments that may be exposed
to various biomolecules, such as proteins, peptides, nucleic acids,
nucleotides and the like, the surfaces exposed to such molecules
may be modified by coating, for example to transform a surface from
a hydrophobic to a hydrophilic surface and/or to decrease
adsorption of molecules to a surface. Surface modification of
common chip materials such as glass, silicon, quartz and/or PDMS is
known in the art (e.g., U.S. Pat. No. 6,263,286). Such
modifications may include, but are not limited to, coating with
commercially available capillary coatings (Supelco, Bellafonte,
Pa.), silanes with various functional groups such as
polyethyleneoxide or acrylamide, or any other coating known in the
art.
[0141] In certain aspects, the systems and apparatus provided
herein are microfluidic devices that include a micromold.
Techniques such as soft lithography and photolithography, which
have been used in the semiconductor industry, can be used to
fabricate micromold of microfluidic device. For example, designs of
micromold can be drawn to scale using CAD software. The designs can
then printed onto transparencies using a high-resolution printer to
form a transparency mask. "Photoresist on Silicon" masters for
micromolding can then prepared by standard photolithographic
techniques using the transparency masks and a photoresist. These
patterned masters can then silanized and used for micromolding with
a silicone material such as poly(dimethyl siloxane) (PDMS). For
example, PDMS precursor can be poured onto the silanized master and
then cured. The cured PDMS containing the channel structure can
then bonded to the supporting surface by applying pressure to
enclose the channels. Typically, the microchannel pathways are
approximately 100 microns in width and between about two
centimeters and about three centimeters in length.
[0142] The substrate can also be prepared using standard
lithographic techniques. For example, a photoresist can be
deposited on substrate support surface and exposed through a mask.
The exposed photoresist can be developed. A suitable heating
element or substrate material can be deposited by, for example,
sputter deposition. In one embodiment, a thin layer of titanium or
chromium having a thickness of about 80 .ANG. is deposited,
followed by subsequent deposition of a thin layer of gold having a
thickness of about 240 .ANG.. The photoresist is then lifted off of
substrate support surface, thereby providing a substrate and/or
heating element on the substrate support surface.
[0143] The nucleotide is detected in the methods provided herein,
by SERS using a Raman detection unit. The Raman detection unit
includes a laser excitation and a wavelength selective detector.
The light source is typically a laser light, as known in the art
and discussed in more detail herein. Light from the light source is
projected at the first specific binding pair member and detected by
the detector.
[0144] The detection unit includes an excitation source, such as a
laser, and a Raman spectroscopy detector. The excitation source
illuminates the reaction chamber or channel with an excitation
beam. The excitation beam interacts with the first specific binding
pair member, resulting in the excitation of electrons to a higher
energy state. As the electrons return to a lower energy state, they
emit a Raman emission signal that is detected by the Raman
detector.
[0145] Data can be collected from a detector, such as a
spectrometer or a monochromator array and provided to an
information processing and control system. The information
processing and control system can perform standard procedures known
in the art, such as subtraction of background signals. Furthermore,
the information processing and control system can analyze the data
to determine nucleotide sequence information from detected signals
and the temporal relationship of these signals.
[0146] The nucleotide detection and/or sequencing reaction of
methods provided herein involves binding of a deconstruction
reagent to the free end of the nucleic acid molecule and removal of
nucleotides one at a time. In certain embodiments the reaction may
be catalyzed by an enzyme, such as an exonuclease. The embodiments
are not limited by the type of exonuclease that may be used.
Non-limiting examples of exonucleases of potential use include E.
coli exonuclease I, III, V or VII, Bal exonuclease, mung bean
exonuclease, S1 nuclease, E. coli DNA polymerase I holoenzyme or
Klenow fragment, RecJ, exonuclease T, T4 or T7 DNA polymerase, Taq
polymerase, exonuclease T7 gene 6, snake venom phosphodiesterase,
spleen phosphodiesterase, Thermococcus litoralis DNA polymerase,
Pyrococcus sp. GB-D DNA polymerase, lambda exonuclease, S. aureus
micrococcal nuclease, DNase I, ribonuclease A, T1 micrococcal
nuclease, or other exonucleases known in the art. Exonucleases are
available from commercial sources such as New England Biolabs
(Beverly, Mass.), Amersham Pharmacia Biotech (Piscataway, N.J.),
Promega (Madison, Wis.), Sigma Chemicals (St. Louis, Mo.) or
Boehringer Mannheim (Indianapolis, Ind.).
[0147] The skilled artisan will realize that enzymes with
exonuclease activity have various properties, for example, they can
remove nucleotides from the 5' end, the 3' end, or either end of
the nucleic acid molecule. They can show specificity for RNA, DNA
or both RNA and DNA. Their activity may depend on the use of either
single or double-stranded nucleic acids. They may be differentially
affected by various characteristics of the reaction medium, such as
salt, temperature, pH, or divalent cations. These and other
properties of the various exonucleases and polymerases are known in
the art.
[0148] The skilled artisan will realize that the rate of
exonuclease activity may be manipulated to coincide with the
optimal rate of analysis of nucleotides by the detection unit.
Various methods are known for adjusting the rate of exonuclease
activity, including adjusting the temperature, pressure, pH, salt
concentration or divalent cation concentration in the reaction
chamber. Methods of optimization of exonuclease activity are known
in the art.
[0149] Surfaces of the reaction chamber, reaction area, and/or flow
path that are opposite the detection unit can be coated with
silver, gold, platinum, copper, aluminum or other materials that
are relatively opaque to the detection unit. In that position, the
opaque material is available to enhance the Raman or other signal,
for example by surface enhanced Raman spectroscopy, while not
interfering with the function of the detection unit. Alternatively,
the reaction chamber and/or flow path can contain a mesh comprising
silver, gold, platinum, copper or aluminum. The skilled artisan
will realize that in embodiments involving a flow path, the
nucleotides will generally be detected while they are in the flow
path. In embodiments without a flow path, the nucleotides will be
detected in the reaction chamber.
[0150] The reaction chamber can have an internal volume of about 1
picoliter, about 2 picoliters, about 5 picoliters, about 10
picoliters, about 20 picoliters, about 50 picoliters, about 100
picoliters, about 250 picoliters, about 500 picoliters, about 1
nanoliter, about 2 nanoliters, 5 nanoliters, about 10 nanoliters,
about 20 nanoliters, about 50 nanoliters, about 100 nanoliters,
about 250 nanoliters, about 500 nanoliters, about 1 microliter,
about 2 microliters, about 5 microliters, about 10 microliters,
about 20 microliters, about 50 microliters, about 100 microliters,
about 250 microliters, about 500 microliters, or about 1
milliliter.
[0151] Free nucleotides after release from the restriction barrier
are moved down a flow path past the detection unit. Non-limiting
example of techniques for transport of free nucleotides includes
microfluidic techniques. The flow path can comprise a
microcapillary (available, e.g., from ACLARA BioSciences Inc.,
Mountain View, Calif.) or a liquid integrated circuit (e.g.,
Caliper Technologies Inc., Mountain View, Calif.). Such
microfluidic platforms require only nanoliter volumes of
sample.
[0152] In certain embodiments, the free nucleotides to be detected
move down the flow path by bulk flow of solvent. In other
embodiments, microcapillary electrophoresis is used to transport
free nucleotides down the flow path and past the detection unit.
Microcapillary electrophoresis generally involves the use of a thin
capillary or channel that may or may not be filled with a
particular separation medium. Electrophoresis of appropriately
charged molecular species, such as negatively charged nucleotides,
occurs in response to an imposed electrical field, negative on the
reaction chamber side of the apparatus and positive on the
detection unit side. Although electrophoresis is often used for
size separation of a mixture of components that are simultaneously
added to the microcapillary, it can also be used to transport
similarly sized nucleotides that are sequentially added to the flow
path. Because the purine nucleotides (A, G) are larger than the
pyrimidine nucleotides (C, T, U) and would therefore migrate more
slowly, the length of the flow path and corresponding transit time
past the detector unit should be kept to a minimum to prevent
differential migration from mixing up the order of nucleotides
released from the nucleic acid. Alternatively, the separation
medium filling the microcapillary may be selected so that the
migration rates of purine and pyrimidine nucleotides down the flow
path are similar or identical. Methods of microcapillary
electrophoresis have been disclosed, for example, by Woolley and
Mathies (Proc. Natl. Acad. Sci. USA 91:11348-352, 1994).
[0153] In various embodiments provided herein, nucleic acid
molecules to be sequenced or otherwise analyzed, or other
biomolecules, can be attached to a solid surface (or immobilized).
Immobilization of nucleic acid molecules can be achieved by a
variety of methods involving either non-covalent or covalent
attachment between the nucleic acid molecule and the surface. For
example, immobilization can be achieved by coating a surface with
streptavidin or avidin and the subsequent attachment of a
biotinylated nucleic acid 13, 102 (Holmstrom et al., Anal. Biochem.
209:278-283, 1993). Immobilization can also occur by coating a
silicon, glass or other surface 14, 103 with poly-L-Lys (lysine) or
poly L-Lys, Phe (phenylalanine), followed by covalent attachment of
either amino- or sulfhydryl-modified nucleic acids using
bifunctional crosslinking reagents (Running et al., BioTechniques
8:276-277, 1990; Newton et al., Nucleic Acids Res. 21:1155-62,
1993). Amine residues can be introduced onto a surface 14, 103
through the use of aminosilane for cross-linking.
[0154] Immobilization can take place by direct covalent attachment
of 5'-phosphorylated nucleic acids to chemically modified surfaces
14, 103 (Rasmussen et al., Anal. Biochem. 198:138-142, 1991). The
covalent bond between the nucleic acid 13, 102 and the surface 14,
103 is formed by condensation with a water-soluble carbodiimide.
This method facilitates a predominantly 5'-attachment of the
nucleic acids 13, 102 via their 5'-phosphates.
[0155] DNA is commonly bound to glass by first silanizing the glass
surface, then activating with carbodiimide or glutaraldehyde.
Alternative procedures can use reagents such as
3-glycidoxypropyltrimethoxysilane (GOP) or
aminopropyltrimethoxysilane (APTS) with DNA linked via amino
linkers incorporated either at the 3' or 5' end of the molecule.
DNA can be bound directly to membrane surfaces using ultraviolet
radiation. Other non-limiting examples of immobilization techniques
for nucleic acids are disclosed in U.S. Pat. Nos. 5,610,287,
5,776,674 and 6,225,068.
[0156] The type of surface to be used for immobilization of the
nucleic acid is not limiting. In various embodiments, the
immobilization surface can be magnetic beads, non-magnetic beads, a
planar surface, a pointed surface, or any other conformation of
solid surface comprising almost any material, so long as the
material is sufficiently durable and inert to allow the nucleic
acid sequencing reaction to occur. Non-limiting examples of
surfaces that can be used include glass, silica, silicate, PDMS,
silver or other metal coated surfaces, nitrocellulose, nylon,
activated quartz, activated glass, polyvinylidene difluoride
(PVDF), polystyrene, polyacrylamide, other polymers such as
poly(vinyl chloride), poly(methyl methacrylate) or poly(dimethyl
siloxane), and photopolymers which contain photoreactive species
such as nitrenes, carbenes and ketyl radicals capable of forming
covalent links with nucleic acid molecules 13, 102 (See U.S. Pat.
Nos. 5,405,766 and 5,986,076).
[0157] Bifunctional cross-linking reagents can be of use in various
embodiments, such as attaching a nucleic acid molecule to a
surface. The bifunctional cross-linking reagents can be divided
according to the specificity of their functional groups, e.g.,
amino, guanidino, indole, or carboxyl specific groups. Of these,
reagents directed to free amino groups are popular because of their
commercial availability, ease of synthesis and the mild reaction
conditions under which they can be applied. Exemplary methods for
cross-linking molecules are disclosed in U.S. Pat. Nos. 5,603,872
and 5,401,511. Cross-linking reagents include glutaraldehyde (GAD),
bifunctional oxirane (OXR), ethylene glycol diglycidyl ether
(EGDE), and carbodiimides, such as
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
Sequence CWU 1
1
1113DNAArtificial SequenceSynthetized Construct 1atcgatacga tcg
13
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