U.S. patent application number 11/443633 was filed with the patent office on 2006-09-28 for methods and systems for detecting biomolecular binding using terahertz radiation.
This patent application is currently assigned to INTEL CORPORATION. Invention is credited to Andrew Berlin, Tae-Woong Koo, Brian Ostrovsky, Ken Salsman.
Application Number | 20060216742 11/443633 |
Document ID | / |
Family ID | 35757837 |
Filed Date | 2006-09-28 |
United States Patent
Application |
20060216742 |
Kind Code |
A1 |
Koo; Tae-Woong ; et
al. |
September 28, 2006 |
Methods and systems for detecting biomolecular binding using
terahertz radiation
Abstract
Provided herein are methods and systems for detecting
biomolecular binding events using gigahertz or terahertz radiation.
The methods and systems use low-energy spectroscopy to detect
biomolecular binding events between molecules in an aqueous
solution. The detected biomolecular binding events include, for
example, nucleic acid hybridizations, antibody/antigen binding, and
receptor/ligand binding.
Inventors: |
Koo; Tae-Woong; (Cupertino,
CA) ; Berlin; Andrew; (San Jose, CA) ;
Salsman; Ken; (Pleasanton, CA) ; Ostrovsky;
Brian; (Portland, OR) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.;DLA PIPER RUDNICK GRAY CARY US LLP
Attorneys for INTEL CORPORATION
4365 Executive Drive, Suite 1100
San Diego
CA
92121-2133
US
|
Assignee: |
INTEL CORPORATION
|
Family ID: |
35757837 |
Appl. No.: |
11/443633 |
Filed: |
May 30, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10911441 |
Aug 4, 2004 |
|
|
|
11443633 |
May 30, 2006 |
|
|
|
Current U.S.
Class: |
435/6.13 ;
435/287.2; 435/7.1 |
Current CPC
Class: |
B01J 2219/0061 20130101;
B01J 2219/00612 20130101; C12Q 1/6825 20130101; G01N 21/7743
20130101; B01J 2219/00605 20130101; B01L 3/5027 20130101; B01J
2219/00637 20130101; C12Q 2545/114 20130101; C12Q 2523/313
20130101; G01N 21/65 20130101; G01N 2021/651 20130101; C12Q 1/6825
20130101; G01N 33/54373 20130101; G01N 21/3581 20130101; B01J
2219/00722 20130101; B01J 2219/00626 20130101; B01J 2219/00639
20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; C12M 1/34 20060101
C12M001/34 |
Claims
1. A system for detecting a biomolecular binding event, the system
comprising: a) a sample containing a first molecule and a second
molecule, wherein the first molecule is suspected of binding the
second molecule; b) a terahertz or gigahertz radiation source
positioned to aim an input radiation toward the sample; and c) a
detector for receiving an exit radiation reflecting from the
sample.
2. The system of claim 1, wherein the system further comprises a
substrate that is transparent to the terahertz radiation, wherein
the first molecule is immobilized on the substrate.
3. The system of claim 2, further comprising a reaction chamber
adjacent to the substrate.
4. The system of claim 3, wherein a solution in the receptacle
comprises the second molecule.
5. The system of claim 4, wherein the solution comprises water.
6. The system of claim 2, wherein the substrate is a surface of a
waveguide.
7. The system of claim 1, further comprising no more than one
diffraction grating positioned between the sample and the
detector.
8. The method of claim 1, wherein the first molecule is a nucleic
acid.
9. The method of claim 1, wherein the second molecule is a nucleic
acid.
10. The method of claim 1, wherein the second molecule is a
protein.
11. The method of claim 1, wherein the first molecule is a receptor
and the second molecule is a ligand.
12. The method of claim 1, wherein the first molecule is a protein
and the second molecule is a protein.
13. An apparatus for detecting a biomolecular binding event in a
sample containing a first molecule and a second molecule, wherein
the first molecule is suspected of binding the second molecule,
comprising: a terahertz radiation source positioned to aim an input
radiation toward the sample; and a detector positioned to receive
an exit radiation reflecting from the sample.
14. The apparatus of claim 13, further comprising an information
processing and control system in communication with the
detector.
15. The apparatus of claim 14, wherein the information processing
and control system is capable of analyzing the data received from
the detector.
16. The apparatus of claim 13, further comprising a spectrum
analyzer.
17. The apparatus of claim 13, further comprising sample
holder.
18. The apparatus of claim 17, wherein the sample holder includes a
substrate that is transparent to the terahertz radiation, wherein
the first molecule is immobilized on the substrate.
19. The apparatus of claim 18, further comprising a reaction
chamber adjacent to the substrate, wherein a solution in the
reaction chamber includes the second molecule.
20. The apparatus of claim 18, wherein the substrate include a
waveguide with an input portion and an output portion, wherein the
first molecule is immobilized on the waveguide.
21. The apparatus of claim 20, wherein the terahertz radiation
source is positioned to aim the input radiation toward the input
portion of the waveguide and the detector positioned to receive the
exit radiation from the output portion of the waveguide.
22. The apparatus of claim 13, further comprising a filter to
select a predetermined bandwidth radiation emitted by the terahertz
radiation source.
23. The apparatus of claim 13, wherein the terahertz radiation
source has a bandwidth between 0.001 and 1000 THz.
24. The apparatus of claim 13, wherein the first molecule is a
nucleic acid.
25. The apparatus of claim 13, wherein the second molecule is a
nucleic acid.
26. The apparatus of claim 13, wherein the second molecule is a
protein.
27. The apparatus of claim 13, wherein the first molecule is a
receptor and the second molecule is a ligand.
28. The apparatus of claim 13, wherein the first molecule is a
protein and the second molecule is a protein.
29. A micro-electro-mechanical system (MEMS) for detecting a
biomolecular binding event in a sample containing a first molecule
and a second molecule, wherein the first molecule is suspected of
binding the second molecule, comprising: one or more fluid
compartments for the sample; a radiation source positioned to aim
an input radiation toward the sample; and a detector positioned to
receive an exit radiation reflecting from the sample.
30. The apparatus of claim 29, further comprising an information
processing and control system in communication with the
detector.
31. The apparatus of claim 30, wherein the fluid compartment
includes a substrate that is transparent to the terahertz
radiation, wherein the first molecule is immobilized on the
substrate.
32. The apparatus of claim 31, wherein the substrate include a
waveguide with an input portion and an output portion, wherein the
first molecule is immobilized on the waveguide.
33. The apparatus of claim 32, wherein the radiation source is
positioned to aim the input radiation toward the input portion of
the waveguide and the detector positioned to receive the exit
radiation from the output portion of the waveguide.
34. The apparatus of claim 29, further comprising a filter to
select a predetermined bandwidth radiation emitted by the radiation
source.
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a divisional application of U.S.
application Ser. No. 10/911,441 filed Aug. 4, 2004, now pending.
The disclosure of the prior application is considered part of and
is incorporated by reference in the disclosure of this
application.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[0002] The invention relates generally to detecting a biomolecular
binding event and more particularly to detecting such events using
spectroscopy.
BACKGROUND INFORMATION
[0003] Many current methods for detecting disease or the risk of
developing a disease rely on detection of one or more biomolecular
interactions between a target molecule in the biological sample and
a detectable probe molecule. The probe molecule is typically
detectable because it is bound to a detectable label. For example,
infection in a subject caused by an infectious agent, such as a
virus, can be detected by detecting binding of a labeled antibody
probe to a viral protein. A plethora of bioassays have been
developed based on this general concept.
[0004] Some more recent methods for detecting disease rely on the
detection or determination of a nucleic acid sequence in a test
sample. Sequence-selective detection of nucleic acid molecules has
become increasingly important as scientists unravel the genetic
basis of disease and use this new information to improve medical
diagnosis and treatment. Nucleic acid hybridization assays are
specific biomolecular binding assays that are commonly used to
detect the presence of specific nucleic acid sequences in a sample.
For example, an infectious agent can be detected by detecting
hybridization of a labeled nucleic acid probe to a nucleic acid of
the virus. Alternatively, the method can base disease detection on
detection or determination of all or a part of the patient's own
nucleic acid sequences. For example, a patient's risk for
developing a disease can be determined by detection of a genetic
mutation.
[0005] Like other methods that detect biomolecular interactions,
nucleic acid hybridization assays typically utilize a labeled
probe. Traditionally, radioisotopes have been used as labels. More
recently, fluorescent, chemiluminescent and bioactive reporter
groups have been used. However, the inclusion of labels in an assay
often makes it more expensive and complicated, and increases the
background signal of the assay.
[0006] Hybridization assays can be used not only to detect the
presence of a nucleic acid molecule, but determine the sequence of
the nucleic acid molecule as well. Traditional approaches for
sequence determination utilize the synthesis of labeled nucleic
acids that are terminated at one of the four nucleotides. However,
these methods are relatively slow and expensive. More recently,
methods have been developed that entail synthesizing
oligonucleotides on a glass support and effecting hybridization
with radioactively or fluorescently-labeled test DNA, and
reconstructing nucleotide sequence on the basis of data analysis
(E. Southern et al., PCT/GB 89/00460, 1989). A device for carrying
out such methods includes a supporting film or glass plate and an
array of nucleotides covalently attached to the surface thereof.
The array includes a set of oligonucleotides of desired length that
are capable of taking part in a hybridization reaction.
[0007] The sequencing-by-hybridization method discussed above,
although providing a less-expensive method with higher-throughput,
has certain disadvantages. For example, it typically requires
labeling of sample or probe nucleic acids. As discussed above, this
increases the cost and complexity of the method and increases
background values, thereby decreasing sensitivity. Furthermore,
inclusion and detection of labels lowers the throughput of the
assay.
[0008] In an attempt to determine nucleic acid sequences
information more efficiently, ultraviolet/visible/near-infrared
spectroscopy has been used to directly detect hybridization.
Although this type of spectroscopy has successfully detected events
for smaller molecules (e.g., CO.sub.2), it failed to provide the
desired level of efficiency and accuracy for biomolecules, which
tend to be larger. The frequency shift in the vibration spectrum
that is experienced by larger biomolecules (e.g., DNA) upon binding
is too small to be accurately and efficiently detected by
UV/visible/near-infrared spectroscopy. Furthermore, the
UV/visible/near-infrared radiation causes the molecules to
fluoresce, creating background noise that interferes with spectrum
signals. Further, the method requires multiple gratings of strong
dispersion to resolve the small frequency change, making the
optical instrument too bulky for convenient use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is an illustration of an embodiment of a system
whereby terahertz radiation 10 impinges a nucleic acid molecule 20
immobilized on a transparent substrate 22. The excitation and
signal collection can be performed, for example, through the
substrate.
[0010] FIG. 2 is a diagram showing frequency transition in Raman
spectroscopy caused by terahertz radiation.
[0011] FIG. 3 is a diagram showing energy absorption caused by
terahertz radiation.
[0012] FIG. 4 is an illustration of another embodiment of a system
whereby total internal reflection geometry is used to deliver
excitation radiation 42 and to collect Raman scattered radiation
46.
[0013] FIG. 5 is a diagram showing a frequency transition in Raman
spectroscopy using ultraviolet/visible/near-infrared radiation to
excite a nucleic acid.
[0014] FIG. 6 is a block diagram of one embodiment of an apparatus
for detecting a bimolecular binding event.
[0015] FIG. 7 is a block diagram of one embodiment of a method to
identify an agent that affects a biomolecular binding.
[0016] FIG. 8 is an illustration of an embodiment of a
micro-electro-mechanical system (MEMS) that incorporates may
incorporate the systems illustrated in FIGS. 1 and/or 4.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The methods and systems provided herein are based on the use
of terahertz radiation to detect biomolecular binding events. The
use of one terahertz radiation to detect molecular binding events
provides numerous advantages over currently available detection
methods. First, because labels are not necessary and because
low-energy terahertz radiation does not cause fluorescence,
background levels are reduced. Second, by using low-energy
terahertz radiation instead of ultraviolet/visible/near-infrared
radiation, only one grating is needed to obtain a meaningful
spectroscopy result. Due to the fact that multiple gratings are not
necessary, the instrument can become more compact and less
expensive. Third, the use of terahertz radiation allows the
biomolecular signature of the sample to be obtained in a dormant
state, without exciting molecules of the sample to an unnecessarily
high energy level, and possibly altering the molecules or their
interactions.
[0018] The invention is founded, at least in part, on the discovery
that terahertz/gigahertz radiation spectroscopy allows efficient
and accurate monitoring of biomolecular binding events without the
inconveniences and high backgrounds associated with the currently
available methods. The terahertz/gigahertz radiation instrument of
the invention specifically excites the molecules in the low
frequency vibrational bands, thereby significantly reducing
background noise in the signals and, at the same time, eliminating
the need for multiple gratings.
[0019] In one embodiment, a method includes preparing a sample
including a first molecule and a second molecule, directing an
input radiation at the sample, whereby the input radiation is
gigahertz or terahertz radiation, and detecting exit radiation
traveling from the sample, is provided. A shift in vibrational
frequency or a change in intensity level of the exit radiation
compared to exit radiation detected from a first molecule not bound
to the second molecule, is indicative of a biomolecular
interaction. At least one of the molecules is typically a
biomolecule. In an illustrative example, the biomolecular binding
event is hybridization of a first nucleic acid molecule to a second
nucleic acid molecule.
[0020] In another embodiment, a method of detecting a biomolecular
binding event by preparing a sample including a first molecule and
a second molecule, directing an input radiation at the sample, is
provided. The input radiation excites the sample without causing
fluorescence. The exit radiation traveling from the sample is
examined for a shift in vibrational frequency, wherein a shift in
vibrational frequency or a change in intensity level of the exit
radiation compared to exit radiation detected from a first molecule
not bound to the second molecule, is indicative of a biomolecular
interaction.
[0021] In another embodiment, an apparatus for detecting a
biomolecular binding event is provided. The apparatus includes a
terahertz light source or terahertz radiation source positioned to
aim an input radiation toward the sample and a detector for
receiving an exit radiation reflecting from the sample. A processor
is also provided to receive the data from the detector and analyze
the data. In some embodiments, the light source, detector and
processor are located in a housing. A filter may also be positioned
between the light source and the sample. An optional sample holder
may also be provided.
[0022] In yet another embodiment, a system for detecting a
biomolecular binding event is provided. The system includes a
sample containing a first molecule and a second molecule, a
gigahertz or terahertz radiation source positioned to aim an input
radiation toward the sample, and a detector for receiving an exit
radiation reflecting from the sample. The system detects exit
radiation traveling from the sample upon excitation by the input
radiation, wherein a shift in vibrational frequency or a change in
intensity level of the exit radiation compared to exit radiation
detected from a first molecule not bound to the second molecule, is
indicative of a biomolecular interaction.
[0023] In methods provided herein, the first molecule is typically
a biomolecule. The second molecule can also be a biomolecule. For
example, a second molecule, which is typically a molecule to be
detected by the method, is a virus, bacteria, or an analyte, such
as, but not limited to, a protein, a nucleic acid, a peptide, a
polysaccharide, or a fatty acid.
[0024] As used herein, "a" or "an" may mean one or more than one of
an item.
[0025] "Nucleic acid" encompasses DNA, RNA, single-stranded,
double-stranded or triple stranded and any chemical modifications
thereof. Virtually any modification of the nucleic acid is
contemplated. As used herein, a single stranded nucleic acid may be
denoted by the prefix "ss", a double stranded nucleic acid by the
prefix "ds", and a triple stranded nucleic acid by the prefix "ts."
For methods provided herein, a biomolecule in illustrative
examples, is a single-stranded nucleic acid molecule. Nucleic acids
in the methods provided herein include viral, bacterial, and
animal, for example mammalian, or, more specifically, human nucleic
acids.
[0026] A "nucleic acid" may be of almost any length, for example
from 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250,
275, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,
3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000,
15,000, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 150,000,
200,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 5,000,000 or
even more bases in length, up to a full-length chromosomal DNA
molecule.
[0027] Nucleic acid molecules to be analyzed can be prepared by any
technique known in the art. In certain embodiments, the nucleic
acids are naturally occurring DNA or RNA molecules. Virtually any
naturally occurring nucleic acid may be prepared and sequenced by
the disclosed methods including, without limit, chromosomal,
mitochondrial and chloroplast DNA and ribosomal, transfer,
heterogeneous nuclear and messenger RNA.
[0028] A "biological sample" includes, for example, urine, blood,
plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid,
tears, mucus, ejaculate, cerebrospinal fluid, pleural fluid,
ascites fluid, or a biopsy sample.
[0029] In certain aspects, the biological sample is from a
mammalian subject, for example a human subject. The biological
sample can be virtually any biological sample, as long as the
sample contains or may contain a second specific binding pair
member. For example, the sample can be suspected of containing a
protein that has an epitope recognized by an antibody included as
the first specific binding pair member. The biological sample can
be a tissue sample which contains, for example, 1 to 10,000,000;
1000 to 10,000,000; or 1,000,000 to 10,000,000 somatic cells. The
sample need not contain intact cells, as long as it contains
sufficient quantity of a specific binding pair member for the
methods provided. According to aspects of the methods provided
herein, wherein the biological sample is from a mammalian subject,
the biological or tissue sample can be from any tissue. For
example, the tissue can be obtained by surgery, biopsy, swab,
stool, or other collection method. In other aspects, the biological
sample contains, or is suspected to contain, or at risk for
containing, a pathogen, for example a virus or a bacterial
pathogen.
[0030] The term "binds specifically" or "specific binding
activity," when used in reference to an antibody means that an
interaction of the antibody and a particular epitope has a
dissociation constant of at least about 1.times.10.sup.-6,
generally at least about 1.times.10.sup.-7, usually at least about
1.times.10.sup.-8, and particularly at least about
1.times.10.sup.-9 or 1.times..sup.-10 or less. As such, Fab,
F(ab').sub.2, Fd and Fv fragments of an antibody that retain
specific binding activity, are included within the definition of an
antibody.
[0031] As used herein, the term "specific binding pair member"
refers to a molecule that specifically binds or selectively
hybridizes to, or interacts with, another member of a specific
binding pair. Specific binding pair members include, for example,
analytes and biomolecules.
[0032] A "biomolecule" is a specific binding pair member found in
nature, or derived from a molecule found in nature. Biomolecules
can include, for example, biomolecules that have a molecular weight
of at least 1 kDa, 2 kDa, 3, kDa, 4, kDa, 5 kDa, 10 kDa, 15 kDa, 20
kDa, 25 kDa, 50 kDa, or 100 kDa. Biomolecules include, for example,
nucleic acid molecules, peptides, polypeptides, proteins, ligands,
lipids, carbohydrates, or polysaccharides, but do not include
water.
[0033] As used herein, the terms "analyte" refer to any atom,
chemical, molecule, compound, composition or aggregate of interest
for detection and/or identification. Non-limiting examples of
analytes include an amino acid, peptide, polypeptide, protein,
glycoprotein, lipoprotein, nucleoside, nucleotide, oligonucleotide,
nucleic acid, sugar, carbohydrate, oligosaccharide, polysaccharide,
fatty acid, lipid, hormone, metabolite, cytokine, chemokine,
receptor, neurotransmitter, antigen, allergen, antibody, substrate,
metabolite, cofactor, inhibitor, drug, pharmaceutical, nutrient,
prion, toxin, poison, explosive, pesticide, chemical warfare agent,
biohazardous agent, radioisotope, vitamin, heterocyclic aromatic
compound, carcinogen, mutagen, narcotic, amphetamine, barbiturate,
hallucinogen, waste product, virus, bacteria, protozoa, and/or
contaminant.
[0034] As used herein, the term "antibody" is used in its broadest
sense to include polyclonal and monoclonal antibodies. The term
antibody as used herein is meant to include intact molecules as
well as fragments thereof, such as Fab and F(ab').sub.2, Fv and SCA
fragments which are capable of binding an epitopic determinant.
[0035] (1) An Fab fragment consists of a monovalent antigen-binding
fragment of an antibody molecule, and can be produced by digestion
of a whole antibody molecule with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain. [0036] (2) An Fab' fragment of an antibody molecule
can be obtained by treating a whole antibody molecule with pepsin,
followed by reduction, to yield a molecule consisting of an intact
light chain and a portion of a heavy chain. Two Fab' fragments are
obtained per antibody molecule treated in this manner. [0037] (3)
An (Fab').sub.2 fragment of an antibody can be obtained by treating
a whole antibody molecule with the enzyme pepsin, without
subsequent reduction. A (Fab').sub.2 fragment is a dimer of two
Fab' fragments, held together by two disulfide bonds. [0038] (4) An
Fv fragment is defined as a genetically engineered fragment
containing the variable region of a light chain and the variable
region of a heavy chain expressed as two chains. [0039] (5) A
single chain antibody ("SCA") is a genetically engineered single
chain molecule containing the variable region of a light chain and
the variable region of a heavy chain, linked by a suitable,
flexible polypeptide linker.
[0040] The term "antibody" as used herein includes naturally
occurring antibodies as well as non-naturally occurring antibodies,
including, for example, single chain antibodies, chimeric,
bifunctional and humanized antibodies, as well as antigen-binding
fragments thereof. Such non-naturally occurring antibodies can be
constructed using solid phase peptide synthesis, can be produced
recombinantly or can be obtained, for example, by screening
combinatorial libraries consisting of variable heavy chains and
variable light chains (see Huse et al., Science 246:1275-1281
(1989). These and other methods of making, for example, chimeric,
humanized, CDR-grafted, single chain, and bifunctional antibodies
are well known to those skilled in the art (Winter and Harris,
Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546,
1989; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring
Harbor Laboratory Press, 1988); Hilyard et al., Protein
Engineering: A practical approach (IRL Press 1992); Borrabeck,
Antibody Engineering, 2d ed. (Oxford University Press 1995.
[0041] Methods for raising polyclonal antibodies, for example, in a
rabbit, goat, mouse or other mammal, are well known in the art
(see, for example, Green et al., "Production of Polyclonal
Antisera," in Immunochemical Protocols (Manson, ed., Humana Press
1992), pages 1-5; Coligan et al., "Production of Polyclonal
Antisera in Rabbits, Rats, Mice and Hamsters," in Curr. Protocols
Immunol. (1992), section 2.4.1). In addition, monoclonal antibodies
can be obtained using methods that are well known and routine in
the art (Harlow and Lane, supra, 1988).
[0042] As used in this invention, the term "epitope" refers to an
antigenic determinant on an antigen, to which the paratope of an
antibody, binds. Antigenic determinants usually consist of
chemically active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics.
[0043] Examples of types of immunoassays of the invention include
competitive and non-competitive immunoassays in either a direct or
indirect format. Those of skill in the art will know, or can
readily discern, other immunoassay formats without undue
experimentation.
[0044] In performing a method of the present invention, "blocking
agents" can be included in the incubation medium. "Blocking agents"
are added to minimize non-specific binding to a surface and between
molecules.
[0045] A "biomolecular binding event" or "biomolecular interaction"
is a specific binding of specific binding pair members, wherein at
least one of the specific binding pair members is a biomolecule.
Methods provided herein can be used to detect molecular interaction
(i.e. binding) of virtually any biomolecule with another molecule.
For example, the methods can detect interaction of an analyte and a
biomolecule.
[0046] The term "receptor" is used to mean a protein, or fragment
thereof, or group of associated proteins that selectively bind a
specific substance called a ligand. Upon binding its ligand, the
receptor triggers a specific response in a cell.
[0047] The term "polypeptide" is used broadly herein to mean two or
more amino acids linked by a peptide bond. The term "fragment" or
"proteolytic fragment" also is used herein to refer to a product
that can be produced by a proteolytic reaction on a polypeptide,
i.e., a peptide produced upon cleavage of a peptide bond in the
polypeptide. A polypeptide of the invention contains at least about
six amino acids, usually contains about ten amino acids, and can
contain fifteen or more amino acids, particularly twenty or more
amino acids. It should be recognized that the term "polypeptide" is
not used herein to suggest a particular size or number of amino
acids comprising the molecule, and that a peptide of the invention
can contain up to several amino acid residues or more. A protein is
a polypeptide that includes other chemical moieties in addition to
amino acids, such as phosphate groups or carbohydrate moieties.
[0048] The term "terahertz (i.e. THz) or gigahertz radiation" is
radiation between the far infrared and high frequency RF ranges.
For example, terahertz radiation can have a bandwidth spanning the
range of 0.1 to 100 THz, which corresponds to about 3 millimeters
to 3 micrometers in wavelength. The pulse of the terahertz
radiation is typically short, and can be in the order of 10.sup.-12
seconds, which therefore has a relatively high peak power, and a
short time resolution.
[0049] An "input radiation," as used herein, is the radiation
coming from the radiation source before impingement on the sample.
An "exit radiation" is the radiation traveling to the detector
after being scattered or reflected by the sample.
[0050] In certain embodiments, a method to detect a biomolecule,
that includes impinging the biomolecule with gigahertz or terahertz
input radiation, and detecting an exit radiation, is provided. A
shift in vibrational frequency of the exit radiation or a change in
intensity level of the exit radiation compared to exit radiation
detected from a control sample not comprising the biomolecule, is
indicative of the presence of the biomolecule. In certain aspects,
the exit radiation is detected by Raman spectroscopy. In other
aspects, absorbance is detected by detecting a decrease in
intensity level of the exit radiation. In certain examples, the
biomolecule is isolated and optionally immobilized, before it is
impinged with the input radiation.
[0051] The method for detecting a biomolecule using gigahertz or
terahertz radiation can be used to detect a biomolecular
interaction, also referred to herein as a biomolecular binding
event. Accordingly, in another embodiment, provided herein is a
method to detect a biomolecular interaction, including: contacting
a first molecule with a second molecule, wherein the first molecule
is a biomolecule that is suspected of binding to the second
molecule; and impinging the first molecule with gigahertz or
terahertz input radiation; and detecting an exit radiation. A shift
in vibrational frequency or a change in intensity level of the exit
radiation compared to exit radiation detected from a first molecule
not bound to the second molecule, is indicative of a biomolecular
interaction.
[0052] FIG. 1 shows one embodiment of the system 5, whereby
terahertz radiation from radiation source 112 impinges upon a
population of immobilized first molecules 20. An arrow 10 indicates
the direction in which the input gigahertz or terahertz radiation
travels, toward the immobilized biomolecules 20. The immobilized
biomolecules 20 are attached to a substrate 22 and immersed in a
reaction solution 24. In certain examples, the input radiation 10
from radiation source 112 has a bandwidth of between 0.001 and 1000
terahertz, for example between 0.01 and 100 terahertz, or between
0.1 and 10 terahertz.
[0053] After the first molecule 20 is impinged by the input
radiation 10, exit radiation 30 is generated and travels toward a
terahertz detector 116, which detects the exit radiation 30. The
detected radiation is analyzed by a processor 118 for any changes
in the vibrational spectrum or changes in intensity levels. When
the immobilized biomolecules 20 experience a binding event with
free molecules in the solution 24, the exit radiation demonstrates
a frequency shift in the vibrational spectrum and/or an energy
absorption compared to the vibrational spectrum or energy
absorption of the immobilized biomolecules 20 when they are not
bound to the second molecule. Thus, a change in the vibrational
spectra and/or an occurrence of absorption, that is different from
that observed or expected for unbound immobilized biomolecules 20,
indicates that the immobilized molecules 20 and the free molecules
bind. For example, a 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02,
0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15,
20, 25, 30, 40, and 50% reduction in intensity level can be
indicative of the occurrence of a biomolecular binding event. In
another example, the frequency shift of 0.05, 0.06, 0.07, 0.08,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,
6, 7, 8, 9 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 THz can be
indicative of occurrence of a biomolecular binding event. Vibration
spectra can be generated and recorded for a first molecule not
bound to a second molecule, and used to compare to vibration
spectra in the presence of a sample suspected of containing the
second molecule.
[0054] As illustrated in FIG. 5, excitation of a biomolecule caused
by ultraviolet/visible/near-infrared radiation 60, excites the
biomolecule to a virtual state 65 and results in the generation of
Raman scattered light 62, but also excites the biomolecule from a
ground state 58 to an unnecessary electronic excited state 66
resulting in fluorescence 68. FIG. 2 is a diagram showing the
molecular energy state transition caused by terahertz radiation 50,
which due to its low energy level excites the biomolecule to lower
and more desirable virtual states 54 and vibrational states 56
while generating Raman scattered light 52. By keeping the
excitation level low, fluorescence 68 is avoided. Since
fluorescence 68 is responsible for background noise in the signal,
use of terahertz radiation results in a signal with reduced
background emissions. Vibrational frequency shift produced as a
result of impingement of a biomolecule with gigahertz or terahertz
radiation can be detected using Raman spectroscopy. FIG. 3 provides
a diagram showing the molecular energy state caused by
biomolecule's absorption 70 of terahertz radiation. Energy of the
terahertz radiation is absorbed 70 when the energy level of the
terahertz radiation matches the energy level of the vibrational
state 59.
[0055] In illustrative examples, absorbance is detected by
detecting a decrease in the intensity level of exit radiation
compared to the intensity level of the corresponding input
radiation. In examples where a decrease in intensity level is
detected, the gigahertz or terahertz radiation can include a target
wavelength, or range of wavelengths, known to be absorbed by the
first molecule, and/or by a complex that includes a first molecule
bound to a second molecule. It will be understood that wavelength
scanning can be performed to identify wavelengths that are absorbed
by the first molecule to identify a target wavelength or target
range of wavelengths. Furthermore, scanning can be performed of
various emission energy levels to identify a vibrational level,
below which Raman transmissions are minimized. In an alternative
embodiment, a broad band terahertz light source can be used in
conjunction with a spectrum analyzer or a similar device that can
analyze the absorbance spectrum over a range of wavelengths.
[0056] In embodiments of the invention directed to detecting
biomolecular interactions, a second molecule known to bind, or
suspected of binding to the first molecule, is found in the
reaction solution 24, where it contacts the immobilized first
molecules 20. Alternatively, the reaction solution can include a
biological sample suspected of containing the second molecule,
which can be an analyte. Accordingly, in another embodiment, a
method is provided to detect an analyte in a biological sample,
including: contacting the biological sample with a first molecule
immobilized on a substrate, wherein the first molecule binds to, or
is suspected of binding to the analyte; impinging the first
molecule with a gigahertz or terahertz input radiation; and
detecting a vibrational spectrum of the first molecule. A shift in
the vibrational spectrum, or decrease in an intensity level of the
vibrational spectrum, compared to a vibrational spectrum of a first
molecule not associated with the analyte, is indicative of the
presence of the analyte in the sample.
[0057] Due to the known water absorption property of terahertz
radiation, in one embodiment of the invention, the radiation source
may be arranged so that the input radiation strikes the immobilized
molecules 20 from the substrate side, where the radiation travels
through the substrate, and therefore travels through minimum
solution depth to reach the immobilized molecules 20. Therefore, in
certain illustrative examples, the gigahertz or terahertz radiation
impinges the first molecule 20 through the substrate. The
radiation, upon striking the immobilized molecules 20, is reflected
or scattered in the direction indicated by an arrow 30.
[0058] Suitable conditions for performing a method of the invention
include any conditions that allow a first molecule to specifically
interact with a second molecule. An advantage of the present
invention is that it allows terahertz radiation to be used to
detect biomolecular binding events in aqueous (i.e.
water-containing) samples. Therefore, the conditions, such as
temperature, and reaction solution composition, can be those
typically used for the type of biomolecular binding being analyzed.
For example, in examples where the method is used to detect binding
of an antibody to an analyte (i.e. an immunoassay) typical
immunoassay conditions can be used.
[0059] In embodiments where nucleic acid hybridization is detected,
conditions for hybridization reactions well known in the art, can
be used. For example, hybridization of a first nucleic acid
molecule with a second nucleic acid molecule can be performed under
moderately stringent or highly stringent physiological conditions,
as are known in the art. An example of progressively higher
stringency conditions that can be used in the methods disclosed
herein are as follows: 2.times.SSC/0.1% SDS at about room
temperature (hybridization conditions); 0.2.times.SSC/0.1% SDS at
about room temperature (low stringency conditions);
0.2.times.SSC/0.1% SDS at about 42.degree. C. (moderate stringency
conditions); and 0.1.times.SSC at about 68.degree. C. (high
stringency conditions). Washing can be carried out using only one
of these conditions, for example, high stringency conditions, or
each of the conditions can be used, for example, for 10 to 15
minutes each, in the order listed above, repeating any or all of
the steps listed.
[0060] Certain illustrative embodiments of the invention are
provided herein in the context of using terahertz radiation to
detect nucleic acid hybridization. However, it will be understood
that the embodiments provided herein are illustrative embodiments,
and the scope of the invention is not limited to the applications
or the embodiments disclosed herein.
[0061] The methods disclosed herein can be used to detect virtually
any biomolecular binding event that demonstrates a frequency shift
in the vibrational spectrum. The biomolecular binding event
typically involves specific binding of a first and second specific
binding pair member, wherein at least one of the specific binding
pair members is a biomolecule. As indicated herein, the first
specific binding pair member is typically a biomolecular with a
molecular weight of at least 1 kDa. In addition to nucleic acids,
the biomolecules also include, for example, a receptor and a
ligand, or an antigen and an antibody. In one aspect, the first
molecule is a protein, such as an antibody molecule, or fragment
thereof, and the second molecule is a protein, that includes an
epitope recognized by the antibody. In another example, the first
molecule is a receptor and the second molecule is a ligand. In an
illustrative aspect, the first or second molecule is a nucleic acid
molecule that interacts with another molecule. Accordingly, this
embodiment can be used to detect binding of nucleic acid molecules
to a protein, or binding of a protein to a nucleic acid
molecule.
[0062] The use of first and second molecules is illustrative.
However, there can be additional molecules. For example, a third
molecule that binds to the first molecule can be included. Binding
of the second molecule can displace the third molecule in a
competitive manner and, as a result, shift the vibrational
frequency or change the intensity level of the exit radiation.
[0063] Accordingly, the immobilized biomolecules 20 can be any
biomolecule capable of forming a specific binding pair with another
molecule. The immobilized biomolecule can be, for example, a
nucleic acid, protein, antigen, antibody, receptor, or ligand. The
reaction solution 24 contains free molecules that may or may not
bind to the immobilized biomolecules 20. Therefore, the invention
can be used to determine whether binding, such as a nucleic acid
hybridization, has occurred by detecting a shift in vibrational
frequency or a change in intensity level of exit radiation.
[0064] As an illustrative example, such as shown in FIG. 1, nucleic
acid hybridization can be detected using the methods provided
herein. If a single stranded nucleic acid with the nucleotide
sequence GGCAAT is immobilized on a substrate 22 and incubated
under highly stringent conditions with a nucleic acid sample, the
vibrational spectrum of the GGCAAT nucleic acid molecule after
irradiation with terahertz radiation will be affected if the
nucleic acid sample includes a nucleic acid that binds to GGCAAT
under highly stringent hybridization conditions. Therefore, the
vibrational spectrum of the GGCAAT nucleic acid molecule after
irradiation with terahertz radiation will be different if the
nucleic acid sample includes a nucleic acid molecule that
hybridizes to GGCAAT, as compared to the vibrational spectrum
generated for the nucleic acid molecule GGCAAT in the absence of a
hybridizing nucleic acid molecule. This difference indicates that
the nucleic acid sample includes a nucleic acid molecule that
hybridizes with GGCAAT, such as a nucleic acid molecule that has
the sequence CCGTTA. As indicated in this example, DNA
hybridization typically results in changes in fundamental
vibrational and rotational modes. As another example, the methods
disclosed herein can be used to detect analytes, such as protein
analytes, in a sample. For example, if the immobilized molecule 20
is an antibody, a change in the vibrational spectrum indicates that
the reaction solution 24, which in this example can be a patient
serum sample, contains a protein that is recognized by the
immobilized antibody. It will understand that the invention is
useful for detecting other types of binding events as well,
provided that they involve a biomolecule and result in a shift in
the vibrational spectrum or a change in the intensity level of the
exit radiation upon binding.
[0065] Since the radiation travels through the substrate 22, the
substrate 22 is preferably made of a material (e.g., glass) that is
transparent to the radiation being used. As long as the substrate
22 is transparent to the radiation, there is no restriction on its
thickness.
[0066] Immobilized biomolecules according to the present invention,
can be immobilized as a single population of identical
biomolecules, or as an array or a desired pattern of immobilized
populations of biomolecules. These arrays can be immobilized, for
example, on biochips, as discussed in more detail herein.
[0067] Immobilized arrays of nucleic acid molecules can be used,
for example, in determining nucleotide sequence information using
sequencing by hybridization reactions. In sequencing by
hybridization reactions, one or more oligonucleotide probes of
known sequence are allowed to hybridize to a target nucleic acid
sequence. Binding of the labeled oligonucleotide to the target
indicates the presence of a complementary sequence in the target
strand. Multiple labeled probes can be allowed to hybridize
simultaneously to the target molecule and detected simultaneously.
In alternative embodiments, bound probes can be identified attached
to individual target molecules, or alternatively multiple copies of
a specific target molecule can be allowed to bind simultaneously to
overlapping sets of probe sequences. Individual molecules or a
subpopulation thereof, can be scanned, for example, using known
molecular combing techniques coupled to a detection method
disclosed herein.
[0068] Regarding the reaction solution 24, it is preferred that the
depth of the reaction solution 24 is not much greater than 100, 50,
40, 30, 25, or 20 microns in aspects where the radiation source
transmits radiation though the reaction solution to impinge the
first molecule. Thirty microns is the approximate penetration limit
of terahertz radiation in water. As each nucleotide base has a
length of approximately 0.3 nm, most samples will be immersed in a
30-micron-deep solution for terahertz spectroscopy. Any well-known
method may be used for binding the immobilized molecules 20 to a
substrate 22.
[0069] FIG. 4 is an illustration of an embodiment of the invention
whereby terahertz radiation impinges upon an immobilized population
of first molecules 20, such as an immobilized population of nucleic
acid molecules, and whereby a waveguide 40 is used to confine input
radiation to create an excitation range 26 which includes the
immobilized nucleic acid molecules 20. As disclosed above in
reference to FIG. 1, the immobilized molecules 20 are immersed in a
reaction solution 24. Unlike the first embodiment shown in FIG. 1,
however, the immobilized biomolecules 20 are attached to a
waveguide 40 through which radiation travels. Since the input
radiation enters the waveguide 40 in a substantially orthogonal
manner with respect to the substrate, from the direction indicated
by the arrow 42, the solution 24 is not limited in depth. For
example, input radiation can enter the waveguide at a 5, 10, 20,
30, 40, 45, 50, 60, 70, 75, 80, or 85.degree. angle with respect to
the waveguide 40. The waveguide 40 is typically made of a material
that has a high index of refraction at the wavelength of the
radiation being used, thereby keeping a vast majority of the
radiation traveling through within the waveguide by total internal
reflection. The exit radiation coming out of the waveguide 40
includes at least one of the Raman scattered radiation and the
totally internally reflected radiation minus any absorption. As the
radiation travels through the waveguide 40, it creates an
excitation region 26 in the solution 24. The excitation region is
typically between 10 and 100 um in depth.
[0070] In certain aspects, where binding of two nucleic acid
molecules with similar length is to be detected, their rotational
bands may appear very close to each other. By labeling one of the
nucleic acid molecules, the rotational bands can be shifted to
obtain different binding signatures. Non-limiting examples of
labels that can be used include TRIT (tetramethyl rhodamine
isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye,
phthalic acid, terephthalic acid, isophthalic acid, cresyl fast
violet, cresyl blue violet, brilliant cresyl blue,
para-aminobenzoic acid, erythrosine, biotin, digoxigenin,
5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein,
5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein,
5-carboxy rhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino
phthalocyanines, azomethines, cyanines, xanthines,
succinylfluoresceins and aminoacridine. Polycyclic aromatic
compounds in general can function as Raman labels, as is known in
the art. These and other labels can be obtained from commercial
sources (e.g., Molecular Probes, Eugene, Oreg.). Other labels that
can be of use include cyanide, thiol, chlorine, bromine, methyl,
phosphorus and sulfur. Carbon nanotubes can also be of use as
labels.
[0071] Labels can be attached directly to the specific binding pair
members or can be attached via various linker compounds. Raman
labels that contain reactive groups designed to covalently react
with other molecules, are commercially available (e.g., Molecular
Probes, Eugene, Oreg.).
[0072] In another embodiment illustrated in FIG. 7, provided herein
is a method to identify an agent that affects a biomolecular
binding, including: contacting under suitable conditions, a first
molecule 200, a second molecule 205, and the agent 210, wherein the
first molecule binds to, or is suspected of binding to, the second
molecule, and wherein the first molecule is immobilized on a
support. The first molecule is then impinged with a gigahertz or
terahertz light source 215; and a vibrational spectrum of the first
molecule is detected 220, wherein a shift in the vibrational
spectrum or a change in intensity level of the vibrational
spectrum, compared to a vibrational spectrum of a first molecule
bound to the second molecule in the absence of the agent,
identifies the agent as an agent that affects the biomolecule
binding.
[0073] The first molecule, the second molecule, and the agent can
be contacted in any order as desired. As such, the screening method
can be used to identify agents that can competitively or non
competitively inhibit binding of the first molecule to the second
molecule, agents that can mediate or enhance binding of the first
molecule to the second molecule, and agents that can induce
dissociation of specifically bound first molecule from specific
bound second molecule. Appropriate control reactions are performed
to confirm that the action of the agent is specific with respect to
the first molecule and the second molecule.
[0074] A screening method of the invention also can be performed
using molecular modeling to identify candidate agents. The
utilization of a molecular modeling method provides a convenient,
cost effective means to identify those agents, among a large
population such as a combinatorial library of potential agents,
that are most likely to interact specifically with a biomolecule,
thereby reducing the number of potential agents that need to be
screened using an assay.
[0075] The term "test agent" or "test molecule" is used broadly
herein to mean any agent that is being examined for agonist or
antagonist activity in a method of the invention. Although the
method generally is used as a screening assay to identify
previously unknown molecules that can act as agonist or antagonist
agents as described herein, the methods also can be used to confirm
that a agent known to have a particular activity in fact has the
activity, for example, in standardizing the activity of the
agent.
[0076] A screening method of the invention provides the advantage
that it can be adapted to high throughput analysis and, therefore,
can be used to screen combinatorial libraries of test agents in
order to identify those agents that can modulate binding of the
first molecule to the second molecule. Methods for preparing a
combinatorial library of molecules that can be tested for a desired
activity are well known in the art and include, for example,
methods of making a phage display library of peptides, which can be
constrained peptides (see, for example, U.S. Pat. No. 5,622,699;
U.S. Pat. No. 5,206,347; Scott and Smith, Science 249:386-390,
1992; Markland et al., Gene 109:13 19, 1991; each of which is
incorporated herein by reference); a peptide library (U.S. Pat. No.
5,264,563, which is incorporated herein by reference); a
peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:83
92, 1995; a nucleic acid library (O=Connell et al., supra, 1996;
Tuerk and Gold, supra, 1990; Gold et al., supra, 1995; each of
which is incorporated herein by reference); an oligosaccharide
library (York et al., Carb. Res., 285:99 128, 1996; Liang et al.,
Science, 274:1520 1522, 1996; Ding et al., Adv. Expt. Med. Biol.,
376:261 269, 1995; each of which is incorporated herein by
reference); a lipoprotein library (de Kruif et al., FEBS Lett.,
399:232 236, 1996, which is incorporated herein by reference); a
glycoprotein or glycolipid library (Karaoglu et al., J. Cell Biol.,
130:567 577, 1995, which is incorporated herein by reference); or a
chemical library containing, for example, drugs or other
pharmaceutical agents (Gordon et al., J. Med. Chem., 37:1385-1401,
1994; Ecker and Crooke, Bio/Technology, 13:351-360, 1995; each of
which is incorporated herein by reference).
[0077] In another embodiment, a terahertz/gigahertz detection unit
apparatus 100 for detecting a biomolecular binding event is
provided. The apparatus 100 includes a terahertz light source or
terahertz radiation source 112 positioned to aim an input radiation
10, 42 toward the sample 120 and a detector 116 for receiving an
exit radiation 30, 46 reflecting from the sample 120. A processor
118 is also provided to receive the data 117 from the detector 116
and analyze the data. In some embodiments, the light source 112,
detector 116 and processor 118 are located in a housing 124. A
filter 114 may also be positioned between the light source 112 and
the sample 120 to select a predetermined bandwidth of the beam of
light 113 emitted by the light source. An optional sample holder
122 may also be provided to position and/or hold the sample 120 in
the proper position during testing. In some embodiments, the
substrate may be part of the sample holder. In other embodiments,
the waveguide may be part of the sample holder. In still other
embodiments, the sample holder secures the sample at a preferred
distance and/or orientation from the light source and detector. In
other embodiments, the light source and/or the detector capable of
being rotated, oriented or adjusted to a preferred orientation.
[0078] In another embodiment, provided herein is a system 150 for
detecting a biomolecular binding event, including a sample 120
containing a first molecule and a second molecule, wherein the
first molecule binds to, or is suspected of binding to the second
molecule; a terahertz or gigahertz radiation source 112 positioned
to aim an input radiation 10, 42 toward the sample 120; and a
detector 116 for receiving an exit radiation 30, 46 reflecting from
the sample. In certain illustrative examples, the system 150
further includes a substrate 22 that is transparent to the
terahertz or gigahertz radiation, wherein the first molecule is
immobilized on the substrate 22.
[0079] The system typically includes a reaction chamber adjacent to
the substrate for containing at least one of the biomolecules and
the reaction solution. The reaction solution can be an organic
solution, but is typically an aqueous solution, which therefore
contains water. The system can also include a waveguide 40, as
illustrated in FIG. 4. The substrate 22 can be a surface of a
waveguide 40. The system can also include diffraction gratings, but
typically includes at most one diffraction grating positioned
between the sample and the detector.
[0080] Systems 150 of the present invention typically include a
terahertz/gigahertz detection unit 100. The terahertz/gigahertz
detection unit 100 includes a terahertz and/or gigahertz radiation
source 112 and a detector 116, which can be wavelength-selective
detector. The excitation source 112 illuminates an immobilized
biomolecule, and optionally a second molecule associated with the
first molecule, in a sample 120 with an excitation beam 10, 42. The
excitation beam 10, 42 interacts with the first molecule, resulting
in the excitation of electrons to a higher energy state. As the
electrons return to a lower energy state, they emit a Raman
emission signal that is detected by the Raman detector. In other
embodiments, as discussed herein, absorbance of the gigahertz
and/or terahertz radiation by the first molecule is detected.
Virtually any type of terahertz and/or gigahertz source can be used
to generate terahertz and/or gigahertz radiation for the systems
150, apparatus 100 and methods of the present invention. For
example, terahertz radiation can be generated using a femtosecond
laser that is directed into an electro-optic crystal such as a zinc
blend crystal (e.g., zinc telluride crystal, gallium arsenide
crystal). In another approach, a photoconductive dipole antenna can
be used (Lai et al, App. Phys. Lett. 72:3100 (1998)). In another
embodiment, terahertz radiation can be obtained by mixing multiple
emissions from near-infrared diode lasers (NASA Jet Propulsion
Laboratory, Pasadena, Calif. reported in NASA TechBriefs, October,
2000). Furthermore, a terahertz or gigahertz transistor, such as
the transistors used in a computer processor, for example a
Pentium.RTM. processor can be used.
[0081] Detectors for detecting radiation in the terahertz/gigahertz
frequency range are known (See e.g., J. Applied Phys. 93, 1897
(2003)). In another approach, nanometer size objects (e.g. quantum
dots, nanowires, and nano channels) can be used to detect
terahertz/gigzhertz radiation (Xing and Liu, Semicon. Sci. Technol.
10:1139 (1995)). Furthermore, the detection of reflected terahertz
radiation can be achieved by using a electro-optic crystal and by
using a photodiode. In this aspect, the detector may include an
electro-optic crystal which in response to terahertz radiation
alters its optical characteristics such that laser radiation
directed upon the photodiode reveals the terahertz radiation. The
optical characteristic can include the polarization experienced by
laser radiation passing through the crystal.
[0082] Data can be collected from a detector 116 and provided to an
information processing and control system 118. The information
processing and control system 118 can perform standard procedures
known in the art, such as subtraction of background signals.
Furthermore, the information processing and control system 118 can
analyze the data to determine nucleotide sequence information from
detected signals and the temporal relationship of these
signals.
[0083] As indicated above, in certain aspects of the invention, the
first molecule is immobilized on an immobilization substrate. The
type of substrate to be used for immobilization of the first
molecule is not limiting as long as it is effective for
immobilizing a first molecule while providing access of the first
molecule to the second molecule and, in certain examples, allowing
terahertz and/or gigahertz radiation to pass through. In
illustrative examples, the substrate is transparent. Non-limiting
examples of surfaces that can be used include glass, silicon,
germanium, gallium arsenide, silica, silicate, silicon oxinitride,
nitrocellulose, nylon, activated quartz, activated glass,
polyvinylidene difluoride (PVDF), polystyrene, polyacrylamide,
other polymers such as poly(vinyl chloride), poly(methyl
methacrylate) or poly(dimethyl siloxane), and photopolymers which
contain photoreactive species such as nitrenes, carbenes and ketyl
radicals. Various methods of attaching specific binding pair
members to surfaces are known in the art and can be employed. For
example cross-linking agents can be used. Furthermore, functional
groups can be covalently attached to cross-linking agents so that
binding interactions between biomolecules can occur without steric
hindrance. Typical cross-linking groups include ethylene glycol
oligomers and diamines. Attachment can be by either covalent or
non-covalent binding. The cross-linking groups for attaching
biomolecules to immobilization surfaces are referred to herein as
immobilization groups.
[0084] As another specific example, immobilization can be achieved
by coating a surface with streptavidin or avidin and the subsequent
attachment of a biotinylated first molecule, such as a biotinylated
antibody (See Holmstrom et al., Anal. Biochem. 209:278-283, 1993
for method using nucleic acids). Immobilization can also involve
coating a silicon, glass or other surface with poly-L-Lys (lysine).
Amine residues can be introduced onto a surface through the use of
aminosilane for cross-linking.
[0085] The first molecule can be bound to glass by first silanizing
the glass surface, then activating with carbodiimide or
glutaraldehyde. Alternative procedures can use reagents such as
3-glycidoxypropyltrimethoxysilane (GOP) or
aminopropyltrimethoxysilane (APTS). Certain specific binding pair
members can be bound directly to membrane surfaces using
ultraviolet radiation.
[0086] Bifunctional cross-linking reagents can be of use for
attaching a biomolecule to a surface. The bifunctional
cross-linking reagents can be divided according to the specificity
of their functional groups, e.g., amino, guanidino, indole, or
carboxyl specific groups. Of these, reagents directed to free amino
groups are popular because of their commercial availability, ease
of synthesis and the mild reaction conditions under which they can
be applied. Exemplary methods for cross-linking molecules are
disclosed in U.S. Pat. Nos. 5,603,872 and 5,401,511. Cross-linking
reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR),
ethylene glycol diglycidyl ether (EGDE), and carbodiimides, such as
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
[0087] In some embodiments of the invention, a method disclosed
herein can be performed in a micro-electro-mechanical system (MEMS)
300, illustrated in FIG. 7. MEMS 300 are integrated systems that
include mechanical elements, sensors, actuators, and electronics.
All of those components can be manufactured by known
microfabrication techniques on a common chip, including a
silicon-based or equivalent substrate (e.g., Voldman et al., Ann.
Rev. Biomed. Eng. 1:401-425, 1999). The sensor components of MEMS
can be used to measure mechanical, thermal, biological, chemical,
optical and/or magnetic phenomena. The electronics can process the
information from the sensors and control actuator components such
pumps, valves, heaters, coolers, filters, etc. thereby controlling
the function of the MEMS.
[0088] The electronic components of MEMS can be fabricated using
integrated circuit (IC) processes (e.g., CMOS, Bipolar, or BICMOS
processes). They can be patterned using photolithographic and
etching methods known for computer chip manufacture. The
micromechanical components can be fabricated using compatible
"micromachining" processes that selectively etch away parts of the
silicon wafer or add new structural layers to form the mechanical
and/or electromechanical components.
[0089] Basic techniques in MEMS manufacture include depositing thin
films of material on a substrate, applying a patterned mask on top
of the films by photolithographic imaging or other known
lithographic methods, and selectively etching the films. A thin
film can have a thickness in the range of a few nanometers to 100
micrometers. Deposition techniques of use may include chemical
procedures such as chemical vapor deposition (CVD),
electrodeposition, epitaxy and thermal oxidation and physical
procedures like physical vapor deposition (PVD) and casting.
[0090] In some embodiments of the invention, MEMS devices 300
include various fluid filled compartments 305, such as microfluidic
channels, nanochannels and/or microchannels. In one embodiment,
systems 5 and 105 may be constructed as a MEMS device, as shown in
FIG. 8. These and other components of the apparatus can formed as a
single unit, for example in the form of a chip as known in
semiconductor chips and/or microcapillary or microfluidic chips.
Alternatively, an immobilization substrate, such as a metal coated
porous silicon substrate, can be removed from a silicon wafer and
attached to other components of an apparatus. Any materials known
for use in such chips may be used in the disclosed apparatus,
including silicon, silicon dioxide, silicon nitride, polydimethyl
siloxane (PDMS), polymethylmethacrylate (PMMA), plastic, glass,
quartz.
[0091] Techniques for batch fabrication of chips are well known in
the fields of computer chip manufacture and/or microcapillary chip
manufacture. Such chips may be manufactured by any method known in
the art, such as by photolithography and etching, laser ablation,
injection molding, casting, molecular beam epitaxy, dip-pen
nanolithography, chemical vapor deposition (CVD) fabrication,
electron beam or focused ion beam technology or imprinting
techniques. Non-limiting examples include conventional molding with
a flowable, optically clear material such as plastic or glass;
photolithography and dry etching of silicon dioxide; electron beam
lithography using polymethylmethacrylate resist to pattern an
aluminum mask on a silicon dioxide substrate, followed by reactive
ion etching. Known methods for manufacture of nanoelectromechanical
systems may be used for certain embodiments of the invention. (See,
e.g., Craighead, Science 290: 1532-36, 2000.) Various forms of
microfabricated chips are commercially available from, e.g.,
Caliper Technologies Inc. (Mountain View, Calif.) and ACLARA
BioSciences Inc. (Mountain View, Calif.).
[0092] In certain embodiments of the invention, part or all of the
apparatus can be selected to be transparent to terahertz and/or
gigahertz radiation, such as glass, silicon, quartz or any other
optically clear material. For fluid-filled compartments that may be
exposed to various biomolecules, such as proteins, peptides,
nucleic acids, nucleotides and the like, the surfaces exposed to
such molecules may be modified by coating, for example to transform
a surface from a hydrophobic to a hydrophilic surface and/or to
decrease adsorption of molecules to a surface. Surface modification
of common chip materials such as glass, silicon, quartz and/or PDMS
is known in the art (e.g., U.S. Pat. No. 6,263,286). Such
modifications may include, but are not limited to, coating with
commercially available capillary coatings (Supelco, Bellafonte,
Pa.), silanes with various functional groups such as
polyethyleneoxide or acrylamide, or any other coating known in the
art.
[0093] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *