U.S. patent application number 13/220214 was filed with the patent office on 2012-02-09 for devices and methods for dual excitation raman spectroscopy.
Invention is credited to Andrew Arthur Berlin, Christopher Marc Gerth, Tae-Woong Koo.
Application Number | 20120034686 13/220214 |
Document ID | / |
Family ID | 32072853 |
Filed Date | 2012-02-09 |
United States Patent
Application |
20120034686 |
Kind Code |
A1 |
Berlin; Andrew Arthur ; et
al. |
February 9, 2012 |
DEVICES AND METHODS FOR DUAL EXCITATION RAMAN SPECTROSCOPY
Abstract
Spectroscopic analysis systems and methods for analyzing samples
are disclosed. An analysis system may contain an electromagnetic
radiation source to provide radiation, a spectroscopic analysis
chamber to perform a coherent Raman spectroscopy (e.g., stimulated
Raman or coherent anti-Stokes Raman spectroscopy), and a radiation
detector to detect radiation based on the spectroscopy. The chamber
may have a resonant cavity to contain a sample for analysis, at
least one window to the cavity to transmit the first radiation into
the cavity and to transmit a second radiation out, a plurality of
reflectors affixed to a housing of the cavity to reflect radiation
of a predetermined frequency, the plurality of reflectors separated
by a distance that is sufficient to resonate the radiation. The
spectroscopic analysis system may be coupled with a nucleic acid
sequencing system to receive a single nucleic acid derivative in
solution and identify the derivative to sequence the nucleic
acid.
Inventors: |
Berlin; Andrew Arthur; (San
Jose, CA) ; Gerth; Christopher Marc; (Santa Clara,
CA) ; Koo; Tae-Woong; (Cupertino, CA) |
Family ID: |
32072853 |
Appl. No.: |
13/220214 |
Filed: |
August 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11404680 |
Apr 14, 2006 |
8009288 |
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13220214 |
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10675884 |
Sep 29, 2003 |
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11404680 |
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10262349 |
Sep 30, 2002 |
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10675884 |
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Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
G01N 21/65 20130101;
G01N 2021/653 20130101; Y10T 436/143333 20150115; G01N 2021/052
20130101; G01N 2021/655 20130101; G01N 21/658 20130101 |
Class at
Publication: |
435/287.2 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Claims
1-21. (canceled)
22. An apparatus for nucleic acid sequencing comprising: a reaction
chamber having an immobilization surface for attachment of a
nucleic acid molecule to be sequenced; a microfluidic channel
coupled to the reaction chamber for transporting nucleotides
released from the nucleic acid molecule; a microchannel coupled to
the microfluidic channel at an entrance for receiving the
nucleotides, a region of nanoparticles positioned within the
microchannel, the region of nanoparticles being configured to allow
passage of the nucleotides; an excitation source proximate the
region of nanoparticles, the excitation source being configured to
excite the nucleotides passing through the region of nanoparticles;
and a detector configured to detect the excited nucleotides.
23. The apparatus of claim 22, further comprising a pair of
electrodes configured to create an electrical potential gradient
proximate the entrance to drive the nucleotides from the
microfluidic channel into the microchannel.
24. The apparatus of claim 22, wherein the nanoparticles are
cross-linked.
25. The apparatus of claim 22, wherein the nanoparticles are gold
nanoparticles.
26. The apparatus of claim 22, wherein the nanoparticles are silver
nanoparticles.
27. The apparatus of claim 22, wherein the size of the
nanoparticles are from 5 nm to 60 mm.
28. The apparatus of claim 22, wherein the excitation source is a
laser.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 10/675,884 filed Sep. 29, 2003, now pending;
which is a continuation-in-part application of U.S. application
Ser. No. 10/262,349 filed Sep. 30, 2002, now pending. The
disclosure of each of the prior applications is considered part of
and is incorporated by reference in the disclosure of this
application.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] Embodiments of the invention relate to analyzing a sample
with Raman spectroscopy. In particular, embodiments relate to
analyzing a sample containing a single nucleic acid derivative of
interest contained within a spectroscopic analysis chamber with
stimulated or coherent anti-Stokes Raman spectroscopy.
[0004] 2. Background Information
[0005] Deoxyribonucleic acid (DNA) sequencing has many commercially
important applications in the fields of medical diagnosis, drug
discovery, and treatment of disease. Existing methods for DNA
sequencing, based on detection of fluorescence labeled DNA
molecules that have been separated by size, are limited by the
length of the DNA that can be sequenced. Typically, only 500 to
1,000 bases of DNA sequence can be determined at one time. This is
much shorter than the length of the functional unit of DNA,
referred to as a gene, which can be tens or even hundreds of
thousands of bases in length. Using current methods, determination
of a complete gene sequence requires that many copies of the gene
be produced, cut into overlapping fragments and sequenced, after
which the overlapping DNA sequences may be assembled into the
complete gene. This process is laborious, expensive, inefficient
and time-consuming. It also typically requires the use of
fluorescent or radioactive labels, which can potentially pose
safety and waste disposal problems.
[0006] More recently, methods for DNA sequencing have been
developed involving hybridization to short oligonucleotides of
defined sequence, attached to specific locations on DNA chips. Such
methods may be used to infer short DNA sequences or to detect the
presence of a specific DNA molecule in a sample, but are not suited
for identifying long DNA sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The invention may best be understood by referring to the
following description and accompanying drawings that are used to
illustrate embodiments of the invention. In the drawings:
[0008] FIG. 1 shows a method for identifying a sample based on a
resonance enhanced spectroscopic analysis, according to embodiments
of the invention.
[0009] FIG. 2 shows a cross-sectional view of a spectroscopic
analysis system containing a chamber in a fluid channel, according
to embodiments of the invention.
[0010] FIG. 3 shows Rayleigh, Stokes, and anti-Stokes
radiation.
[0011] FIG. 4 shows Stokes Raman spectra for dilute aqueous
solutions of four DNA nucleotides, according to embodiments of the
invention.
[0012] FIG. 5 shows a cross-sectional view of a spectroscopic
analysis system containing a spheroidal chamber coupled with a
fluid channel, according to embodiments of the invention.
[0013] FIG. 6 shows another cross-sectional view of a spheroidal
chamber coupled with a fluid channel, according to embodiments of
the invention.
[0014] FIG. 7A shows a view of the top of the chamber shown in FIG.
6 along the section lines 7A-7A, according to embodiments of the
invention.
[0015] FIG. 7B shows a view of the bottom of the chamber shown in
FIG. 6 along the section lines 7B-7B, according to embodiments of
the invention.
[0016] FIG. 8 shows a top-plan view of a spectroscopic analysis
chamber configured to receive excitation radiation in the form of
aligned frequency-matched seed radiation having the same frequency
as the scattered radiation, and transverse frequency-unmatched
radiation having a different frequency than the scattered
radiation, according to embodiments of the invention.
[0017] FIG. 9 shows an energy level diagram for a coherent
anti-Stokes Raman Spectroscopy process used to analyze a sample,
according to embodiments of the invention.
[0018] FIG. 10 shows a cross-beam configuration for implementing
coherent anti-Stokes Raman spectroscopy, according to embodiments
of the invention.
[0019] FIG. 11 shows momentum conservation for a cross-beam
configuration similar to that shown in FIG. 10, according to
embodiments of the invention.
[0020] FIG. 12 shows a forward-scattered configuration for
implementing coherent anti-Stokes Raman spectroscopy, according to
embodiments of the invention.
[0021] FIG. 13 shows a back-scattered configuration for
implementing coherent anti-Stokes Raman spectroscopy, according to
embodiments of the invention.
[0022] FIG. 14 shows a nucleic acid sequencing system in which
embodiments of the invention may be implemented.
[0023] FIG. 15 shows an exemplary apparatus and method for nucleic
acid sequencing by surface enhanced Raman spectroscopy (SERS),
surface enhanced resonance Raman spectroscopy (SERRS), resonance
enhanced Raman spectroscopy (e.g., stimulated Raman spectroscopy),
and/or coherent anti-Stokes Raman spectroscopy (CARS)
detection.
[0024] FIG. 16 shows the Raman spectra of all four deoxynucleoside
monophosphates (dNTPs) at 100 mM concentration, using a 100
millisecond data collection time.
[0025] FIG. 17 shows SERS detection of 1 nM guanine, obtained from
dGMP by acid treatment according to Nucleic Acid Chemistry, Part 1,
L. B. Townsend and R. S. Tipson (Eds.), Wiley-Interscience, New
York, 1978.
[0026] FIG. 18 shows SERS detection of 100 nM cytosine.
[0027] FIG. 19 shows SERS detection of 100 nM thymine.
[0028] FIG. 20 shows SERS detection of 100 pM adenine, obtained
from dAMP by acid treatment.
[0029] FIG. 21 shows a comparative SERS spectrum of a 500 nM
solution of deoxyadenosine triphosphate covalently labeled with
fluorescein (upper trace) and unlabeled dATP (lower trace).
[0030] FIG. 22 shows a plot comparing the energies of Raman
scattered light generated from single molecules respectively
contained inside an optical resonance chamber (curves A and B) or
positioned in free space without resonance enhancement (curve C),
according to one embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Described herein are spectroscopic analysis systems and
methods for analyzing samples with Raman spectroscopy. In the
following description, numerous specific details are set forth.
However, it is understood that embodiments of the invention may be
practiced without these specific details. For example, a
practitioner may use a spectroscopic analysis chamber other than
the specific chambers disclosed herein. As another example, a
practitioner may use another spectroscopy technique than the
specific stimulated and coherent anti-Stokes Raman spectroscopies
disclosed herein. In other instances, well-known structures and
techniques have not been shown in detail in order to avoid
obscuring the understanding of this description. It is recognized
that there is a generally high level of skill in the arts of
spectroscopy and in fabricating chambers as disclosed herein.
[0032] Embodiments of the invention may be used in conjunction with
DNA sequencing. One exemplary method for DNA sequencing may involve
the ordered deconstruction of a DNA molecule into smaller
nucleotide components, for example through the use of enzymes, and
then analyzing and identifying the nucleotides to determine the
ordered sequence of the nucleotides in the original DNA molecule.
Spontaneous Raman spectroscopy has been attempted for
identification of nucleotides. During spontaneous Raman
spectroscopy, the sample containing the nucleotide is exposed to
radiation, and a small portion of the radiation that interacts with
the nucleotide is scattered due to the spontaneous Raman scattering
effect according to vibrational characteristics of the
nucleotide.
[0033] Unfortunately, in spontaneous Raman spectroscopy, the
optical signals scattered from nucleotides in dilute solution are
generally very weak, and the resulting generally low probability of
detecting the optical signals makes the approach insensitive,
inaccurate, and unreliable. There have been efforts to improve the
accuracy of this approach by making multiple measurements and
performing data averaging, although this takes more time, and may
not be desirable in a rapid DNA sequencing environment. There have
also been efforts to improve the strength of the optical signal by
attaching chemical moieties, such as fluorescent chromophores, to
the nucleotides in order to enhance their luminescent or
fluorescent properties. Reliably and consistently attaching these
moieties remains problematic. The moieties or their buffer
solutions may be unstable. In some instances, the moieties fail to
attach altogether. In other instances the moieties that do attach
have different characteristics and cause their nucleotides to have
a different spectroscopic response. Such problems make it difficult
to accurately and reliably identify the nucleotides. In addition,
the processes to attach the chemical moieties add additional time
and complexity to the analysis. These problems may be overcome by
the methods and systems disclosed herein.
[0034] FIG. 1 shows a method for identifying a sample based on a
resonance enhanced stimulated Raman spectroscopic analysis,
according to embodiments of the invention. The method allows
identifying a sample based on spectroscopic data that serves as a
fingerprint or signature for the sample. In brief, the method
includes adding a sample, such as a single molecule of interest in
solution, to a resonant spectroscopic analysis chamber at block
110, analyzing the sample with a resonance enhanced stimulated
Raman spectroscopy at blocks 120-160, and identifying the sample
based on the analysis at block 170. The analysis of the single
molecule is an aspect of some applications, and not a limitation.
Other applications may involve analyzing a plurality if molecules.
In some embodiments of the invention, the resonance enhanced
stimulated Raman spectroscopy may include irradiating a sample
contained in a resonance chamber at block 120, scattering radiation
from the sample at block 130, resonating the scattered radiation in
the chamber at block 140, irradiating or transmitting the scattered
radiation from the chamber at block 150, and detecting the
irradiated scattered radiation at block 160. In one aspect, the
method may be used in coordination with nucleic acid sequencing and
may include identifying a single nucleic acid derivative in a
sample received from a nucleic acid sequencing system in an effort
to sequence a DNA or RNA molecule.
[0035] FIG. 2 shows a cross-sectional view of a spectroscopic
analysis system 200 suitable to perform a resonance enhanced
stimulated Raman spectroscopic analysis of a sample in order to
identify the sample, according to embodiments of the invention. The
system includes an electromagnetic radiation source 210, a fluid
channel 270, a spectroscopic analysis chamber 250 (shown by dashed
lines), and an electromagnetic radiation detector 290. A sample 230
is contained within the chamber. During resonance enhanced
spectroscopic analysis of the sample, the electromagnetic radiation
source provides input or excitation radiation 215 to irradiate the
sample within the chamber in order to generate an output of Raman
inelastically scattered radiation 285 that contains information
that may be used to characterize and identify the sample. In the
case of stimulated Raman spectroscopy, the input radiation may be
visible, ultraviolet, or near infrared spectrum radiation from an
electromagnetic radiation source such as a laser and the output
radiation may be corresponding stimulated Raman inelastically
scattered radiation. The output radiation may be detected by the
detector and used to identify the sample or one or more components
thereof.
[0036] Initially, with reference to block 110 of the method 100,
the sample 230, which may include a liquid, gas, or mixed fluid, is
added to the chamber 250. In the system illustrated in FIG. 2, the
sample is added to the chamber by flowing through the fluid
channel. The channel is a void or hollowed-out space within a solid
material and may be, for example, any tube, pipe, duct, or conduit
to convey a fluid. The cross section of the channel may have a
circular, oval, square, rectangular, or other shape. The fluid
channel has a sample inlet 275 that is coupled with a sample source
to receive a sample for analysis and a sample outlet 280 that is
coupled with a sample destination to discharge an analyzed sample.
The sample may be received at the inlet, flowed through the
channel, flowed into the chamber, analyzed, subsequently flowed out
of the chamber, and flowed through the outlet to the proper
destination. Flow may be achieved by providing an appropriate
driving force, such as a pressure differential between the inlet
and the outlet or a pump within the channel. As one example, an
aqueous solution of diluted nucleic acid derivative may be received
from a pressurized nucleic acid sequencing system by pressure
injection and added to the chamber by flowing through the channel
in order to identify the particular nucleic acid derivative.
Alternatively, the sample may be added with a fluid pump, such as a
syringe, through an opening to the chamber, rather than by flowing
through a fluid channel, and subsequently removed with the syringe,
in still other implementations, if the sample is electrically
charged it may be added to the chamber under the force provided by
an electric field, or if the sample is magnetic (e.g., has a
magnetic moiety attached) it may be added to the chamber by the
force provided by a magnetic field.
[0037] Although not required, the channel may be micro-sized. The
term micro-sized channel, microfluidic channel, and the like will
be used to refer to a channel having a cross-sectional length, for
example a diameter in the case of a tubular pipe, which is less
than approximately one millimeter (mm, one-thousandth of a meter).
Often, the micro-sized channel may have a cross-sectional length
that is in the range of approximately 10-500 micrometers (urn, one
millionth of a meter). To help put these lengths in proper
perspective, the cross-sectional diameter of a human hair is
approximately 100 micrometers. These miniaturized channels are
often useful for handling small sized samples and allow many
channels to be constructed in a small substrate, although this is
not a requirement.
[0038] Often, the channel and chamber may be disposed in a solid
substrate. Suitable materials for solid substrates include but are
not limited to ceramics (e.g., alumina), semiconductors (e.g.,
silicon or gallium arsenide), glasses, quartz, metals (e.g.,
stainless steel or aluminum), polymers (e.g., polycarbonate,
polymethylmethacrylate (PMMA), polymethylsiloxine,
polydimethylsiloxane (PDMS), or polytetrafluoroethylene
(Teflon.RTM.)), and combinations of these materials. Of course it
will be appreciated that many other materials may also be used. Of
these particular materials certain glasses, quartz, and polymers
(e.g., PMMA and polycarbonate) are known to be substantially
transparent at least in the visible spectrum. The transparency of
these and other materials in the near infrared or ultraviolet is
available in the literature or may easily be determined without
undue experimentation. These transparent materials may be used as
the substrate. Alternatively, non-transparent materials may be used
as the substrate and transparent materials may be used as windows
to allow sufficient transmittance of the relevant radiation.
Materials that are suitable for windows include the transparent
materials previously mentioned as well as other materials such as
sapphire, magnesium fluoride, and calcium fluoride crystals. One
exemplary substrate contains a channel and cavity formed in a
stainless steel substrate, the channel and the cavity surfaces
lined with an inert material such as Teflon.RTM. (for improved
chemical compatibility), and one or more transparent windows of
these or other transparent materials formed into the chamber to
allow transmittance of radiation into the chamber. Alternatively,
the stainless steel substrate may be replaced with glass, quartz,
or ceramic, which should have sufficient compatibility with samples
that omitting the Teflon coating would be appropriate. As yet
another example, it may be desirable to use a polymer to help
reduce fabrication costs and take advantage of molding, hot
embossing, and other fabrication techniques. An exemplary substrate
may include polydimethylsiloxane containing a channel and chamber
housing molded or embossed therein into which one or more windows
and reflectors may be inserted and affixed.
[0039] Various micromachining methods, such as micromilling, laser
ablation, and focused ion beam milling may be used to form channels
in ceramic, glass quartz, semiconductor, metal, and polymeric
substrates. Approaches for fabricating microfluidic channels are
described for example in U.S. Pat. Nos. 5,904,824, 6,197,503,
6,379,974, 6,409,900, and 6,425,972. Additionally,
photolithographic etches that are commonly used in the
semiconductor processing arts may be used to form channels in
semiconductor, quartz, glass, and certain ceramic and metal
substrates. For example, reactive plasma may be used to etch a
channel having a depth of a few hundred microns and smooth vertical
sidewalls in a silicon substrate. Photolithographic etches based on
different chemistries may similarly be used to form channels in
polymeric materials. In addition, molding, injection molding,
stamping, hot embossing, and other approaches may be used to form
channels in polymeric materials, as well as certain metals. Other
techniques that are suitable for forming channels, as well as
further background information on microfabrication is available in
"The MEMS Handbook" (ISBN/ISSN: 0849300770), by Mohamed Gad-el-Hak,
published on Sep. 27, 2001, containing pages 1-1368, and available
from the University of Notre Dame.
[0040] A chamber according to embodiments of the invention contains
a resonant cavity to contain a sample for analysis, at least one
window to the cavity to transmit a first radiation having a first
frequency (e.g., an input excitation radiation from a laser) into
the cavity and to transmit a second electromagnetic radiation
having a second frequency (e.g., an output beam of stimulated Raman
radiation) out of the cavity, and a plurality of reflectors (e.g.,
multi-layer dielectric mirrors) affixed to a housing of the cavity
to reflect radiation of a predetermined frequency (e.g., the
stimulated radiation).
[0041] Referring to FIG. 2 and the particular chamber 250, the
resonant cavity is a region within the fluid channel between a
reflector 235 and a partial reflector 245. The chamber has an inlet
opening, at the leftmost edge of the reflectors 235, 245 (as
viewed), to allow addition of a sample, and an outlet opening, at
the rightmost edge of the reflectors 235, 245 (as viewed), to allow
removal of the analyzed sample. The sample may either be flowed
continuously through the chamber or stopped within the chamber.
Alternatively, the chamber may be located at a dead-end of the
channel and may utilize a common inlet-outlet opening to add and
remove a sample.
[0042] Once the sample is contained within the chamber, with
reference to block 120 of the method 100 (FIG. 1), the sample is
irradiated, hi the system illustrated in FIG. 2, the source 210
provides input radiation 215 to the chamber and irradiates the
sample positioned therein. Suitable sources include coherent light
sources, lasers, light emitting diodes (LEDs), lamps, and fiber
optic cables coupled with such a source of radiation. Radiation
sources that provide strong monochromatic or quasimonochromatic
radiation will often be favored over those that provide weaker or
broad-spectrum radiation, since such monochromatic radiation
facilitates detection of the relevant offset spectroscopic signals.
The source 210 may contain filters or monochromators to reduce
certain frequencies. The source may also contain lenses, mirrors,
or other devices to redirect and focus the radiation on the
chamber. These devices are commercially available from numerous
sources, including vendors that are listed in "The Photonics
Directory.TM.: The Photonics Buyers' Guide To Products and
Manufacturers". This directory contains vendor listings for
radiation sources, radiation detectors, spectrometers,
spectroscopic analysis software, as well as numerous other
accessories (e.g., lenses, wavelength selection devices, etc.). The
Photonics Directory.TM. is available from Laurin Publishing, and is
presently available online at the website website:
www.photonics.com/directory/index.asp.
[0043] A laser (light amplification by stimulated emission of
radiation) may be used to provide a high intensity beam of coherent
and monochromatic light having wavelengths suitable for resonance
enhanced Raman spectroscopy. Many different types of lasers are
suitable including tunable lasers, gas lasers, solid-state lasers,
semiconductor lasers, laser diodes, VCSEL (Vertical-Cavity
Surface-Emitting Laser), and quantum cavity lasers. The laser may
be used to provide radiation at frequencies or wavelengths that are
suitable for Raman spectroscopy, including radiation in the
visible, often in the green, red, or near infrared, and ultraviolet
regimes. Many molecules of potential interest, including many
nucleic acid derivatives, have their resonance wavelengths in or
near the ultraviolet spectrum. For example, adenine, a DNA base,
has a near maximum resonance wavelength at approximately 267
nanometers (nm, one billionth of a meter). As such, the ultraviolet
spectrum as an excitation wavelength may provide comparatively
strong signals. Potential drawbacks to using ultraviolet as
excitation radiation is that depending on the intensity it may
damage the molecule of interest and may induce background noise
from other system components and compounds that also have resonance
wavelengths in the ultraviolet. Lasers and associated optics for
the ultraviolet range also tend to be more costly. Another option
is the use of near infrared or visible excitation radiation. While
the signals may be slightly less intense compared to ultraviolet,
there may be comparatively less background noise, since most
materials have their resonance wavelengths outside of these
spectral frequencies. Although this particular laser is not
required, the present inventors have utilized an argon-ion laser to
provide a radiation at about 514 nanometers wavelength and have
found that such a laser is suitable for differentiating the Raman
spectra of DNA nucleotides. This particular laser is commercially
available from Coherent Inc. of Santa Clara, Calif. and
Spectra-Physics, Inc. of Mountain View, Calif. The laser may be
operated at a sufficient intensity that allows detection and avoids
damaging the molecule. As an example, the laser may be operated at
energy in the range of approximately 100 W to 100 kW, depending
upon the stability of the molecule. Other suitable lasers include a
10 mW helium-neon laser providing radiation at a wavelength of 633
nanometers, a linearly polarized 50 mW diode laser providing
radiation in the near infrared at a wavelength of 785 nanometers,
and other lasers.
[0044] Referring to FIG. 2, the chamber receives input radiation
from the source through at least one window into the cavity that
allows the sample positioned therein to be irradiated. The
particular chamber illustrated receives transmitted radiation 225,
which is transmitted through the fluid channel walls, through an
inlet window 220 opening to the cavity at a left edge of the
reflectors 235, 245. The inlet window may be a portion of the
cavity housing that is at least partially transparent to the
particular excitation radiation, a position adjacent to a
reflector, a space or gap between reflectors, an incomplete or
finite reflector through which a portion of the radiation may pass,
a lens, an electro-optic device, or a waveguide (e.g., a fiber
optic material or wispering gallery mode) that confines directs and
guides the radiation into or out of the cavity. Suitable
electro-optic devices include laser modulators and Pockel cells. A
Pockel cell is a solid state electro-optic device that is often
used as a Q-switch. An electrical field or other signal is applied
to the device in order to modify or switch the birefringence of the
cell. This allows modifying the transmittance of radiation through
the cell and switching it on or off Accordingly, the
electro-optical device may act as a window that may be opened or
closed by applying appropriate electrical current in order to allow
transmittance of light through the device into and/or out of the
chamber. Electro-optic devices and Pockel cells are available
commercially from Conoptics, Inc. of Danbury Conn., among other
vendors. It is also contemplated that an actual moving shutter may
be opened and closed to allow light into and/or out of the chamber.
Hinged members commonly used in MEMS (micro-electromechanical)
devices may be used for the shutter.
[0045] As previously discussed, the transmitted input radiation 225
is introduced into the cavity and some of the input radiation, or
some of the reflected input radiation, or both, irradiates the
sample. With reference to block 130 of the method 100 of FIG. 1,
some of the radiation is scattered by the sample. As used herein,
the terms "scattered radiation" and the like will be used to refer
to a photon of light or other radiation that has collided with and
been absorbed by sample matter, such as a molecule, and has been
released or emitted. Most of the scattered photons will be released
elastically in which the scattered radiation has no change of
energy or frequency. This radiation is known as Rayleigh scattered
radiation. Some of the scattered photons, often a small fraction,
will be released inelastically in which there is an exchange of
energy and a change of frequency. These are well-known phenomenon.
Stokes scattered radiation refers to scattering where the molecule
loses energy and the frequency is reduced whereas anti-Stokes
scattered radiation refers to scattering where the molecule gains
energy and the frequency is increased. This scattering of light by
matter is known as Raman spectroscopy. FIG. 3 conceptually
illustrates Rayleigh, Stokes, and anti-Stokes radiation. Stokes and
anti-Stokes radiation typically have lower intensity than Rayleigh
radiation, and respectively exist at lower and higher frequencies
than Rayleigh radiation. As used herein, Stokes and anti-Stokes
radiation will be referred to collectively as inelastically
scattered radiation.
[0046] The change in frequency or wavelength of the inelastically
scattered radiation is based on the particular characteristics of
the sample or molecule of interest contained therein. That is, the
difference between the frequencies of the input radiation and the
inelastically scattered radiation is characteristic of the sample.
For example, the frequency of a photon scattered from an inelastic
collision with a molecule may depend upon the polarization,
vibration, rotation, and orientation characteristics of a molecule.
The reasons for this are explained by quantum mechanics. Briefly,
the photon may excite the molecule from its ground vibrational
state to a high-energy or virtual vibrational state, from which it
may relax to a lower-energy vibrational state by emission of
inelastically scattered radiation, and ultimately relax back to the
ground state. Further background information on this process as
well as further background information on other vibrational
spectroscopy topics is available in "The Handbook of Vibrational
Spectroscopy", by John Chalmers and Peter R. Griffiths, published
by John Wiley & Sons, Ltd. In any event, the frequency shifts
in the light that is inelastically scattered by a molecule of
interest incorporate information comprising a fingerprint or
signature for the molecules polarization, vibration, rotation, and
orientation characteristics.
[0047] As previously discussed, it is often difficult and
improbable to detect, or reliably detect, spontaneously scattered
radiation from a dilute molecule in solution. Accordingly, with
reference to block 140 of the method 100 of FIG. 1, the present
inventors have discovered systems and methods for resonating
radiation in a cavity in order to perform a resonance enhanced
spectroscopic analysis of a sample. The resonated signal is
stronger and easier to detect than the spontaneous signal.
Advantageously, this may allow improved probability and reliability
of accurately and reliably detecting and identifying dilute
molecules of interest in solution.
[0048] The particular chamber 250 of FIG. 2 is a resonate chamber
containing the reflector and the partial reflector to confine and
resonate a radiation within the cavity in order to increase or
amplify its intensity. The inelastically scattered radiation may be
resonated in order to increase its intensity and aid detection and
identification of a sample positioned within the chamber. The
reflectors may be incorporated into or affixed to the housing of
the channel to internally reflect radiation within the cavity.
Radiation that is inelastically scattered by the sample or a
portion of the input radiation 225 that is reflected by the
reflector 235 may be reflected by the partial reflector 245 and
resonated in the cavity. Portions of the radiation that are not
reflected sufficiently parallel to the optical axis normal to the
reflectors may leave the chamber quickly. The reflectors may have
lengths that are smaller than the distance of separation between
the reflectors to allow rapid removal of misaligned radiation. In
this way, in addition to resonating a particular frequency or range
of frequencies, the chamber effectively selects a direction of
radiation that accumulates in the chamber, thereby allowing an
intense, coherent beam of radiation.
[0049] The cavity may have a shape and size that facilitates
resonance. As-used herein, unless specified otherwise, resonance
refers to the intensification of radiation in a chamber due to
reflection, and is not to be confused with excited states of
electrons within a molecule. As illustrated, chamber 250 has the
reflectors opposite and parallel to one another, centered along a
common optical axis, and separated by a particular predetermined
distance that is proportional to or at least based on a wavelength
of radiation to be resonated in the chamber, and that provides a
non-destructive relationship between the phases of incident and
reflected radiation. When the distance between the reflectors is a
multiple of approximately half the wavelength of the radiation
field, the partial waves may overlap substantially constructively,
otherwise they may overlap less constructively or destructively.
The particular reflectors may be separated by a distance that is
approximately one half the product of the mode of the resonance
frequency, times the speed of light, divided by the index of
refraction of the medium filling the cavity, divided by the
frequency of the particular radiation to be resonated in the
chamber. Since coherent Raman spectroscopy may utilize excitation
radiation in the visible, near infrared, and ultraviolet regimes,
and since the inelastically scattered radiation is merely offset
from the excitation radiation, the cavity may be designed to
resonate inelastically scattered radiation having virtually any
wavelength in the visible, ultraviolet, and near infrared regimes,
depending upon the particular excitation radiation desired. The
particular choice of excitation radiation may depend upon the cost
of the laser and detectors, the capability of the detectors to
detect a particular radiation, the resonance and stability of the
molecule of interest, the false signals or noise caused by other
absorbing molecules or species that are not of interest, etc.
[0050] A chamber may be designed to resonate an inelastically
scattered radiation for a molecule of interest (e.g., a particular
nucleotide) or set of molecules of interest (e.g., a set of
nucleotides). As one example, consider for a moment the Stokes
spectra shown in FIG. 4 (which will be discussed in more detail
below) generated from excitation with a 514 nanometer argon-ion
laser. The bottommost spectra for the molecule deoxythymidine
monophosphate has a pronounced peak at a Stokes Raman shift of
approximately 1350 cm-1. This shift is offset from the excitation
wavenumber of 19455 cm .sup.-1 (i.e., 10.sup.7/514=19455) for the
514 nanometer excitation radiation, and translates into an actual
Stokes Raman wavenumber of approximately 18105 cm.sup.-1 (i.e.,
19455-1350). This corresponds to a wavelength of 552 nanometers
(i.e., 10.sup.7/18105). This Stokes scattered radiation may be
resonated in the chamber in order to amplify the optical signal and
improve detection. The particular reflectors shown in FIG. 2 may be
separated by a distance that is approximately one half the product
of the mode of the resonance frequency times the speed of light
divided by the index of refraction of the medium filling the cavity
divided by the frequency of the particular radiation to be
resonated in the chamber for this 552 nanometer scattered
radiation. As another example, the chamber may be designed to
resonate this wavelength of radiation as well as wavelengths of
inelastically scattered radiation from other molecules of interest.
In one embodiment of the invention, the predetermined distance
between the reflectors is a function of wavelengths corresponding
to prominent peaks in the Raman spectra of a plurality of molecules
of interest (see e.g., the peaks in FIG. 4). The function may be an
average, a weighted average, or other combinatorial functions.
[0051] The reflector 235 and the partial reflector 245 may be
multiple layer dielectric mirrors, metal mirrors, or other
reflectors that are commonly utilized in the spectroscopy, laser,
optics, or fiber optic arts. A multi-layer dielectric mirror, of
which a Distributed Bragg Reflector (DBR) is one example, is an
interference based mirror structure containing a stack or laminate
of alternating layers of materials having different indices of
refraction. The thickness of the layers may be proportional to the
wavelength of the radiation to be reflected such that the reflected
waves from the layers are in phase with one another and
superimpose. The reflector may contain alternating layers of low
and high refractive index materials. A dielectric or insulating
material is often used for at least one of the alternating layers
and the other layer may be a different dielectric material, a
non-dielectric material, a semiconductor, or other materials. One
non-limiting example of a dielectric mirror includes a plurality of
alternating layers of silicon (Si) and an oxide of silicon (e.g.,
silicon dioxide, SiO.sub.2), for example SiO.sub.2/Si/SiO.sub.2
that each have a thickness approximately quarterwavelength (i.e.,
1/4 the wavelength) of the reflected radiation (e.g., inelastically
scattered radiation). More pairs of alternating layers may be added
to improve reflectance. The partial reflector 235 may have less
total layers than the reflector 245 in order to transmit relatively
more incident radiation than the reflector 245. Another
non-limiting example of a dielectric mirror includes a
quarterwavelength thickness of gallium arsenide (GaAs), a stress
matched SiO.sub.2/Si.sub.3N.sub.4/SiO.sub.2 trilayer (alternating
dielectric layers), and about 1500 Angstroms of gold. That is, this
mirror contains both a dielectric interference based mirror as well
as a metal mirror. Numerous other examples of multi-layer
dielectric mirrors abound in the literature. Multi-layer dielectric
mirrors may achieve high reflectance that are often in the range of
approximately 99-99.9%, or higher. These mirrors may be used to
rapidly achieve good gains and increased intensities.
[0052] A multi-layer dielectric mirror may contain layers that have
a thickness that is proportional to a wavelength of an
inelastically scattered radiation for a molecule of interest (e.g.,
a particular nucleotide) or set of molecules of interest (e.g., a
set of nucleotides). For example, the layers may have a thickness
that is approximately quarterwavelength of the average anti-Stokes
or Stokes scattered radiation for a set of monobasic nucleic acid
derivatives for DNA. This may allow a single resonant cavity to be
used to perform resonance enhanced spectroscopic analysis of the
entire set of derivatives, for example in the context of DNA
sequencing, and allow identification of a particular derivative of
the set based on a resonance intensified beam incorporating
derivative specific frequency shift information that is transmitted
from the cavity. As another example, the layers may have a
thickness that is approximately quarterwavelength of the 552
nanometer Stokes scattered radiation for molecule deoxythymidine
monophosphate exposed to 514 nanometer radiation from an argon-ion
laser. Other thicknesses may apply for other molecules and
excitation radiations.
[0053] Multi-layer dielectric mirrors are available commercially
from a number of sources or may be fabricated. SuperMirrors.TM. are
examples of suitable multi-layer dielectric mirrors that are
available from Newport Corporation of Irvine, Calif. The
SuperMirrors.TM. may be obtained and affixed to the housing of the
chamber. Alternatively, a multi-layer dielectric mirror may be
fabricated by techniques that are commonly used in the
semiconductor processing arts (see e.g., U.S. Pat. Nos. 6,320,991
or 6,208,680). As another example, a dielectric mirror containing
alternating layers of Si and SiO.sub.2 may be deposited by a
conventional chemical vapor deposition or physical vapor deposition
technique that is commonly used in the semiconductor processing
arts. For example, a low pressure chemical vapor deposition (LPCVD)
technique may be used to provides good quality layers of controlled
thickness. Alternatively, a physical vapor deposition technique
such as evaporation, sputtering, or molecular beam epitaxy may be
used to deposit the layers. As another variation, a thicker silicon
layer may be deposited by a chemical vapor deposition and a silicon
dioxide layer may be thermally grown from the silicon layer to a
similar thickness. Of course, the chemical and physical deposition
techniques may also be used to deposit other materials.
[0054] The reflector and partial reflector may alternatively be
metal mirrors. Suitable metals include, among others, aluminum,
gold, silver, chrome, and alloys, mixtures, or combinations
thereof. Hereinafter the term metal will include pure metals as
well as alloys, mixtures, or combinations. Suitable metal mirrors
are available from Newport Corporation. Metal mirrors may also be
fabricated by conventional chemical vapor deposition and physical
vapor deposition techniques. For example, a gold layer may be
deposited on a cavity wall by molecular beam epitaxy evaporative
deposition. Metal mirrors may provide reflectance in the range of
approximately 90-95%, or slightly higher but usually not greater
than about 99%.
[0055] The reflected radiation may be resonated within the cavity
and may stimulate or induce other inelastic scattering events. The
stimulated events may occur more rapidly or more probabilistically
than spontaneous scattering events and the intensity of the
inelastically scattered radiation may build within the chamber
until saturation or equilibrium is achieved wherein round trip gain
due to resonance matches loses. It is anticipated that intensities
up to on the order of a hundred milliwatts may be obtained. Such
resonance enhanced stimulated Raman spectroscopy may provide an
amplified, intense, coherent, frequency-shifted beam that should
allow probable, accurate, and reliable detection and identification
of even a single molecule of interest in dilute solution. It is not
required that a single molecule of interest be analyzed and in
other embodiments a sample containing any desired number of
molecules of interest may be analyzed.
[0056] By now it should be apparent that different types of samples
may be analyzed. As used herein, the term sample will refer to one
or more molecules, atoms, or ions to be analyzed either alone or
with a vapor, gas, solution, solid, or sorbent (e.g., a metal
particle or colloidal aggregate). The sample may contain a dilute
solution containing one or more molecules of interest. Virtually
any molecule may be of interest, depending on the particular
implementation, such as a biologic molecule, a molecule associated
with nucleic acid sequencing, a pharmaceutical, pesticide, an
herbicide, a polymer, a pollutant, or others. For example in the
case of analyzing a biological molecule of interest associated with
nucleic acid sequencing, an aqueous or other solution containing a
protein, protein fragment, or nucleic acid derivative may be
analyzed. The term nucleic acid derivative will be used to refer to
a nucleic acid, nucleic acid fragment, nucleotide, nucleoside (e.g.
adenosine, cytidine, guanosine, thymidine, uridine), base (e.g.
adenine, cytosine, guanine, thymine, uracil), purine, pyrimidine,
or a derivative of one of these molecules. One exemplary sample
contains a solution of a single nucleic acid derivative having a
single base that is selected from the group consisting of adenine,
cytosine, guanine, thymine, and uracil.
[0057] Proteins provide a number of critical reactions, structures,
and controls for cells. Nucleic acids provide cells information
about which proteins to synthesize. Nucleic acids are linear
polymers of nucleotides connected by phosphodiester bonds. Nucleic
acids may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
Nucleotides consist of three parts, a phosphate group, a pentose (a
five carbon sugar molecule), and a base. Nucleosides are bases and
sugars without a phosphate group. The bases of both nucleotides and
nucleosides are adenine (A), cytosine (C), guanine (G), thymine
(T), and uracil (U). The bases adenine, cytosine, and guanine are
found in both DNA and RNA, thymine usually found only in DNA, and
uracil is usually found only in RNA. Accordingly, the nucleosides
for DNA include deoxyadenosine, deoxyguanosine, deoxycytidine, and
deoxythymidine, whereas the nucleosides for RNA include adenosine,
guanosine, cytidine, and uridine. Likewise, the nucleotides for DNA
include deoxyadenosine monophosphate, deoxyguanosine monophosphate,
deoxycytidine monophosphate, deoxythymidine monophosphate (or in
the salt form, deoxyadenylate, deoxyguanylate, deoxycytidylate, and
thymidylate), whereas the nucleotides for RNA include adenosine
monophosphate, guanosine monophosphate, cytidine monophosphate,
uridine monophosphate (or in the salt form, adenylate, guanylate,
cytidylate, and uridylate). In RNA the pentose is ribose whereas in
DNA the pentose is deoxyribose. The bases cytosine, thymine, and
uracil are pyrimidine bases and contain a single heterocyclic ring
containing carbon and other atoms, in this case nitrogen. The bases
adenine and guanine are purine bases and contain a pair of fused
heterocyclic rings that also contain nitrogen. The ability to
determine a nucleic acid and/or protein sequence may have
tremendous commercial importance in the fields of medical
diagnosis, treatment of disease, and drug discovery. Of course,
these are just examples of the types of samples and molecules that
may be analyzed by the systems and methods developed by the present
inventors.
[0058] With continued reference to the method 100 of FIG. 1, and to
block 150, after resonating, the scattered radiation may be
irradiated or transmitted from the chamber. Referring to FIG. 2,
the chamber may contain at least one window to allow the radiation
to exit the chamber and be detected. The particular chamber 250
contains an outlet window 260, which in this particular instance
comprises the partial reflector 245. The partial reflector is an
incomplete or finite reflector that has a sufficient reflectivity
to achieve resonance and a sufficient transmittance to allow a
detectable level of incident radiation to be transmitted to the
detector 290. The reflector 235 may have a higher total reflectance
than the partial reflector 245 in order to provide good
amplification of intensity in the cavity, although this is not
required. In one instance, the reflector 235 may have a high
reflectivity for both the input excitation radiation and the
inelastically scattered radiation, for example greater than
approximately 99% (e.g., approximately 99.9%) for both, whereas the
partial reflector 245 may have a sufficient transmittance for the
inelastically scattered radiation, for example a transmittance that
is not less than approximately 1% (or a reflectance that is not
greater than approximately 99%). Of course alternate types of
outlet windows may also be used, such as a transparent material
incorporated into the cavity housing, a Pockel cell, a space, gap,
slit, pinhole, or other opening in the reflector 235, or a shutter.
Additionally, as another alternative, a single inlet-outlet window
may be utilized to transmit radiation into and out of the
chamber.
[0059] At least a portion of the resonated radiation 240 that is
incident on the partial reflector 245 leaves the cavity and chamber
as output radiation 285. The output radiation contains
inelastically scattered radiation that incorporates frequency-shift
information about the sample in the chamber. The intensity of the
output radiation may depend upon the reflectivity of the partial
reflector, the resonance characteristics of the chamber, and upon
losses due to absorption, scattering and diffraction. Often, the
chamber will be designed to achieve high intensities of the output
radiation to allow accurate detection and identification of
samples.
[0060] With reference again to the method 100 of FIG. 1, at least
some of the inelastically scattered radiation in the output
radiation 285 is detected at block 160. The electromagnetic
radiation detector 290 is shown in simplified format and is to be
interpreted broadly. Often, the output radiation may be passed
through a lens and a wavelength selection device. The lens may
collect, direct, and focus the radiation emitted from the chamber.
The light from the lens or from the chamber may be passed through a
wavelength selection device that emphasizes, selects, separates, or
isolates a particular frequency or range of frequencies. The
wavelength selector may be used to distinguish radiation of
interest (e.g., inelastically scattered radiation) from other
radiation (e.g., elastically scattered radiation and excitation
radiation). Suitable wavelength selection devices include among
others prisms, monochromators, filters (e.g., absorbance, bandpass,
interference, or Fourier), dichroic filters, dichroic mirrors, and
demultiplexers.
[0061] After passing the radiation from the chamber through any
lenses, wavelength selection devices, and the like, the resultant
radiation may be passed to a spectrometer, optical multichannel
analyzer (OMA), or other radiation detection device. These
radiation detection devices will often employ an optical transducer
to convert received radiation into a corresponding electrical
signal that captures and represents at least some of the same
information. Exemplary optical transducers include a charge coupled
device (CCD), phototransistor, photomultiplier tube, photo diode,
or an array of one or more of these devices.
[0062] The radiation detection device may comprise a Raman
spectrometer. Spectrometers are well known radiation detection
devices that measure the wavelength and intensity of light.
Spectrometers are commercially available from a number of sources.
One suitable Raman spectrometer is SpectraPro 300i Spectometer
available from Acton Research Corporation of Acton Mass. Another
spectrometer that is suitable is the RAMANRXN1 Analyzer available
from Kaiser Optical Systems, Inc. (website: www.kosi.comi) of Ann
Arbor, Mich. This spectrometer uses a thermoelectrically cooled
Charge Coupled Device (CCD) array to provide high sensitivity
detection of radiation. Optionally, this spectrometer may also be
obtained with an Invictus NIR Laser as a radiation source, a
SuperNotch.RTM. Filter, a Holographic Laser Bandpass Filter, and a
HoloSpec holographic imaging spectrograph. Further background
information about this spectrometer including its operation is
available from the vendor. Of course other spectrometers may also
be used.
[0063] Often, the spectrometer may generate spectra showing
frequency or wavelength versus intensity. FIG. 4 shows Stokes Raman
spectra for highly diluted aqueous samples of each of the four DNA
nucleotides, according to embodiments of the invention. For
clarity, the four spectra have been offset or displaced from one
another along the vertical axis. From top to bottom, the spectra
are for deoxyadenosine monophosphate, deoxycytidine monophosphate,
deoxyguanosine monophosphate, and deoxythymidine monophosphate. The
spectra were generated by performing spontaneous Raman
spectroscopic analysis on the samples including irradiating the
samples with 514 nanometer radiation from a laser and detecting the
output inelastically scattered radiation with a Raman spectrometer.
The spontaneous spectra should be substantially similar to
resonance enhanced Raman spectra except that the signals would be
correspondingly weaker in intensity. The figure shows intensity in
arbitrary units versus Raman wavelength shift (Stokes offset from
the excitation radiation) in reciprocal centimeters. As shown, each
of the spectra have distinct spectral characteristics, prominent
peaks, or fingerprint bands, that identify the corresponding
nucleotide. Exemplary prominent peaks include 1630 cm.sup.-1 for
the top spectra, 1440 cm.sup.-1 for the next spectra down, 800
cm.sup.-1 for the second spectra from the bottom, and 1350
cm.sup.-1 for the bottom spectra.
[0064] Finally, with reference to the method 100 of FIG. 1, the
sample may be identified at block 170. The sample may be identified
by comparing the determined spectrum with a spectral library
containing many predetermined spectrum for known samples and
identifying the sample as one of the known samples if the
determined spectrum sufficiently approximates the corresponding
predetermined spectrum in the library. The electrical signals
generated by the radiation detector as a result of the output
radiation may be provided to a computer system that is
appropriately programmed with spectroscopic analysis instructions
and the library (e.g., a database) that allows the Raman
fingerprint or signature represented in the electrical signals to
be correlated to a specific fingerprint or signature in the
library. For example, in the case of a spectrometer, a spectrum (or
certain bands thereof) for a sample may be compared or contrasted
to spectroscopic data for other identified samples to determine
whether the fingerprints are sufficiently identical (i.e., the
sample has the same identity as the identified sample in the
database). Various methods for identification of nucleotides by
Raman spectroscopy are known in the art (see e.g., U.S. Pat. Nos.
5,306,403, 6,002,471, or 6,174,677). The identity of the sample may
be stored in memory, further analyzed, or used for other
purposes.
[0065] With reference again to the method 100 of FIG. 1, another
way of implementing this method may include adding a sample to a
chamber at block 110, irradiating the sample with a short and
accurately-known pulse of a radiation having substantially the same
frequency or wavelength as an inelastically scattered wavelength
relevant to the sample at block 120, then scattering, resonating,
and irradiating the radiation from the cavity at blocks 130-150,
respectively, and then detecting the radiation as well as some
indication of the decay time of the inelastically scattered light
in the chamber at block 160. The decay time may be used as a
fingerprint or signature for the identity of the sample. Of course
other methods are also contemplated.
[0066] FIG. 5 shows a cross-sectional view of a spectroscopic
analysis system 500 suitable to perform a resonance enhanced
spectroscopic analysis of a sample in order to identify the sample,
according to embodiments of the invention. The system includes a
radiation source 510, a substrate 505, a fluid channel 570, a
spectroscopic analysis chamber 550, and a radiation detector 590. A
sample 530 is contained within the chamber. The channel and the
chamber are formed within the substrate and are coupled. The
particular chamber contains a spheroidal resonant cavity, an inlet
window 520, a first, second, third and fourth curved reflectors
535A-D, and an outlet window 560. The sample may be added to the
channel at an inlet 575 thereof, then flowed into a cavity of the
chamber and spectroscopically analyzed, and then flowed out of the
chamber and through the sample outlet 580.
[0067] The spheroidal resonant cavity is formed of concave surfaces
that may assist with reflecting radiation toward the interior of
the cavity. The term spheroidal will be used to refer to a shape
that resembles or approximates a sphere but that is not necessarily
a perfect sphere. Of course, the spheroidal shape is not required
and in other cavities only a portion of the chamber housing may be
concave (e.g., spherically or parabolically concave), or the cavity
may be a cylindrical void (e.g., having a diameter greater than
that of the fluid channel), a polyhedron void, a hexahedron void, a
cubic void, or a void having any other regular or irregular
shape.
[0068] The cavity serves as a wide spot in the fluid channel and
may have a volume that is convenient for the particular
implementation. Typically, the cavity will have a volume that is
not greater than about a milliliter (mL, one-thousandth of a
liter). Alternatively, if a smaller cavity is favored, the cavity
may be a micro-sized cavity or microcavity having a volume that is
not greater than approximately a microliter (.mu.L, one-millionth
of a liter). For example, the volume may be approximately 500
nanoliters (nL, one billionth of a liter), approximately 100 nL,
approximately 1 nL, or less.
[0069] An input excitation radiation 515 from the source 510 is
directed through the substrate, through the inlet window 520, and
into the cavity. The particular inlet window 520 comprises a region
of the cavity housing between the second and third reflectors
535B-C that is at least partially transparent to the inlet
radiation 515. The inlet window may be a space between the second
and third reflectors, for example a portion of sufficiently
transparent substrate, or a partial reflector between the second
and third reflectors or a transparent material. At least some of
the input radiation irradiates the sample and is inelastically
scattered. The reflectors 535A-D confine the inelastically
scattered radiation, and in this particular embodiment much of the
input radiation, inside the chamber by internal reflection. Some of
the reflected radiation may stimulate further inelastically
scattered radiation by re-irradiating the sample and again being
inelastically scattered. In another embodiment of the invention,
such as that discussed in FIG. 8, an outlet window may be used to
remove the input excitation radiation from the cavity.
[0070] Output radiation 585 leaves the cavity through the outlet
window 560 and contains at least some resonance enhanced stimulated
inelastically scattered radiation. The outlet window is positioned
away from the path of the input radiation, and primary reflections
thereof, to reduce transmission of the inlet radiation through the
outlet window. Of course, this is not required, and a wavelength
selection device may be used to remove any such radiation that is
transmitted.
[0071] FIG. 6 shows another cross-sectional view of a spheroidal
chamber 650, according to embodiments of the invention. The chamber
shows the features of the chamber 550 as well as alternate
locations 676, 677, 678, and 679 for either the inlet window 620 or
outlet window 660. By way of example, the outlet window may be
re-positioned at one of the locations 676-679, or the inlet window
may be re-positioned at one of the locations 676-679. A sample 630
is positioned within the chamber. FIG. 6 also shows section lines
7A-7A and 7B-7B that respectively show the views of FIGS. 7A and
7B.
[0072] FIGS. 7A-7B show fabrication of the chamber 650 of FIG. 6,
according to embodiments of the invention. FIG. 7A shows a top-plan
view of the top of the chamber 650 along the section lines 7A-7A
shown in FIG. 6. The hemi-spheroid void 750A of the top of the
chamber 650 and the hemi-cylindrical voids 770A (half of a
cylindrical channel void) may be formed within a substrate 705A. In
one example, these voids 750A, 770A may be formed in a quartz or
glass substrate by photolithography and etching. An anisotropic
etch with agitation may be used to form the nearly spherical
concave surfaces of the chamber 750A and the concave cylindrical
surfaces of the channel 770A by etching along crystal planes. In
another example, these voids may be formed in a polymeric material,
such as PDMS by molding or embossing.
[0073] The interior surface of the hemi-spheroid void 750A contains
reflectors 735B-D (which correspond to the reflectors 635B-D in the
view of FIG. 6). The reflector may be formed by subsequent
alternating deposition of layers of appropriate thickness of
materials having different index of refraction (e.g., Si and
SiO.sub.2). The depositions may be over the whole hemisphere in
which case an inlet window 720 and an exit window 760 may be formed
by removing a portion of these deposited layers to form a partial
reflector, or by removing all of these layers to form a transparent
quartz window. These layers may be removed by laser ablation, by
etching, or by other conventional techniques for removing layers.
Alternatively, a mask patterned with the sizes and positions of the
windows 720 and 760 may be used to selectively deposit the
multi-layer dielectric mirror layers over the non-window portions.
In the case of the PDMS polymer, a sapphire, quartz, or other
transparent window may be molded into the polymer or the polymer
drilled and the window fixedly inserted.
[0074] FIG. 7B shows a top-plan view of the bottom of the chamber
650 along the section lines 7B-7B shown in FIG. 6. The
hemi-spheroid void 750B of the bottom of the chamber 650, the
hemi-cylindrical voids 770B, and the reflector 735B may be formed
within a substrate 705B, such as a silicon substrate, as described
for FIG. 7A. After forming these structures, the substrate 705A may
be bonded to the substrate 705B by a wafer bonding approach, for
example by a high temperature fusion of oxidized surfaces thereof,
or with an adhesive.
[0075] FIG. 8 shows a top-plan view of a spectroscopic analysis
chamber 850 configured to receive input excitation radiation in the
form of aligned seed radiation 815B and transverse radiation 815A,
according to embodiments of the invention. The input transverse
excitation radiation may have any suitable frequency. The direction
of the transverse radiation is crosswise, perpendicular, to the
direction of the output radiation 885, and to the direction of
resonance. This allows a portion of the transverse radiation 886
that does not irradiate a molecule 831, to leave the chamber. This
may aid in detecting the output inelastically scattered radiation
885. The seed radiation is added to the chamber through a partial
reflector 845. The seed radiation may have the same frequency as
radiation inelastically scattered from a particular sample and may
stimulate scattering if that sample is contained within the
chamber. The direction of the seed radiation is opposite, but
aligned with, the direction of output inelastically scattered
radiation 885. This allows the seed radiation transmitted through
the partial reflector to be reflected and resonated. The radiation
detector may be configured to determine the gain or increase in
intensity of the output radiation over the known intensity of the
input seed radiation, which gain or increase may be attributed to
stimulated scattering.
[0076] Accordingly, with continued reference to the method 100 of
FIG. 1, irradiating a sample at block 110 may include irradiating a
sample with an input transverse radiation and an input seed
radiation having different frequencies. Likewise, detecting
radiation at block 160 may include detecting a gain over the
intensity of the seed radiation. The seed radiation frequency may
be varied over a set of predetermined frequencies corresponding to
different molecules to determine whether the sample within the
chamber contains a molecule corresponding to one of these
frequencies. For example, the seed radiation may be provided at an
inelastically scattering frequency for adenine. If the gain over
the seed radiation is sufficiently zero then there was no simulated
scattering and it may be concluded that the sample did not contain
adenine. Then the frequency of the seed radiation may be adjusted
to that corresponding to other nucleotide bases until the gain is
sufficiently nonzero indicating that the sample may contain that
particular base. If none of the frequencies cause the gain to
become sufficiently nonzero, the sample may be removed from the
chamber and another sample added thereto.
[0077] The concepts of seed and transverse input radiations
discussed in FIG. 8 may also be applied to a spectroscopic analysis
system similar to the ones shown in FIGS. 5-6. For example, input
transverse excitation radiation may be transmitted in through
window 620, input seed radiation may be transmitted in through
window 660, and scattered radiation may be transmitted out through
window 660. Portions of the input transverse radiation that do not
irradiate the sample may be removed through a window at location
679.
[0078] Accordingly, in embodiments of the invention employing a
stimulated Raman analysis, a single molecule of interest may be
positioned within a resonant chamber that is capable of optically
amplifying the intensity of inelastically scattered stimulated
Raman radiation by causing resonance enhanced stimulated scattering
events that aid in increasing or amplifying the intensity of the
signal to an extent that allows detection and accurate
identification of the single molecule of interest. As an example, a
single nucleic acid derivative of interest may be positioned in a
resonant chamber having two opposing reflectors. The first
reflector has a high reflectivity for both an excitation radiation
and an inelastically scattered Raman radiation, while the second
reflector has a high reflectivity for the excitation radiation and
has a sufficient transmittance for the inelastically scattered
Raman radiation. In one aspect, a high reflectivity may be greater
than approximately 99% (e.g., 99.5%-99.9%) while a sufficient
transmittance may be a reflectivity not greater than 99%. The two
opposing reflectors are separated by a distance that is sufficient
to resonate the inelastically scattered Raman radiation. The
distance may be determined based on inelastically scattered Raman
radiation for the single nucleic acid derivative, or for a
plurality of nucleic acid derivatives so that the chamber may be
used for identifying different derivatives.
[0079] Accordingly, a sample containing a single molecule of
interest may be analyzed with stimulated Raman spectroscopy. In
addition to stimulated Raman spectroscopy, another suitable form of
coherent Raman spectroscopy is Coherent Anti-Stokes Raman
Spectroscopy (CARS), which employs a coherent anti-Stokes Raman
scattering phenomenon. Many other forms of Raman, such as
spontaneous Raman and surface enhanced Raman spectroscopy (SERS),
involve spontaneous emission of random and non-coherent
inelastically scattered radiation. In contrast, CARS inherently
produces a highly directional and coherent output inelastically
scattered radiation signal. Rather than due to resonance, as in
stimulated Raman, the coherency is provided by coherent build up of
amplitudes for phase matched radiations. As used herein, CARS will
encompass various implementations of CARS, such as time-resolved
CARS, scanned CARS, OPO CARS, MAD CARS, PARS, and others. The CARS
radiation signal is sufficiently coherent and intense to allow
detection of even a single molecule of interest. The spectral shape
and intensity of the CARS output signal contains the spectral
characteristics of the sample and may be used to identify the
sample. Accordingly, the present inventors contemplate employing
CARS to analyze and identify a single nucleic acid derivative in
conjunction with nucleic acid sequencing.
[0080] CARS is a four-wave mixing spectroscopy. During CARS a
sample in a chamber is irradiated and excited with a first
radiation having a first wavelength (w.sub.1) and often a second
radiation having a second wavelength (W.sub.2) that overlap and are
phase matched (i.e., they spatially and temporally overlap) in
order to induce the sample to irradiate coherent anti-Stokes
scattered radiation having a third wavelength (W.sub.3) that is
related to the first and second frequencies according to the
relationship, W.sub.3=2w.sub.1-W.sub.2.
[0081] FIG. 9 shows an energy diagram for a CARS process used to
analyze a molecule of a sample, according to embodiments of the
invention. Energy of the molecule is plotted on the y-axis and the
progression of the CARS process is plotted on the x-axis. For
convenience, the discussion will refer to a photon, although often
in practice a radiation containing at least one but often many
photons will be used to analyze the sample. Initially, the molecule
is excited coherently with a first photon at wavelength (w.sub.1)
910 in order to induce an emission of a second photon at wavelength
(w2) 920. The first photon is often provided as a laser beam pulse
directed at the chamber to irradiate the molecule. As shown, the
first photon excites the molecule from a ground vibrational state
950 to a lower virtual state 970. The emitted second photon brings
the molecule from the virtual state 870 down to a lower excited
vibrational state 960. The excited vibrational state differs from
the ground vibrational state by vibratory transition of Raman shift
(W.sub.4). The molecule may emit the second photon by either
spontaneous or stimulated emission however coherent stimulated
emission is used in CARS. Often, the molecule will be exposed by
another photon also of wavelength (W.sub.2) in order to induce
stimulated emission of the second photon. Stimulated emission is
relatively more probable than spontaneous emission and may lead to
higher CARS signal intensities that facilitate detection and
molecule identification. The stimulated emission is often induced
by irradiating the chamber with a second radiation having the
second wavelength (W.sub.2), concurrently with the irradiation with
the first radiation (the first photon).
[0082] The molecule in the lower excited vibrational state 960 is
subsequently irradiated with a third photon at wavelength (w.sub.1)
930. Irradiation with a laser beam pulse may also provide this
photon. The third photon excites the molecule from the excited
vibrational state 960 to a higher virtual state 980. The molecular
vibrations oscillate in phase and interfere constructively. The
molecule emits a fourth photon at wavelength (W.sub.3) to drop from
the higher virtual state to the ground vibrational state. The
wavelength of the fourth photon is twice the wavelength of the
first and third photons minus the wavelength of the emitted second
photon (i.e., w.sub.3=2w1-w.sub.2). In essence, CARS
disproportionates or divides two (w.sub.1) photons into a (w.sub.2)
photon and a (W.sub.3) photon. A phase matching condition
determines the direction of the coherent CARS signal. As will be
discussed further below, the present inventors contemplate
employing various geometric configurations of beams to take
advantage of this property to help spatially isolate the coherent
CARS signal from input excitation beams, in order to facilitate
detection and identification of molecules. The intensity of the
emitted CARS signal is directly related to the intensity of the
excitation radiations.
[0083] Different ways of exposing the sample to these photons are
known in the arts. In one approach, the sample may be concurrently
irradiated with a first laser beam pulse of the wavelength w.sub.1,
a second laser beam pulse of the wavelength W.sub.2, and a third
laser beam pulse of the wavelength (w.sub.1). In practice, the
first laser beam pulse can also serve as a third laser beam pulse
as they have the same wavelength, (w.sub.1). Electronics to provide
concurrent laser beam pulses from multiple lasers are commercially
available. Such synchronization electronics include SynchroLock
from Coherent (Santa Clara, Calif.) or Lok-to-Clok from
Spectra-Physics (Mountain View, Calif.). Alternatively, in another
approach the sample may be continually irradiated with a laser beam
of the wavelength (w.sub.1) and intermittently irradiated with a
pulse or scan of a laser beam of the wavelength (w.sub.2). When the
difference (w.sub.1-w.sub.2) substantially coincides or matches the
wavelength of a molecular vibration of the sample, for example the
vibratory transition (w.sub.4), the intensity of the CARS signal is
often significantly enhanced. A spectrum may be generated for a
range of the wavelength (w.sub.2) or the difference
(w.sub.1-w.sub.2). Often, (w.sub.2) is continually changed, varied,
or scanned, while holding (w.sub.1) fixed, and simultaneously
recording the corresponding CARS signals. Alternatively, (w.sub.1)
is continually changed, varied, or scanned, while holding (w.sub.2)
fixed, and simulateneously recording the corresponding CARS
signals. In another embodiment, both (w.sub.1) and (w.sub.2) are
continually changed, varied, or scanned, to obtain the desired
(w.sub.1-w.sub.2), while recording the corresponding CARS signals.
In one approach a tunable broadband dye laser is scanned over the
desired range of wavelength. Another approach involves using a
broadband Stokes laser covering the whole range of frequencies
concurrently. Alternatively, signal and idler beams from optical
parametric oscillator can be used to scan (w.sub.1-w.sub.2).
[0084] In order for a CARS process to occur efficiently energy and
momentum conservation need to occur. During CARS the output signal
is built by coherent addition of amplitudes. In the CARS four-wave
mixing process, it will often be desirable to impose a phase
matching condition on the wave vectors of the input excitation
radiations and output coherent anti-Stokes radiation in order to
achieve good intensities of the output coherent radiation. The
amplitudes should be substantially in phase, that is the beams
aligned to provide phase matching between the sum of two incoming
waves at wavelength (w.sub.1) and the sum of output waves at
wavelength (w.sub.2) and (w.sub.3). When the phase matching
condition is satisfied, the strength or intensity of the output
anti-Stokes wave should be near maximum. This results in the sample
irradiating or emitting a coherent resonance enhanced anti-Stokes
scattered radiation, often as a coherent, collimated,
highly-directional beam with a predetermined direction, rather than
as a weak and randomly scattered signal as would be expected in
spontaneous Raman spectroscopy. The predetermined direction of the
CARS signal may allow various configurations that allow it to be
geometrically or configurationally separated from the excitation
radiation.
[0085] A number of prior art phase matching beam configurations or
geometries are known in the arts including a collinear CARS
geometry, a BOX CARS geometry, a Folded BOX CARS geometry, and a
USED CARS geometry. Any of these may potentially be used.
Additionally, the inventors contemplate using configurations that
help separate or isolate the CARS radiation signal from input
excitation radiation. FIGS. 11-13 show exemplary configurations of
excitation radiation beams that when phase matched may help to
spatially separate and isolate the CARS signal from the excitation
beams at the radiation detection end, according to various
embodiments of the invention. Advantageously, in these exemplary
configurations, the CARS signal beam is spatially separated and
isolated from the excitation beams at the radiation detection end,
which may facilitate sample identification. Other configurations
will be apparent to those having an ordinary level of skill in the
art and the benefit of the present teachings.
[0086] FIG. 10 shows a cross-beam configuration, according to
embodiments of the invention. In the cross-beam configuration, an
input first (w.sub.1) and second radiation (w.sub.2) are provided
to a chamber 1050 with an angle relative to one another so that
they irradiate a sample 1030 within the chamber and subsequently
separate from one anther and from a coherent anti-stokes scattered
radiation (w.sub.3) that is irradiated from the sample. In this
way, the configuration helps separate the input radiation from the
output coherent scattered radiation, and wavelength selection or
filtering may be avoided. The chamber may be similar to other
chambers disclosed herein. The chamber may, but need not, be a
resonant chamber and may, but need not, contain reflectors or have
a size or shape to facilitate resonance.
[0087] FIG. 11 shows momentum conservation for a cross-beam
configuration similar to that shown in FIG. 10. The phase matching
condition determines the direction of the coherent anti-Stokes
radiation. Because of the conservation of energy and momentum in a
four-wave mixing process, a phase matching condition is imposed on
the wave vectors of the input and anti-Stokes output waves. When
the phase matching condition is sufficiently satisfied the output
strength is near maximum and the direction and momentum of the
coherent anti-Stokes photons are related to the direction and
momentum of the excitation photons. This is shown graphically in
FIG. 11 wherein two vectors for the first excitation wavelength
(2k.sub.w1) and one vector for the second excitation wavelength
(k.sub.w2) are combined to give a vector for the coherent
anti-Stokes radiation (k.sub.w3), or mathematically
k.sub.w3=2k.sub.w1-k.sub.w2. The length of the vectors represents
the photons momentum. The configuration of the direction of the
excitation radiations allows the CARS signal photon to be
geometrically separated from the input excitation photons. This may
facilitate detection of the emitted CARS photon.
[0088] FIG. 12 shows a forward-scattered configuration, according
to embodiments of the invention. In this configuration, the
coherent radiation is not separated from the input radiations, and
a wavelength selection device, such as a filter, may be used to
isolate the coherent radiation.
[0089] FIG. 13 shows a back-scattered configuration, according to
embodiments of the invention. In this configuration, as in the
cross-beam configuration, the coherent radiation is separated from
the input radiations. The intensity of the coherent radiation in
this configuration is often comparable with that of the
forward-scattered configuration when the sample size is less than
the excitation wavelength, although in other instances it may be
desirable to use a forward-scattered configuration and appropriate
wavelength selection device to obtain a stronger signal.
[0090] FIG. 14 shows a nucleic acid sequencing system 1401 in which
embodiments of the invention may be implemented. A nucleic acid may
be provided to a sampling system 1402. The sampling system may
include enzymes or other cleavers of nucleic acids to remove or
derive a single nucleic acid derivative from the nucleic acid. In
one instance, the system may include an exonuclease enzyme that
breaks down a nucleic acid chain by removing single nucleotides one
by one from the end of a chain. The sampling system may also
include equipment to prepare the sample, for example to dilute the
nucleic acid derivative in an aqueous solution and supply any
additives. The sample may be added to the spectroscopic analysis
system 1400, for example though a fluid channel 1470. The analysis
system performs a Raman spectroscopic analysis of the sample, as
described elsewhere herein, in order to identify the nucleic acid
derivative. For example, the analysis system may determine whether
the sample contains a single base selected from the group
comprising adenine, cytosine, guanine, thymine, and uracil. The
identity of the nucleic acid derivative may be stored in a memory
as part of an ordered sequence for the input nucleic acid, analyzed
as part of the sequence to solve a medical problem, for example to
diagnose or treat a disease, or used for other purposes.
[0091] Having been generally described, the following examples are
given as particular embodiments of the invention, to illustrate
some of the properties and demonstrate the practical advantages
thereof, and to allow one skilled in the art to utilize the
invention. It is understood that these examples are to be construed
as merely illustrative.
EXAMPLES
Example 1
Nucleic Acid Sequencing Using Raman Detection and Nanoparticles
[0092] Certain embodiments of the invention, exemplified in FIG.
15, involve sequencing of one or more single-stranded nucleic acid
molecules 1509 that may be attached to an immobilization surface in
a reaction chamber 1501. The reaction chamber 1501 may contain one
or more exonucleases that sequentially remove one nucleotide 1510
at a time from the unattached end of the nucleic acid molecule
1509.
[0093] As the nucleotides 1510 are released, they may move down a
microfluidic channel 1502 and into a nanochannel 1503 or
microchannel 1503, past a detection unit. The detection unit may
comprise an excitation source 1506, such as a laser, that emits an
excitatory beam. The excitatory beam may interact with the released
nucleotides 1510 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 may be detected by a Raman
spectroscopic detector 1507, such as a spectrometer, a
monochromator or a charge coupled device (CCD), such as a CCD
camera.
[0094] The excitation source 1506 and detector 1507 may be arranged
so that nucleotides 1510 are excited and detected as they pass
through a region of nanoparticles 1511 in a nanochannel 1503 or
microchannel 1503. The nanoparticles 1511 may be cross-linked to
form "hot spots" for Raman detection. By passing the nucleotides
1510 through the nanoparticle 1511 hot spots, the sensitivity of
Raman detection may be increased by many orders of magnitude.
Alternatively, the nucleotides may be passed into a resonant
chamber.
[0095] Preparation of Reaction Chamber, Microfluidic Channel and
MicroChannel
[0096] Borofloat glass wafers (Precision Glass & Optics, Santa
Ana, Calif.) may be pre-etched for a short period in concentrated
HF (hydrofluoric acid) and cleaned before deposition of an
amorphous silicon sacrificial layer in a plasma-enhanced chemical
vapor deposition (PECVD) system (PEII-A, Technics West, San Jose,
Calif.). Wafers may be primed with hexamethyldisilazane (HMDS),
spin-coated with photoresist (Shipley 1818, Marlborough, Mass.) and
soft-baked. A contact mask aligner (Quintel Corp. San Jose, Calif.)
may be used to expose the photoresist layer with one or more mask
designs, and the exposed photoresist may be removed using a mixture
of Microposit developer concentrate (Shipley) and water. Developed
wafers may be hard-baked and the exposed amorphous silicon removed
using CF.sub.4 (carbon tetrafluoride) plasma in a PECVD reactor.
Wafers may be chemically etched with concentrated HF to produce the
reaction chamber 1501, microfluidic channel 1502 and microchannel
1503. The remaining photoresist may be stripped and the amorphous
silicon may be removed.
[0097] Nanochannels 1503 may be formed by a variation of this
protocol. Standard photolithography may be used to form the micron
scale features of the integrated chip. A thin layer of resist may
be coated onto the chip. An atomic force microscopy/scanning
tunneling probe tip may be used to remove a 5 to 10 nm wide strip
of resist from the chip surface. The chip may be briefly etched
with dilute HF to produce a nanometer scale groove on the chip
surface. In the present non-limiting example, a channel 1503 with a
diameter of between 500 nm and 1 .mu.m may be prepared.
[0098] Access holes may be drilled into the etched wafers with a
diamond drill bit (Crystalite, Westerville, Ohio). A finished chip
may be prepared by thermally bonding two complementary etched and
drilled plates to each other in a programmable vacuum furnace
(Centurion VPM, J. M. Ney, Yucaipa, Calif.). Alternative exemplary
methods for fabrication of a chip incorporating a reaction chamber
1501, microfluidic channel 1502 and nanochannel 1503 or
microchannel 1503 are disclosed in U.S. Pat. Nos. 5,867,266 and
6,214,246. A nylon filter with a molecular weight cutoff of 2,500
daltons may be inserted between the reaction chamber 1501 and the
microfluidic channel 1502 to prevent exonuclease and/or nucleic
acid 1509 from leaving the reaction chamber 1501.
[0099] Nanoparticle Preparation
[0100] Silver nanoparticles 1511 may be prepared according to Lee
and Meisel (J. Phys. Chem. 86:3391-3395, 1982). Gold nanoparticles
1511 may be purchased from Polysciences, Inc. (Warrington, Pa.),
Nanoprobes, Inc. (Yaphank, N.Y.) or Ted-pella Inc. (Redding,
Calif.). In a non-limiting example, 60 nm gold nanoparticles 1511
may be used. The skilled artisan will realize that other sized
nanoparticles 1511, such as 5, 10, or 20 nm, may also be used.
[0101] Gold nanoparticles 1511 may be reacted with alkane dithiols,
with chain lengths ranging from 5 nm to 50 nm. The linker compounds
may contain thiol groups at both ends of the alkane to react with
gold nanoparticles 1511. An excess of nanoparticles 1511 to linker
compounds may be used and the linker compounds slowly added to the
nanoparticles 1511 to avoid formation of large nanoparticle
aggregates. After incubation for two hours at room temperature,
nanoparticle 1511 aggregates may be separated from single
nanoparticles 1511 by ultracentrifugation in 1 M sucrose. Electron
microscopy reveals that aggregates prepared by this method contain
from two to six nanoparticles 1511 per aggregate. The aggregated
nanoparticles 1511 may be loaded into a microchannel 1503 by
microfluidic flow. A constriction or filter at the end of the
microchannel 1503 may be used to hold the nanoparticle aggregates
1511 in place.
[0102] Nucleic Acid Preparation and Exonuclease Treatment
[0103] Human chromosomal DNA may be purified according to Sambrook
et al. (1989). Following digestion with Bam H1, the genomic DNA
fragments may be inserted into the multiple cloning site of the
pBluescript.RTM. II phagemid vector (Stratagene, Inc., La Jolla,
Calif.) and grown up in E. coli. After plating on
ampicillin-containing agarose plates a single colony may be
selected and grown up for sequencing. Single-stranded DNA copies of
the genomic DNA insert may be rescued by co-infection with helper
phage. After digestion in a solution of proteinase K:sodium dodecyl
sulphate (SDS), the DNA may be phenol extracted and then
precipitated by addition of sodium acetate (pH 6.5, about 0.3 M)
and 0.8 volumes of 2-propanol. The DNA containing pellet may be
resuspended in Tris-EDTA buffer and stored at -20.degree. C. until
use.
[0104] M13 forward primers complementary to the known
pBluescript.RTM. sequence, located next to the genomic DNA insert,
may be purchased from Midland Certified Reagent Company (Midland,
Tex.). The primers may be covalently modified to contain a biotin
moiety attached to the 5' end of the oligonucleotide. The biotin
group may be covalently linked to the 5'-phosphate of the primer
via a (CH.sub.2).sub.6 spacer. Biotin-labeled primers may be
allowed to hybridize to the ssDNA template molecules prepared from
the pBluescript.RTM. vector. The primer-template complexes may be
attached to streptavidine coated beads according to Done et al.
(Bioimaging 5: 139-152, 1997). At appropriate DNA dilutions, a
single primer-template complex is attached to a single bead. A bead
containing a single primer-template complex may be inserted into
the reaction chamber 1501 of a sequencing apparatus 1500.
[0105] The primer-template may be incubated with modified T7 DNA
polymerase (United States Biochemical Corp., Cleveland, Ohio). The
reaction mixture may contain unlabeled
deoxyadenosine-5'triphosphate (dATP) and
deoxyguanosine-5'-triphosphate (dGTP), digoxigenin-labeled
deoxyuridine-5-triphosphate (digoxigenin-dUTP) and
rhodamine-labeled deoxycytidine-5'-triphosphate (rhodamine-dCTP).
The polymerization reaction may be allowed to proceed for 2 hours
at 37.degree. C. After synthesis of the digoxigenin and rhodamine
labeled nucleic acid, the template strand may be separated from the
labeled nucleic acid, and the template strand, DNA polymerase and
unincorporated nucleotides washed out of the reaction chamber 1501.
Alternatively, all deoxynucleoside triphosphates used for
polymerization may be unlabeled. In other alternatives, single
stranded nucleic acids may be directly sequenced without
polymerization of a complementary strand.
[0106] Exonuclease activity may be initiated by addition of
exonuclease m to the reaction chamber 1501. The reaction mixture
may be maintained at pH 8.0 and 37.degree. C. As nucleotides 1510
are released from the 3' end of the nucleic acid, they may be
transported by microfluidic flow down the microfluidic channel
1502. At the entrance to the microchannel 1503, an electrical
potential gradient created by a pair of electrodes 1504, 1505 may
be used to drive the nucleotides 1510 out of the microfluidic
channel 1502 and into the microchannel 1503. As the nucleotides
1510 pass through the nanoparticles 1511, or alternatively into a
resonant chamber, they may be exposed to excitatory radiation from
a laser 1506. Raman emission spectra may be detected by the Raman
detector 1507 as disclosed below.
[0107] Raman Detection of Nucleotides
[0108] A Raman detection unit as disclosed in Example 2 may be
used. The Raman detector 1507 may be capable of detecting and
identifying single nucleotides 1510 of dATP, dGTP, rhodamine-dCTP
and digoxigenin-dUTP moving past the detector 1507. Data on the
time course for labeled nucleotide detection may be compiled and
analyzed to obtain the sequence of the nucleic acid. In alternative
embodiments, the detector 1507 may be capable of detecting and
identifying single unlabeled nucleotides.
Example 2
Raman Detection of Nucleotides
[0109] Methods and Apparatus
[0110] In a non-limiting example, the excitation beam of a Raman
detection unit was generated by a titanium: sapphire laser (Mira by
Coherent) at a near-infrared wavelength (750-50 nm) or a gallium
aluminum arsenide diode laser (PI-ECL series by Process
Instruments) at 785 nm or 830 nm. Pulsed laser beams or continuous
beams were used. The excitation beam was transmitted through a
dichroic mirror (holographic notch filter by Kaiser Optical or a
dichromatic interference filter by Chroma or Omega Optical) into a
collinear geometry with the collected beam. The transmitted beam
passed through a microscope objective (Nikon LU series), and was
focused onto the Raman active substrate, or into the resonant
chamber, where target analytes (nucleotides or purine or pyrimidine
bases) were located.
[0111] The Raman scattered light from the analytes was collected by
the same microscope objective, and passed the dichroic mirror to
the Raman detector. The Raman detector comprised a focusing lens, a
spectrograph, and an array detector. The focusing lens focused the
Raman scattered light through the entrance slit of the
spectrograph. The spectrograph (Acton Research) comprised a grating
that dispersed the light by its wavelength. The dispersed light was
imaged onto an array detector (back-illuminated deep-depletion CCD
camera by RoperScientific). The array detector was connected to a
controller circuit, which was connected to a computer for data
transfer and control of the detector function.
[0112] For surface-enhanced Raman spectroscopy (SERS), the Raman
active substrate consisted of metal-coated nanostructures. Silver
nanoparticles, ranging in size from 5 to 200 nm, were made by the
method of Lee and Meisel (J. Phys. Chem., 86:3391, 1982).
Alternatively, samples were mixed with metallic nanoparticles and
were placed on an aluminum substrate under the microscope
objective. The following discussion assumes a stationary sample on
the aluminum substrate. The number of molecules detected was
determined by the optical collection volume of the illuminated
sample and the average concentration of the illuminated sample.
[0113] Single nucleotides may also be detected by SERS using
microfluidic channels. In various embodiments of the invention,
nucleotides may be delivered to a Raman active substrate through a
microfluidic channel (between about 5 and 200 .mu.m wide).
Microfluidic channels can be made by molding polydimethylsiloxane
(PDMS), using the technique disclosed in Anderson et al.
("Fabrication of topologically complex three-dimensional
microfluidic systems in PDMS by rapid prototyping," Anal. Chem.
72:3158-3164, 2000).
[0114] Where SERS was performed in the presence of silver
nanoparticles, the nucleotide, purine or pyrimidine analyte was
mixed with LiCl (90 .mu.M final concentration) and nanoparticles
(0.25 M final concentration silver atoms). SERS data were collected
using room temperature analyte solutions.
[0115] Results
[0116] Nucleoside monophosphates, purines and pyrimidines were
analyzed by SERS, using the system disclosed above. Table 1 shows
exemplary detection limits for various analytes of interest.
TABLE-US-00001 TABLE 1 SERS Detection of Nucleoside Monophosphates,
Purines and Pyrimidines Number of Molecules Analyte Final
Concentration Detected dAMP 9 picomolar (pM) ~1 molecule Adenine 9
pM ~1 molecule dGMP 90 .mu.M 6 .times. 10.sup.6 Guanine 909 pM 60
dCMP 909 .mu.M 6 .times. 10.sup.7 Cyotosine 90 nM 6 .times.
10.sup.3 dTMP 9 .mu.M 6 .times. 10.sup.5 Thymine 90 nM 6 .times.
10.sup.3
[0117] Conditions were optimized for adenine nucleotides only. LiCl
(90 .mu.M final concentration) was determined to provide optimal
SERS detection of adenine nucleotides. Detection of other
nucleotides may be facilitated by use of other alkali-metal halide
salts, such as NaCl, KCl, RbCl or CsCl. The claimed methods are not
limited by the electrolyte solution used, and it is contemplated
that other types of electrolyte solutions, such as MgCl.sub.2,
CaCl.sub.2, NaF, KBr, LiI, etc. may be of use. The skilled artisan
will realize that electrolyte solutions that do not exhibit strong
Raman signals will provide minimal interference with SERS detection
of nucleotides. The results demonstrate that the Raman detection
system and methods disclosed above were capable of detecting and
identifying single molecules of nucleotides and purine bases. This
demonstrates Raman detection of unlabeled nucleotides at the single
nucleotide level.
Example 3
Raman Emission Spectra of Nucleotides, Purines and Pyrimidines
[0118] The Raman emission spectra of various analytes of interest
was obtained using the protocol of Example 2, with the indicated
modifications. FIG. 16 shows the Raman emission spectra of a 100 mM
solution of each of the four nucleoside monophosphates, in the
absence of surface enhancement and without Raman labels. No LiCl
was added to the solution. A 10 second data collection time was
used. Lower concentrations of nucleotides may be detected with
longer collection times, with surface enhancement, using labeled
nucleotides and/or with added electrolyte solution. Excitation
occurred at 514 nm. For each of the following figures, a 785 nm
excitation wavelength was used. As shown in FIG. 16, the unenhanced
Raman spectra showed characteristic emission peaks for each of the
four unlabeled nucleoside monophosphates.
[0119] FIG. 17 shows the SERS spectrum of a 1 nm solution of
guanine, in the presence of LiCl and silver nanoparticles. Guanine
was obtained from dGMP by acid treatment, as discussed in Nucleic
Acid Chemistry, Part 1, L. B. Townsend and R. S. Tipson (eds.),
Wiley-Interscience, New York, 1978. The SERS spectrum was obtained
using a 100 msec data
[0120] FIG. 18 shows the SERS spectrum of a 10 nM cytosine
solution, obtained from dCMP by acid hydrolysis. Data were
collected using a 1 second collection time.
[0121] FIG. 19 shows the SERS spectrum of a 100 nM thymine
solution, obtained by acid hydrolysis of dTMP. Data were collected
using a 100 msec collection time.
[0122] FIG. 20 shows the SERS spectrum of a 100 pM adenine
solution, obtained by acid hydrolysis of dAMP. Data were collected
for 1 second.
[0123] FIG. 21 shows the SERS spectrum of a 500 nM solution of dATP
(lower trace) and fluorescein-labeled dATP (upper trace).
dATP-fluorescein was purchased from Roche Applied Science
(Indianapolis, Ind.). The Figure shows a strong increase in SERS
signal due to labeling with fluorescein.
Example 4
Resonance Enhanced Raman Detection of Single Molecules
[0124] This example demonstrates that the energies of Raman
scattered light generated from single molecules in resonant
chambers may be detected by various known radiation detection
devices. The results are based on simulations performed by the
inventors using MATLAB.RTM. available from The MathWorks, Inc. of
Natick, Mass.
[0125] FIG. 22 shows a plot comparing the energies of Raman
scattered light generated from single molecules respectively
contained inside an optical resonance chamber (curves A and B), or
positioned in free space without resonance enhancement (curve C),
according to one embodiment of the invention. The energies, in
Joules, are plotted on the y-axis, against time, in seconds, on the
x-axis.
[0126] The curves A and B plot the energy of Raman scattered light
stored in the optical resonance chamber as a function of time. For
both curves, the optical resonance chamber is capable of resonating
the excitation light as well as the Raman scattered light in order
to provide even greater resonant enhancement and stronger signals
for detection. The simulations assumed a cavity quality factor of
about 10.sup.6 for both excitation light and Raman scattered light.
Even higher cavity quality factors are known in the arts. For
example, cavity factors of about 10.sup.9 have been reported by
Spillane et al., in Nature, 415:621-623 (2002). The simulations
assumed a Raman cross-section of about 10.sup.-29 cm.sup.2. The
excitation light was provided as a 1 W continuous-wave. The time
steps were 100 nanoseconds.
[0127] Different hypothetical molecules with widely differing Raman
gain coefficients were used to generate the curves A and B. The
Raman gain coefficients essentially quantify the molecules
likelihood of generating stimulated Raman emissions. The curve A is
based on a molecule with a relatively high Raman gain coefficient
of about 20.times.10.sup.-8 cm/W, which is typical for silicon. The
curve B is based on a molecule with zero Raman gain coefficient. As
such, the curves A and B respectively provide near upper and lower
bounds for the stimulated Raman behavior of most molecules,
including nucleic acid derivatives. The energy of the cumulative
Raman scattered light generated during the given period of time is
plotted in curve C. There is no resonance enhancement in curve
C.
[0128] As shown, the energies of Raman scattered light generated
from the single molecules in the resonant chambers are
significantly greater than the energy of the molecule positioned in
free space (without resonance enhancement). The greatest energies
were observed when the stimulated Raman process was induced (curve
A). The energies for the stimulated Raman (curve A) were around
eight orders of magnitude larger than the energies without
resonance (curve C). Even when the stimulated Raman process was not
induced, the energies for the resonated spontaneous Raman process
(curve B) were still about six orders of magnitude larger than the
energies without resonance (curve C). Such enhancement in the
energy available for detection is quite significant.
[0129] A dashed line at 10.sup.-20 J indicates a typical detection
level for a variety of commercially available high-quality
radiation detection devices. Avalanche photodiodes, photomultiplier
tubes, and intensified charge-coupled devices typically have at
least such sensitivities. The Raman scattered light generated
inside the optical resonance chamber in both curves A and B exceed
the indicated detection level within about 100 microseconds. In
contrast, the energy of Raman scattered light from the molecule in
free space without optical resonance (curve C), remains below the
detection level for more than one second.
[0130] Accordingly, as demonstrated by these simulations, the
energies of Raman scattered light generated from single molecules
in resonant chambers may be detected by various known radiation
detection devices. Significantly, this is true for both spontaneous
Raman (curve B) and stimulated Raman (Curve A).
Example 5
Resonance Enhanced Raman Detection of Multiple Molecules
[0131] This prospective example demonstrates how to achieve even
greater detection energies than those reported in Example 4 by
using a plurality of molecules, instead of just a single molecule.
In this approach, a plurality of molecules may be introduced into
the resonant chamber. For example, at least 100, or at least 1000
molecules are introduced into the resonant chamber. In one aspect,
enough molecules may be introduced to allow detection of a
particular type of molecule with a desired radiation detection
device. To aid in identification, it may be appropriate to include
a majority, or a vast majority, of the molecules being of the same
type.
[0132] The total detection energy generally increases for each
additional molecule in the resonant chamber. Accordingly, each
additional molecule should further increase the total energy of
Raman scattered light in the resonant chamber. Often, the total
energy of Raman scattered light generated in the resonant chamber
may initially increase substantially linearly for small numbers of
molecules, and then increase even more dramatically (nonlinearly)
for larger numbers of molecules. In this way, a plurality of
molecules may be used to provide even stronger Raman emission
signals.
[0133] Thus, spectroscopic analysis chambers and methods for
analyzing samples within those chambers are disclosed. While the
invention has been described in terms of several embodiments, those
skilled in the art will recognize that the invention is not limited
to the embodiments described, but may be practiced with
modification and alteration within the spirit and scope of the
claims. It is to be realized that the optimum dimensional
relationships for the parts of the invention, to include variations
in size, materials, shape, form, function and manner of operation,
assembly and use, are deemed readily apparent to one of ordinary
skill in the art, and all equivalent relationships to those
illustrated in the drawings and described in the specification are
intended to be encompassed by the present invention. The
description is thus to be regarded as illustrative instead of
limiting.
* * * * *
References