U.S. patent application number 12/147243 was filed with the patent office on 2008-10-23 for multiplex data collection and analysis in bioanalyte detection.
Invention is credited to Richard Jones, Tae-Woong Koo, Qing Ma, Xing Su, Lei Sun, Mineo Yamakawa, Jingwu Zhang.
Application Number | 20080262989 12/147243 |
Document ID | / |
Family ID | 37804682 |
Filed Date | 2008-10-23 |
United States Patent
Application |
20080262989 |
Kind Code |
A1 |
Su; Xing ; et al. |
October 23, 2008 |
MULTIPLEX DATA COLLECTION AND ANALYSIS IN BIOANALYTE DETECTION
Abstract
Method and device to collect multiplex data simultaneously in
analyte detection and analyze the data by experimentally trained
software (machine-learning) is disclosed. Various ways (magnetic
particles and microcoils) are disclosed to collect multiple
reporter (tag) signals. Multiplex detection can increase the
biomolecule analysis efficiency by using small sample size and
saving assay reagents and time. Machine learning and data analysis
schemes are also disclosed. Multiple affinity binding partners,
each labeled by a unique reporter, are contacted with a sample and
a single spectrum is taken to detect multiple reporter signals. The
spectrum is deconvoluted by experimentally trained software to
identify multiple analytes.
Inventors: |
Su; Xing; (Cupertino,
CA) ; Sun; Lei; (Santa Clara, CA) ; Yamakawa;
Mineo; (Campbell, CA) ; Zhang; Jingwu; (San
Jose, CA) ; Ma; Qing; (San Jose, CA) ; Koo;
Tae-Woong; (Cupertino, CA) ; Jones; Richard;
(Santa Clara, CA) |
Correspondence
Address: |
Client 21058;c/o DARBY & DARBY P.C.
P.O. BOX 770, CHURCH STREET STATION
NEW YORK
NY
10008-0770
US
|
Family ID: |
37804682 |
Appl. No.: |
12/147243 |
Filed: |
June 26, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11216112 |
Sep 1, 2005 |
7410763 |
|
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12147243 |
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Current U.S.
Class: |
706/16 |
Current CPC
Class: |
B82Y 30/00 20130101;
G01N 33/53 20130101; G01N 33/5438 20130101; G01N 33/54366 20130101;
B82Y 15/00 20130101 |
Class at
Publication: |
706/16 |
International
Class: |
G06F 17/50 20060101
G06F017/50 |
Claims
1-40. (canceled)
41. A computer implemented system comprising a first algorithm to
simulate spectral features produced by a hypothetical composition
comprising a plurality of reporters mixed in different ratios, a
second algorithm to compare the simulated spectral features with
experimentally obtained spectral features produced by an actual
composition comprising a plurality of reporters in different
ratios, and a third algorithm to determine a goodness-of-fit
between the simulated spectral features and the experimentally
obtained spectral features and to iteratively adjust the simulated
spectral features by adjusting the hypothetical composition to
maximize the goodness-of-fit to meet a pre-set statistical
criteria.
42. The computer implemented system of claim 41, wherein the first,
second and third algorithms are bundled into one or more software
programs or one or more hardware components.
43. The computer implemented system of claim 41, wherein the
plurality of reporters in the actual composition is associated with
a plurality of analytes of a biological sample.
44. The computer implemented system of claim 41, wherein the
goodness-of-fit is maximized by minimizing the difference between
the simulated spectral features and the experimentally obtained
spectral features.
45. The computer implemented system of claim 44, wherein the
difference between the between the simulated spectral features and
the experimentally obtained spectral features is determined by a
genetic algorithm that qualitatively optimizes the genetic
algorithm, by a neural network that optimizes a set of selected
parameters for a selected neural patterns or circuits, or by a
principal component analysis that statistically decomposes
components with maximum likelihood.
46-68. (canceled)
69. A computer implemented method comprising simulating spectral
features product by a hypothetical composition comprising a
plurality of reporters mixed in different ratios, comparing the
stimulated spectral features with experimentally obtained spectral
features produced by an actual composition comprising a plurality
of reporters in different ratios, determining a goodness-of-fit
between the simulated spectral features and the experimentally
obtained spectral features and iteratively adjusting the stimulated
spectral features by adjusting the hypothetical composition to
maximize the goodness-of-fit to meet a pre-set statistical
criteria.
70. The computer implemented method of claim 69, wherein the
goodness-of-fit is maximized by minimizing the difference between
the simulated spectral features and the experimentally obtained
spectral features.
Description
RELATED APPLICATIONS
[0001] This application is related to U.S. patent application Ser.
No. 10/748,336, filed Dec. 29, 2003, entitled "Composite
Organic-Inorganic Nanoparticles (COIN) as SERS Tags for Analyte
Detection," U.S. patent application Ser. No. 10/916,710, filed Aug.
11, 2004, entitled "Multiplex Detection of Analytes in Fluid
Systems," U.S. patent application Ser. No. 10/927,996, filed Aug.
26, 2004, entitled "Biomolecule Analysis Using Raman Surface
Scanning", and U.S. patent application Ser. No. 11/027,470, filed
Dec. 30, 2004, entitled "Biomolecule Analysis Using Raman Surface
Scanning."
FIELD OF INVENTION
[0002] The embodiments of the invention relate to methods and
devices for complex data collection and analysis in multiplexed
biomolecule detection. The invention transcends several scientific
disciplines such as polymer chemistry, biochemistry, molecular
biology, medicine and medical diagnostics.
BACKGROUND
[0003] The molecular-level origins of disease are being elucidated
at a rapid pace, potentially ushering in a new era of personalized
medicine in which a specific course of therapy is developed for
each patient. To fully exploit this expanding knowledge of disease
phenotype, new methods for detecting multiple biomolecules (e.g.,
DNA and proteins) simultaneously are required. The multiplex
biomolecule detection methods must be rapid, sensitive, highly
parallel, and ideally capable of diagnosing cellular phenotype in
vivo.
[0004] Some biomolecule detection methods have been developed based
upon the unique electrochemical and photoelectrochemical properties
of metal particles. In one assay method, gold nanoparticles (10 nm
diameter) are tagged with ssDNA probe strands and a photoactive dye
molecule. A metal electrode of a microarray chip (also called gene
chip) is also modified with ssDNA probe strands. If a target (the
analyte or bioanalyte) mRNA or ssDNA is complementary to the probe
on the particle and the substrate, hybridization will occur which
brings the particle in contact with the electrode. A laser is then
rastered across the surface. When the laser addresses a spot in
which nanoparticles are bound, the dye molecule is electronically
excited, and the excited electron is injected into the electrode.
The electron is collected as a current, signifying the presence of
a particular DNA analyte.
[0005] Synthesis of a functionalized electrode having polymer
arrays on an electrode of a microarray chip is known. Examples of
such polymer arrays include nucleic acid arrays, peptide arrays,
and carbohydrate arrays.
[0006] One method of preparing functionalized electrodes of polymer
arrays on microarray chips involves photolithographic techniques
using photocleavable protecting groups. Briefly, the method
includes attaching photoreactive groups to the surface of a
substrate, exposing selected regions of the substrate to light to
activate those regions, attaching a monomer with a photoremovable
group to the activated regions, and repeating the steps of
activation and attachment until macromolecules of a length and
sequence are synthesized.
[0007] Additional methods and techniques applicable to prepare a
functionalized electrode include electrochemical synthesis. One
example includes providing a porous substrate with an electrode
therein, placing a molecule having a protected chemical group in
proximity of the porous substrate, placing a buffering solution in
contact with the electrode and the porous substrate to prevent
electrochemically generated reagents from leaving the locality of
the electrode (the use of confinement electrodes to prevent
reagents from diffusing away have also been described), applying a
potential to the electrode to generate electrochemical reagents
capable of deprotecting the protected chemical functional group of
the molecule, attaching the deprotected chemical functional group
to the porous substrate or a molecule on the substrate, and
repeating the above steps until polymers of a length and sequence
are synthesized.
[0008] The biomolecules on microarray chip typically are detected
through optical readout of fluorescent labels attached to a target
molecule that is specifically attached or hybridized to a probe
molecule. These optical methods are difficult to implement and
miniaturize because they rely on the use of optical labels and
require large or expensive instrumentation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows a schematic of the methods and devices to
create sub-set of binding complexes.
[0010] FIG. 2 shows the sample preparation method for a micro-fluid
channel based multiplex analyzer system.
[0011] FIG. 3 shows the detection methodology for a micro-fluid
channel based multiplexed analyzer systems.
[0012] FIG. 4 shows the sample preparation method for a micro-array
based multiplexed analyzer systems.
[0013] FIG. 5 shows the detection methodology for a micro-array
based multiplexed analyzer systems.
[0014] FIG. 6 shows a schematic of the magnetic COIN as reporter
and analyte carrier.
[0015] FIG. 7 shows a schematic of the microcoil for concentrating
magnetic-COIN sandwich complexes.
[0016] FIG. 8 shows a schematic of a system for machine learning
and analyte quantification.
[0017] FIG. 9 shows Raman spectra of multiplexed COIN mixtures.
DETAILED DESCRIPTION
[0018] A biological sample often contains many thousands or even
more types of biomolecules and clinical diagnosis needs to measure
multiple analytes for disease confirmation. Currently, each analyte
is measured separately, which requires multiple samples from a
patient. The procedure is time consuming and labor intensive. The
embodiments of the invention allow for multiple analyte detection
from a single sample and a single test, which could be of great
interest to clinical diagnosis, and biomedical research as
well.
[0019] Analytes include nucleic acids (DNA and RNA), which can form
double-stranded molecules by hybridization, that is, complementary
base pairing. The specificity of nucleic acid hybridization is such
that the detection of molecular and/or nanomaterials binding events
can be done through electrical readout of polarization changes
caused by the interaction of charged target molecules (DNA, RNA,
proteins, for example.) and chemically modified nanomaterials
(carbon nanotubes, nanowires, nanoparticles functionalized with
DNA, for example) with complementary molecular probes (DNA, RNA,
anti-body, for example) attached to the electrodes (such as Au, Pt,
for example). This specificity of complementary base pairing also
allows thousands of hybridization to be carried out simultaneously
in the same experiment on a DNA chip (also called a DNA array).
[0020] Molecular probes are immobilized on the surface of
individually addressable electrode arrays through the surface
functionalization techniques. Electrodes allow polarization changes
to be electrically detected. The polymer arrays of the embodiment
of the invention could be a DNA array (collections of DNA probes on
a shared base) comprising a dense grid of spots (often called
elements or pads) arranged on a miniature support. Each spot could
represent a different gene.
[0021] The probe in a DNA chip is usually hybridized with a complex
RNA or cDNA target generated by making DNA copies of a complex
mixture of RNA molecules derived from a particular cell type
(source). The composition of such a target reflects the level of
individual RNA molecules in the source. The intensities of the
signals resulting from the binding events from the DNA spots of the
DNA chip after hybridization between the probe and the target
represent the relative expression levels of the genes of the
source.
[0022] The DNA chip could be used for differential gene expression
between samples (e.g., healthy tissue versus diseased tissue) to
search for various specific genes (e.g., connected with an
infectious agent) or in gene polymorphism and expression analysis.
Particularly, the DNA chip could be used to investigate expression
of various genes connected with various diseases in order to find
causes of these diseases and to enable accurate treatments.
[0023] Using embodiments of the invention, one could find a
specific segment of a nucleic acid of a gene, i.e., find a site
with a particular order of bases in the examined gene. This
detection could be performed by using a diagnostic polynucleotide
made up of short synthetically assembled single-chained
complementary polynucleotide--a chain of bases organized in a
mirror order to which the specific segment of the nucleic acid
would attach (hybridize) via A-T or G-C bonds.
[0024] The practice of the embodiments of the invention may employ,
unless otherwise indicated, conventional techniques of organic
chemistry, polymer technology, molecular biology (including
recombinant techniques), cell biology, biochemistry, and
immunology, which are within the skill of the art. Such
conventional techniques include polymer array synthesis,
hybridization, ligation, detection of hybridization using a label.
Specific illustrations of suitable techniques can be had by
reference to the example herein below. However, other equivalent
conventional procedures can, of course, also be used.
[0025] As used in the specification and claims, the singular forms
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "an array" may
include a plurality of arrays unless the context clearly dictates
otherwise.
[0026] An "array" is an intentionally created collection of
molecules which can be prepared either synthetically or
biosynthetically. The molecules in the array can be identical or
different from each other. The array can assume a variety of
formats, e.g., libraries of soluble molecules; libraries of
compounds tethered to resin beads, silica chips, or other solid
supports. The array could either be a macroarray or a microarray,
depending on the size of the sample spots on the array. A
macroarray generally contains sample spot sizes of about 300
microns or larger and can be easily imaged by gel and blot
scanners. A microarray would generally contain spot sizes of less
than 300 microns.
[0027] "Solid support," "support," and "substrate" refer to a
material or group of materials having a rigid or semi-rigid surface
or surfaces. In some aspects, at least one surface of the solid
support will be substantially flat, although in some aspects it may
be desirable to physically separate synthesis regions for different
molecules with, for example, wells, raised regions, pins, etched
trenches, or the like. In certain aspects, the solid support(s)
will take the form of beads, resins, gels, microspheres, or other
geometric configurations.
[0028] The term "probe" or "probe molecule" refers to a molecule
attached to the substrate of the array, which is typically cDNA or
pre-synthesized polynucleotide deposited on the array. Probes
molecules are biomolecules capable of undergoing binding or
molecular recognition events with target molecules. (In some
references, the terms "target" and "probe" are defined opposite to
the definitions provided here.) The polynucleotide probes require
only the sequence information of genes, and thereby can exploit the
genome sequences of an organism. In cDNA arrays, there could be
cross-hybridization due to sequence homologies among members of a
gene family. Polynucleotide arrays can be specifically designed to
differentiate between highly homologous members of a gene family as
well as spliced forms of the same gene (exon-specific).
Polynucleotide arrays of the embodiment of this invention could
also be designed to allow detection of mutations and single
nucleotide polymorphism.
[0029] The term "target" or "target molecule" refers to a small
molecule, biomolecule, or nanomaterial such as but not necessarily
limited to a small molecule that is biologically active, nucleic
acids and their sequences, peptides and polypeptides, as well as
nanostructure materials chemically modified with biomolecules or
small molecules capable of binding to molecular probes such as
chemically modified carbon nanotubes, carbon nanotube bundles,
nanowires and nanoparticles. The target molecule may be
fluorescently labeled DNA or RNA.
[0030] The terms "die," "polymer array chip," "DNA array," "array
chip," "DNA array chip," "bio-chip" or "chip" are used
interchangeably and refer to a collection of a large number of
probes arranged on a shared substrate which could be a portion of a
silicon wafer, a nylon strip or a glass slide.
[0031] The term "molecule" generally refers to a chemical made up
of two or more atoms and includes a macromolecule, biomolecule or
polymer as described herein. However, arrays comprising single
molecules, as opposed to macromolecules or polymers, are also
within the scope of the embodiments of the invention. The term
"biomolecule" refers to any organic molecule that is part of a
living organism. A "complex of a biomolecule" refers to a structure
made up of two or more types of biomolecules. Examples of a complex
of biomolecule include a cell or viral particles. A cell can
include bacteria, fungi, animal mammalian cell, for example.
[0032] "Predefined region," "spot" "binding area" or "pad" refers
to a localized area on a solid support which is, was, or is
intended to be used for the formation of a selected molecule and is
otherwise referred to herein in the alternative as a "selected"
region. The predefined region may have any convenient shape, e.g.,
circular, rectangular, elliptical, wedge-shaped, etc. For the sake
of brevity herein, "predefined regions" are sometimes referred to
simply as "regions" or "spots." In some embodiments, a predefined
region and, therefore, the area upon which each distinct molecule
is synthesized is smaller than about 1 cm.sup.2 or less than 1
mm.sup.2, and still more preferably less than 0.5 mm.sup.2. In most
preferred embodiments the regions have an area less than about
10,000 .mu.m.sup.2 or, more preferably, less than 100 .mu.m.sup.2.
Additionally, multiple copies of the polymer will typically be
synthesized within any preselected region. The number of copies can
be in the thousands to the millions. More preferably, a die of a
wafer contains at least 400 spots in, for example, an at least
20.times.20 matrix. Even more preferably, the die contains at least
2048 spots in, for example, an at least 64.times.32 matrix, and
still more preferably, the die contains at least 204,800 spots in,
for example, an at least 640.times.320 array. A spot could contain
an electrode to generate an electrochemical reagent, a working
electrode to synthesize a polymer and a confinement electrode to
confine the generated electrochemical reagent. The electrode to
generate the electrochemical reagent could be of any shape,
including, for example, circular, flat disk shaped and hemisphere
shaped.
[0033] A "microcoil" refers to a localized microelectromagnet on or
in a solid support which is, was, or is intended to be used for the
formation of a selected molecule under the influence of magnetic
field. Integrated microcoils in an array may have any convenient
shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
In some embodiments of the invention, the microcoil could be
smaller than about 1 cm.sup.2 or less than 1 mm.sup.2, and still
more preferably less than 0.5 mm.sup.2. In most preferred
embodiments the microcoil could have an area less than about 10,000
.mu.m.sup.2 or, more preferably, less than 100 .mu.m.sup.2. For
independent magnetic field control, each microcoil is connected to
its own on-chip current source. The operating principle of the
microcoil array for cell manipulation is to create and move
magnetic field peaks by modulating currents in the microcoils. For
instance, by activating only one microcoil in the array, a magnetic
bead suspended in fluid will be attracted to the field peak at the
center of the microcoil on the surface of the IC having the
microcoil. Subsequently, by turning off the microcoil while
activating an adjacent one, the magnetic field peak is moved to the
center of the adjacent microcoil, transporting the magnetic bead to
the new peak location. The spatial resolution of the manipulation
is determined by the spacing between two neighboring coils. For
precise spatial control of individual magnetic beads, the microcoil
could be carefully designed to generate a single magnetic field
peak on the chip surface. Note that while the microcoil generally
produces a single magnetic peak on the chip surface, multiple
magnetic peaks can exist below the surface.
[0034] An "electrode" is a body or a location at which an
electrochemical reaction occurs. The term "electrochemical" refers
to an interaction or interconversion of electric and chemical
phenomena. A "functionalized electrode" is an electrode of a
microchip array having a probe molecule that has a specific
chemical affinity to a target molecule. An "unfunctionalized
electrode" is an electrode of a microchip array having no probe
molecule or having a probe molecule that has no specific chemical
affinity to a target molecule.
[0035] The electrodes used in embodiments of the invention may be
composed of, but are not limited to, metals such as iridium and/or
platinum, and other metals, such as, palladium, gold, silver,
copper, mercury, nickel, zinc, titanium, tungsten, aluminum, as
well as alloys of various metals, and other conducting materials,
such as, carbon, including glassy carbon, reticulated vitreous
carbon, basal plane graphite, edge plane graphite and graphite.
Doped oxides such as indium-tin oxide and semiconductors such as
silicon oxide and gallium arsenide are also contemplated.
Additionally, the electrodes may be composed of conducting
polymers, metal doped polymers, conducting ceramics and conducting
clays. Among the metals, platinum and palladium are especially
preferred because of the advantageous properties associated with
their ability to absorb hydrogen, i.e., their ability to be
"preloaded" with hydrogen before being used in the methods of the
invention.
[0036] The electrodes may be connected to an electric source in any
known manner. Preferred ways of connecting the electrodes to the
electric source include CMOS (complementary metal oxide
semiconductor) switching circuitry, radio and microwave frequency
addressable switches, light addressable switches, direct connection
from an electrode to a bond pad on the perimeter of a semiconductor
chip, and combinations thereof. CMOS switching circuitry involves
the connection of each of the electrodes to a CMOS transistor
switch. The switch could be accessed by sending an electronic
address signal down a common bus to SRAM (static random access
memory) circuitry associated with each electrode. When the switch
is "on", the electrode is connected to an electric source. Radio
and microwave frequency addressable switches involve the electrodes
being switched by a RF or microwave signal. This allows the
switches to be thrown both with and/or without using switching
logic. The switches can be tuned to receive a particular frequency
or modulation frequency and switch without switching logic. Light
addressable switches are switched by light. In this method, the
electrodes can also be switched with and without switching logic.
The light signal can be spatially localized to afford switching
without switching logic. This could be accomplished, for example,
by scanning a laser beam over the electrode array; the electrode
being switched each time the laser illuminates it.
[0037] In some aspects, a predefined region can be achieved by
physically separating the regions (i.e., beads, resins, gels, etc.)
into wells, trays, etc.
[0038] A "protecting group" is a moiety which is bound to a
molecule and designed to block one reactive site in a molecule, but
may be spatially removed upon selective exposure to an activator or
a deprotecting reagent. Several examples of protecting groups are
known in the literature. The proper selection of protecting group
(also known as protective group) for a particular synthesis would
be governed by the overall methods employed in the synthesis.
Activators include, for example, electromagnetic radiation, ion
beams, electric fields, magnetic fields, electron beams, x-ray, and
the like. A deprotecting reagent could include, for example, an
acid, a base or a free radical. Protective groups are materials
that bind to a monomer, a linker molecule or a pre-formed molecule
to protect a reactive functionality on the monomer, linker molecule
or pre-formed molecule, which may be removed upon selective
exposure to an activator, such as an electrochemically generated
reagent. Protective groups that may be used in accordance with an
embodiment of the invention preferably include all acid and base
labile protecting groups. For example, peptide amine groups are
preferably protected by t-butyloxycarbonyl (BOC) or
benzyloxycarbonyl (CBZ), both of which are acid labile, or by
9-fluorenylmethoxycarbonyl (FMOC), which is base labile.
Additionally, hydroxyl groups on phosphoramidites may be protected
by dimethoxytrityl (DMT), which is acid labile. Exocyclic amine
groups on nucleosides, in particular on phosphoramidites, are
preferably protected by dimethylformamidine on the adenosine and
guanosine bases, and isobutyryl on the cytidine bases, both of
which are base labile protecting groups. This protection strategy
is known as fast oligonucleotide deprotection (FOD).
[0039] Any unreacted deprotected chemical functional groups may be
capped at any point during a synthesis reaction to avoid or to
prevent further bonding at such molecule. Capping groups "cap"
deprotected functional groups by, for example, binding with the
unreacted amino functions to form amides. Capping agents suitable
for use in an embodiment of the invention include: acetic
anhydride, n-acetylimidizole, isopropenyl formate, fluorescamine,
3-nitrophthalic anhydride and 3-sulfoproponic anhydride. Of these,
acetic anhydride and n-acetylimidizole are preferred.
[0040] Additional protecting groups that may be used in accordance
with an embodiment of the invention include acid labile groups for
protecting amino moieties:
tertbutyloxycarbonyl,-tert-amyloxycarbonyl, adamantyloxycarbonyl,
1-methylcyclobutyloxycarbonyl, 2-(p-biphenyl)propyl(2)oxycarbonyl,
2-(p-phenylazophenylyl)propyl(2)oxycarbonyl,
alpha.,.alpha.-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl,
2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl,
benzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl (trityl),
p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl,
diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl,
o-nitrophenylsulfenyl, and 1-naphthylidene; as base labile groups
for protecting amino moieties: 9-fluorenylmethyloxycarbonyl,
methylsulfonylethyloxycarbonyl, and
5-benzisoazolylmethyleneoxycarbonyl; as groups for protecting amino
moieties that are labile when reduced: dithiasuccinoyl, p-toluene
sulfonyl, and piperidino-oxycarbonyl; as groups for protecting
amino moieties that are labile when oxidized: (ethylthio)carbonyl;
as groups for protecting amino moieties that are labile to
miscellaneous reagents, the appropriate agent is listed in
parenthesis after the group: phthaloyl (hydrazine), trifluoroacetyl
(piperidine), and chloroacetyl (2-aminothiophenol); acid labile
groups for protecting carboxylic acids: tert-butyl ester; acid
labile groups for protecting hydroxyl groups: dimethyltrityl; and
basic labile groups for protecting phosphotriester groups:
cyanoethyl.
[0041] An "electrochemical reagent" refers to a chemical generated
at a selected electrode by applying a sufficient electrical
potential to the selected electrode and is capable of
electrochemically removing a protecting group from a chemical
functional group. The chemical group would generally be attached to
a molecule. Removal of a protecting group, or "deprotection," in
accordance with the invention, preferably occurs at a particular
portion of a molecule when a chemical reagent generated by the
electrode acts to deprotect or remove, for example, an acid or base
labile protecting group from the molecule. This electrochemical
deprotection reaction may be direct, or may involve one or more
intermediate chemical reactions that are ultimately driven or
controlled by the imposition of sufficient electrical potential at
a selected electrode.
[0042] Electrochemical reagents that can be generated
electrochemically at an electrode fall into two broad classes:
oxidants and reductants. Oxidants that can be generated
electrochemically, for example, include iodine, iodate, periodic
acid, hydrogen peroxide, hypochlorite, metavanadate, bromate,
dichromate, cerium (IV), and permanganate ions. Reductants that can
be generated electrochemically, for example, include chromium (II),
ferrocyanide, thiols, thiosulfate, titanium (III), arsenic (III)
and iron (II) ions. The miscellaneous reagents include bromine,
chloride, protons and hydroxyl ions. Among the foregoing reagents,
protons, hydroxyl, iodine, bromine, chlorine and thiol ions are
preferred.
[0043] The generation of and electrochemical reagent of a type of
chemical species requires that the electric potential of the
electrode that generates the electrochemical reagent have a certain
value, which may be achieved by specifying either the voltage or
the current. There are two ways to achieve the potential at this
electrode: either the voltage may be specified at a value or the
current can be determined such that it is sufficient to provide a
voltage. The range between the minimum and maximum potential values
could be determined by the type of electrochemical reagent chosen
to be generated.
[0044] An "activating group" refers to those groups which, when
attached to a particular chemical functional group or reactive
site, render that site more reactive toward covalent bond formation
with a second chemical functional group or reactive site.
[0045] A "polymeric brush" ordinarily refers to polymer films
comprising chains of polymers that are attached to the surface of a
substrate. The polymeric brush could be a functionalized polymer
films which comprise functional groups such as hydroxyl, amino,
carboxyl, thiol, amide, cyanate, thiocyanate, isocyanate and
isothio cyanate groups, or a combination thereof, on the polymer
chains at one or more predefined regions. The polymeric brushes of
the embodiment of the invention are capable of attachment or
stepwise synthesis of macromolecules thereon.
[0046] A "linker" molecule refers to any of those molecules
described supra and preferably should be about 4 to about 40 atoms
long to provide sufficient exposure. The linker molecules may be,
for example, aryl acetylene, ethylene glycol oligomers containing
2-10 monomer units, diamines, diacids, amino acids, among others,
and combinations thereof. Alternatively, the linkers may be the
same molecule type as that being synthesized (i.e., nascent
polymers), such as polynucleotides, oligopeptides, or
oligosaccharides.
[0047] The linker molecule or substrate itself and monomers used
herein are provided with a functional group to which is bound a
protective group. Generally, the protective group is on the distal
or terminal end of a molecule. Preferably, the protective group is
on the distal or terminal end of the linker molecule opposite the
substrate. The protective group may be either a negative protective
group (i.e., the protective group renders the linker molecules less
reactive with a monomer upon exposure) or a positive protective
group (i.e., the protective group renders the linker molecules more
reactive with a monomer upon exposure). In the case of negative
protective groups, there could be an additional step of
reactivation. In some embodiments, this will be done by
heating.
[0048] The polymeric brush or the linker molecule may be provided
with a cleavable group at an intermediate position, which group can
be cleaved with an electrochemically generated reagent. This group
is preferably cleaved with a reagent different from the reagent(s)
used to remove the protective groups. This enables removal of the
various synthesized polymers or nucleic acid sequences following
completion of the synthesis. The cleavable group could be acetic
anhydride, n-acetylimidizole, isopropenyl formate, fluorescamine,
3-nitrophthalic anhydride and 3-sulfoproponic anhydride. Of these,
acetic anhydride and n-acetylimidizole are preferred.
[0049] The polymeric brush or the linker molecule could be of
sufficient length to permit polymers on a completed substrate to
interact freely with binding entities (monomers, for example)
exposed to the substrate. The polymeric brush or the linker
molecule, when used, could preferably be long enough to provide
sufficient exposure of the functional groups to the binding entity.
The linker molecules may include, for example, aryl acetylene,
ethylene glycol oligomers containing from 2 to 20 monomer units,
diamines, diacids, amino acids, and combinations thereof. Other
linker molecules may be used in accordance with the different
embodiments of the present invention and will be recognized by
those skilled in the art in light of this disclosure. In one
embodiment, derivatives of the acid labile 4,4'-dimethyoxytrityl
molecules with an exocyclic active ester can be used in accordance
with an embodiment of the invention. More preferably,
N-succinimidyl-4[bis-(4-methoxyphenyl)-chloromethyl]-benzoate is
used as a cleavable linker molecule during DNA synthesis.
Alternatively, other manners of cleaving can be used over the
entire array at the same time, such as chemical reagents, light or
heat.
[0050] "Monomer" as used herein refers to those monomers that are
used to a form a polymer. However, the meaning of the monomer will
be clear from the context in which it is used. The monomers in a
given polymer or macromolecule can be identical to or different
from each other. A monomer can be a small or a large molecule,
regardless of molecular weight. Furthermore, each of the monomers
may be protected members which are modified after synthesis.
[0051] The monomers for forming the polymers of the embodiments of
the invention, e.g., a polymeric brush or a linker molecule, have
for example the general structure:
##STR00001##
[0052] wherein R.sup.1 is hydrogen or lower alkyl; R.sub.2 and
R.sub.3 are independently hydrogen, or -Y-Z, wherein Y is lower
alkyl, and Z is hydroxyl, amino, or C(O)--R, where R is hydrogen,
lower alkoxy or aryloxy.
[0053] The term "alkyl" refers to those groups such as methyl,
ethyl, propyl, butyl etc, which may be linear, branched or
cyclic.
[0054] The term "alkoxy" refers to groups such as methoxy, ethoxy,
propoxy, butoxy, etc., which may be linear, branched or cyclic.
[0055] The term "lower" as used in the context of lower alkyl or
lower alkoxy refers to groups having one to six carbons.
[0056] The term "aryl" refers to an aromatic hydrocarbon ring to
which is attached an alkyl group. The term "aryloxy" refers to an
aromatic hydrocarbon ring to which is attached an alkoxy group. One
of ordinary skill in the art would readily understand these
terms.
[0057] Other monomers for preparing macromolecules of the
embodiments of the invention are well-known in the art. For
example, when the macromolecule is a peptide, the monomers include,
but are not restricted to, for example, amino acids such as the
L-amino acids, the D-amino acids, and the synthetic and/or natural
amino acids. When the macromolecule is a nucleic acid, or
polynucleotide, the monomers include any nucleotide. When the
macromolecule is a polysaccharide, the monomers can be any pentose,
hexose, heptose, or their derivatives.
[0058] A "macromolecule" or "polymer" comprises two or more
monomers covalently joined. The monomers may be joined one at a
time or in strings of multiple monomers, ordinarily known as
"oligomers." Thus, for example, one monomer and a string of five
monomers may be joined to form a macromolecule or polymer of six
monomers. Similarly, a string of fifty monomers may be joined with
a string of hundred monomers to form a macromolecule or polymer of
one hundred and fifty monomers. The term polymer as used herein
includes, for example, both linear and cyclic polymers of nucleic
acids, polynucleotides, polynucleotides, polysaccharides,
oligosaccharides, proteins, polypeptides, peptides, phospholipids
and peptide nucleic acids (PNAs). The peptides include those
peptides having either .alpha.-, .beta.-, or .omega.-amino acids.
In addition, polymers include heteropolymers in which a known drug
is covalently bound to any of the above, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or
other polymers which will be apparent upon review of this
disclosure.
[0059] A "nanomaterial" as used herein refers to a structure, a
device or a system having a dimension at the atomic, molecular or
macromolecular levels, in the length scale of approximately 1-500
nanometer range. Preferably, a nanomaterial has properties and
functions because of the size and can be manipulated and controlled
on the atomic level.
[0060] A "carbon nanotube" refers to a fullerene molecule having a
cylindrical or toroidal shape. A "fullerene" refers to a form of
carbon having a large molecule consisting of an empty cage of sixty
or more carbon atoms.
[0061] The term "nucleotide" includes deoxynucleotides and analogs
thereof. These analogs are those molecules having some structural
features in common with a naturally occurring nucleotide such that
when incorporated into a polynucleotide sequence, they allow
hybridization with a complementary polynucleotide in solution.
Typically, these analogs are derived from naturally occurring
nucleotides by replacing and/or modifying the base, the ribose or
the phosphodiester moiety. The changes can be tailor-made to
stabilize or destabilize hybrid formation, or to enhance the
specificity of hybridization with a complementary polynucleotide
sequence as desired, or to enhance stability of the
polynucleotide.
[0062] The term "polynucleotide" or "nucleic acid" as used herein
refers to a polymeric form of nucleotides of any length, either
ribonucleotides or deoxyribonucleotides, that comprise purine and
pyrimidine bases, or other natural, chemically or biochemically
modified, non-natural, or derivatized nucleotide bases.
Polynucleotides of the embodiments of the invention include
sequences of deoxyribopolynucleotide (DNA), ribopolynucleotide
(RNA), or DNA copies of ribopolynucleotide (cDNA) which may be
isolated from natural sources, recombinantly produced, or
artificially synthesized. A further example of a polynucleotide of
the embodiments of the invention may be polyamide polynucleotide
(PNA). The polynucleotides and nucleic acids may exist as
single-stranded or double-stranded. The backbone of the
polynucleotide can comprise sugars and phosphate groups, as may
typically be found in RNA or DNA, or modified or substituted sugar
or phosphate groups. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and nucleotide analogs.
The sequence of nucleotides may be interrupted by non-nucleotide
components. The polymers made of nucleotides such as nucleic acids,
polynucleotides and polynucleotides may also be referred to herein
as "nucleotide polymers.
[0063] An "oligonucleotide" is a polynucleotide having 2 to 20
nucleotides. Phosphoramidites protected in this manner are known as
FOD phosphoramidites.
[0064] Analogs also include protected and/or modified monomers as
are conventionally used in polynucleotide synthesis. As one of
skill in the art is well aware, polynucleotide synthesis uses a
variety of base-protected nucleoside derivatives in which one or
more of the nitrogens of the purine and pyrimidine moiety are
protected by groups such as dimethoxytrityl, benzyl, tert-butyl,
isobutyl and the like.
[0065] For instance, structural groups are optionally added to the
ribose or base of a nucleoside for incorporation into a
polynucleotide, such as a methyl, propyl or allyl group at the 2'-O
position on the ribose, or a fluoro group which substitutes for the
2'-O group, or a bromo group on the ribonucleoside base.
2'-O-methyloligoribonucleotides (2'-O-MeORNs) have a higher
affinity for complementary polynucleotides (especially RNA) than
their unmodified counterparts. Alternatively, deazapurines and
deazapyrimidines in which one or more N atoms of the purine or
pyrimidine heterocyclic ring are replaced by C atoms can also be
used.
[0066] The phosphodiester linkage or "sugar-phosphate backbone" of
the polynucleotide can also be substituted or modified, for
instance with methyl phosphonates, O-methyl phosphates or
phosphororthioates. Another example of a polynucleotide comprising
such modified linkages for purposes of this disclosure includes
"peptide polynucleotides" in which a polyamide backbone is attached
to polynucleotide bases, or modified polynucleotide bases. Peptide
polynucleotides which comprise a polyamide backbone and the bases
found in naturally occurring nucleotides are commercially
available.
[0067] Nucleotides with modified bases can also be used in the
embodiments of the invention. Some examples of base modifications
include 2-aminoadenine, 5-methylcytosine, 5-(propyn-1-yl)cytosine,
5-(propyn-1-yl)uracil, 5-bromouracil, 5-bromocytosine,
hydroxymethylcytosine, methyluracil, hydroxymethyluracil, and
dihydroxypentyluracil which can be incorporated into
polynucleotides in order to modify binding affinity for
complementary polynucleotides.
[0068] Groups can also be linked to various positions on the
nucleoside sugar ring or on the purine or pyrimidine rings which
may stabilize the duplex by electrostatic interactions with the
negatively charged phosphate backbone, or through interactions in
the major and minor groves. For example, adenosine and guanosine
nucleotides can be substituted at the N.sup.2 position with an
imidazolyl propyl group, increasing duplex stability. Universal
base analogues such as 3-nitropyrrole and 5-nitroindole can also be
included. A variety of modified polynucleotides suitable for use in
the embodiments of the invention are described in the
literature.
[0069] When the macromolecule of interest is a peptide, the amino
acids can be any amino acids, including .alpha., .beta., or
.omega.-amino acids. When the amino acids are .alpha.-amino acids,
either the L-optical isomer or the D-optical isomer may be used.
Additionally, unnatural amino acids, for example, .beta.-alanine,
phenylglycine and homoarginine are also contemplated by the
embodiments of the invention. These amino acids are well-known in
the art.
[0070] An "antibody" is any of various bodies or substances in the
blood which act in antagonism to harmful foreign bodies, as toxins
or the bacteria producing the toxins. Normal blood serum apparently
contains various antibodies, and the introduction of toxins or of
foreign cells also results in the development of their specific
antibodies. For example, an antibody is a Y-shaped protein on the
surface of B cells that is secreted into the blood or lymph in
response to an antigenic stimulus, such as a bacterium, virus,
parasite, or transplanted organ, and that neutralizes the antigen
by binding specifically to it; an immunoglobulin.
[0071] A "peptide" is a polymer in which the monomers are amino
acids and which are joined together through amide bonds and
alternatively referred to as a polypeptide. In the context of this
specification it should be appreciated that the amino acids may be
the L-optical isomer or the D-optical isomer. Peptides are two or
more amino acid monomers long, and often more than 20 amino acid
monomers long.
[0072] A "protein" is a long polymer of amino acids linked via
peptide bonds and which may be composed of two or more polypeptide
chains. More specifically, the term "protein" refers to a molecule
composed of one or more chains of amino acids in a specific order;
for example, the order as determined by the base sequence of
nucleotides in the gene coding for the protein. Proteins are
essential for the structure, function, and regulation of the body's
cells, tissues, and organs, and each protein has unique functions.
Examples are hormones, enzymes, and antibodies.
[0073] A "carbohydrate" is a compound with carbon, hydrogen and
oxygen usually in a proportion to form water with the general
formula C.sub.n(H.sub.2O).sub.n. Carbohydrates can also be called
chemically as neutral compounds of carbon, hydrogen and oxygen.
Carbohydrates are mainly sugars and starches, together constituting
one of the three principal types of nutrients used as energy
sources (calories) by the body. Carbohydrates come in simple forms
such as sugars and in complex forms such as starches and fiber. The
body breaks down most sugars and starches into glucose, a simple
sugar that the body can use to feed its cells. Complex
carbohydrates are derived from plants. Dietary intake of complex
carbohydrates can lower blood cholesterol when they are substituted
for saturated fat. Carbohydrates are classified into mono, di, tri,
poly and heterosaccharides. The smallest carbohydrates are
monosaccharides such as glucose whereas polysaccharides such as
starch, cellulose and glycogen can be large and even indeterminate
in length.
[0074] A "lipid" is defined as a substance such as a fat, oil or
wax that dissolves in alcohol but not in water. Lipids contain
carbon, hydrogen and oxygen but have far less oxygen proportionally
than carbohydrates. Lipids are an important part of living cells.
Together with carbohydrates and proteins, lipids are the main
constituents of plant and animal cells. Cholesterol and
triglycerides are lipids. Lipids are easily stored in the body.
They serve as a source of fuel and are an important constituent of
the structure of cells. Lipids include fatty acids, neutral fats,
waxes and steroids (like cortisone). Compound lipids (lipids
complexed with another type of chemical compound) comprise the
lipoproteins, glycolipids and phospholipids.
[0075] An "antigen" a substance that is capable of causing the
production of an antibody. For example, when an antigen is
introduced into the body, it stimulates the production of an
antibody. Antigens include toxins, bacteria, foreign blood cells,
and the cells of transplanted organs.
[0076] The term "sequence" refers to the particular ordering of
monomers within a macromolecule and it may be referred to herein as
the sequence of the macromolecule.
[0077] The term "hybridization" refers to the process in which two
single-stranded polynucleotides bind non-covalently to form a
stable double-stranded polynucleotide; triple-stranded
hybridization is also theoretically possible. The resulting
(usually) double-stranded polynucleotide is a "hybrid." The
proportion of the population of polynucleotides that forms stable
hybrids is referred to herein as the "degree of hybridization." For
example, hybridization refers to the formation of hybrids between a
probe polynucleotide (e.g., a polynucleotide of the invention which
may include substitutions, deletion, and/or additions) and a
specific target polynucleotide (e.g., an analyte polynucleotide)
wherein the probe preferentially hybridizes to the specific target
polynucleotide and substantially does not hybridize to
polynucleotides consisting of sequences which are not substantially
complementary to the target polynucleotide. However, it will be
recognized by those of skill that the minimum length of a
polynucleotide desired for specific hybridization to a target
polynucleotide will depend on several factors: G/C content,
positioning of mismatched bases (if any), degree of uniqueness of
the sequence as compared to the population of target
polynucleotides, and chemical nature of the polynucleotide (e.g.,
methylphosphonate backbone, phosphorothiolate, etc.), among
others.
[0078] Methods for conducting polynucleotide hybridization assays
have been well developed in the art. Hybridization assay procedures
and conditions will vary depending on the application and are
selected in accordance with the general binding methods known in
the art.
[0079] It is appreciated that the ability of two single stranded
polynucleotides to hybridize will depend upon factors such as their
degree of complementarity as well as the stringency of the
hybridization reaction conditions.
[0080] As used herein, "stringency" refers to the conditions of a
hybridization reaction that influence the degree to which
polynucleotides hybridize. Stringent conditions can be selected
that allow polynucleotide duplexes to be distinguished based on
their degree of mismatch. High stringency is correlated with a
lower probability for the formation of a duplex containing
mismatched bases. Thus, the higher the stringency, the greater the
probability that two single-stranded polynucleotides, capable of
forming a mismatched duplex, will remain single-stranded.
Conversely, at lower stringency, the probability of formation of a
mismatched duplex is increased.
[0081] The appropriate stringency that will allow selection of a
perfectly-matched duplex, compared to a duplex containing one or
more mismatches (or that will allow selection of a particular
mismatched duplex compared to a duplex with a higher degree of
mismatch) is generally determined empirically. Means for adjusting
the stringency of a hybridization reaction are well-known to those
of skill in the art.
[0082] A "ligand" is a molecule that is recognized by a particular
receptor. Examples of ligands that can be investigated by this
invention include, but are not restricted to, agonists and
antagonists for cell membrane receptors, toxins and venoms, viral
epitopes, hormones, hormone receptors, peptides, enzymes, enzyme
substrates, cofactors, drugs (e.g. opiates, steroids, etc.),
lectins, sugars, polynucleotides, nucleic acids, oligosaccharides,
proteins, and monoclonal antibodies. Ligands to cells or
cell-derived molecules, which can include both known and unknown
ligands as well as putative drug candidates that are either
unattached to other solid supports or attached to surfaces or
particle-like structures, could interact with other cell-derived
molecules in a manner such that binding between two binding
partners occurs and can be detected. One of the binding partners or
its attached support can additionally be derivatized with a
substance that can be recognized and quantified by a detection
apparatus. This complex (through interaction) is then brought into
the presence of the detection apparatus using characteristics of
the associated complex that differentiate it from the unassociated
binding partners.
[0083] An "affinity binding partner" or "binding partner" could be
a probe or a ligand defined above.
[0084] A "receptor" is molecule that has an affinity for a given
ligand. Receptors may-be naturally-occurring or manmade molecules.
Also, they can be employed in their unaltered state or as
aggregates with other species. Receptors may be attached,
covalently or noncovalently, to a binding member, either directly
or via a specific binding substance. Examples of receptors which
can be employed by this invention include, but are not restricted
to, antibodies, cell membrane receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, polynucleotides, nucleic
acids, peptides, cofactors, lectins, sugars, polysaccharides,
cells, cellular membranes, and organelles. Receptors are sometimes
referred to in the art as anti-ligands. However, as the term
receptor is used herein, no difference in meaning is intended. A
"ligand receptor pair" is formed when two macromolecules have
combined through molecular recognition to form a complex. Other
examples of receptors which can be investigated by this invention
include but are not restricted to:
[0085] a) Microorganism receptors: Determination of ligands which
bind to receptors, such as specific transport proteins or enzymes
essential to survival of microorganisms, is useful in developing a
new class of antibiotics. Of particular value would be antibiotics
against opportunistic fungi, protozoa, and those bacteria resistant
to the antibiotics in current use.
[0086] b) Enzymes: For instance, one type of receptor is the
binding site of enzymes such as the enzymes responsible for
cleaving neurotransmitters; determination of ligands which bind to
certain receptors to modulate the action of the enzymes which
cleave the different neurotransmitters is useful in the development
of drugs which can be used in the treatment of disorders of
neurotransmission.
[0087] c) Antibodies: For instance, the invention may be useful in
investigating the ligand-binding site on the antibody molecule
which combines with the epitope of an antigen of interest;
determining a sequence that mimics an antigenic epitope may lead to
the-development of vaccines of which the immunogen is based on one
or more of such sequences or lead to the development of related
diagnostic agents or compounds useful in therapeutic treatments
such as for auto-immune diseases (e.g., by blocking the binding of
the "anti-self" antibodies).
[0088] d) Nucleic Acids: Sequences of nucleic acids may be
synthesized to establish DNA or RNA binding sequences.
[0089] e) Catalytic Polypeptides: Polymers, preferably
polypeptides, which are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products. Such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, which
functionality is capable of chemically modifying the bound
reactant.
[0090] f) Hormone receptors: Examples of hormones receptors
include, e.g., the receptors for insulin and growth hormone.
Determination of the ligands which bind with high affinity to a
receptor is useful in the development of, for example, an oral
replacement of the daily injections which diabetics take to relieve
the symptoms of diabetes. Other examples are the vasoconstrictive
hormone receptors; determination of those ligands which bind to a
receptor may lead to the development of drugs to control blood
pressure.
[0091] g) Opiate receptors: Determination of ligands which bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0092] By "analyte" is meant any molecule or compound. An analyte
can be in the solid, liquid, gaseous or vapor phase. By "gaseous or
vapor phase analyte" is meant a molecule or compound that is
present, for example, in the headspace of a liquid, in ambient air,
in a breath sample, in a gas, or as a contaminant in any of the
foregoing. It will be recognized that the physical state of the gas
or vapor phase can be changed by pressure, temperature as well as
by affecting surface tension of a liquid by the presence of or
addition of salts etc.
[0093] The term analyte further includes polynucleotide analytes
such as those polynucleotides defined below. These include m-RNA,
r-RNA, t-RNA, DNA, DNA-RNA duplexes, etc. The term analyte also
includes receptors that are polynucleotide binding agents, such as,
for example, peptide nucleic acids (PNA), restriction enzymes,
activators, repressors, nucleases, polymerases, histones, repair
enzymes, chemotherapeutic agents, and the like.
[0094] The analyte may be a molecule found directly in a sample
such as a body fluid from a host. The sample can be examined
directly or may be pretreated to render the analyte more readily
detectible. Furthermore, the analyte of interest may be determined
by detecting an agent probative of the analyte of interest such as
a specific binding pair member complementary to the analyte of
interest, whose presence will be detected only when the analyte of
interest is present in a sample. Thus, the agent probative of the
analyte becomes the analyte that is detected in an assay. The body
fluid can be, for example, urine, blood, plasma, serum, saliva,
semen, stool, sputum, cerebral spinal fluid, tears, mucus, and the
like.
[0095] The analyte can further be a member of a specific binding
pair (sbp) and may be a ligand, which is monovalent (monoepitopic)
or polyvalent (polyepitopic), usually antigenic or haptenic, and is
a single compound or plurality of compounds which share at least
one common epitopic or determinant site. The analyte can be a part
of a cell such as bacteria or a cell bearing a blood group antigen
such as A, B, D, etc., or an HLA antigen or a microorganism, e.g.,
bacterium, fungus, protozoan, or virus. Also, the analyte could be
charged. A member of a specific binding pair ("sbp member") is one
of two different molecules, having an area on the surface or in a
cavity which specifically binds to and is thereby defined as
complementary with a particular spatial and polar organization of
the other molecule. The members of the specific binding pair are
referred to as ligand and receptor (antiligand) or analyte and
probe. Therefore, a probe is a molecule that specifically binds an
analyte. These will usually be members of an immunological pair
such as antigen-antibody, although other specific binding pairs
such as biotin-avidin, hormones-hormone receptors, nucleic acid
duplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA,
DNA-RNA, and the like are not immunological pairs but are included
in the invention and the definition of sbp member.
[0096] Bioanalyte can also be complex of molecules or compounds in
organized or random fashion, such cells, virus, bacteria, fungi,
etc.
[0097] "Specific binding" is the specific recognition of one of two
different molecules for the other compared to substantially less
recognition of other molecules. Generally, the molecules have areas
on their surfaces or in cavities giving rise to specific
recognition between the two molecules. Exemplary of specific
binding are antibody-antigen interactions, enzyme--substrate
interactions, polynucleotide hybridization interactions, and so
forth.
[0098] "Non-specific binding" is non-covalent binding between
molecules that is relatively independent of specific surface
structures. Non-specific binding may result from several factors
including hydrophobic interactions between molecules.
[0099] The polyvalent ligand analytes will normally be poly(amino
acids), i.e., polypeptides and proteins, polysaccharides, nucleic
acids, and combinations thereof. Such combinations include
components of bacteria, viruses, chromosomes, genes, mitochondria,
nuclei, cell membranes and the like.
[0100] For the most part, the polyepitopic ligand analytes can have
a molecular weight of at least about 5,000, more usually at least
about 10,000. In the poly(amino acid) category, the poly(amino
acids) of interest will generally be from about 5,000 to 5,000,000
molecular weight, more usually from about 20,000 to 1,000,000
molecular weight; among the hormones of interest, the molecular
weights will usually range from about 5,000 to 60,000 molecular
weight.
[0101] The monoepitopic ligand analytes can generally be from about
100 to 2,000 molecular weight, more usually from 125 to 1,000
molecular weight. The analytes include drugs, metabolites,
pesticides, pollutants, and the like. Included among drugs of
interest are the alkaloids. Among the alkaloids are morphine
alkaloids, which includes morphine, codeine, heroin,
dextromethorphan, their derivatives and metabolites; cocaine
alkaloids, which include cocaine and benzyl ecgonine, their
derivatives and metabolites; ergot alkaloids, which include the
diethylamide of lysergic acid; steroid alkaloids; iminazoyl
alkaloids; quinazoline alkaloids; isoquinoline alkaloids; quinoline
alkaloids, which include quinine and quinidine; diterpene
alkaloids, their derivatives and metabolites.
[0102] The term "reporter" means a detectable moiety. The reporter
can be detected, for example, by Raman spectroscopy. Generally, the
reporter and any molecule linked to the reporter can be detected
without a second binding reaction. The reporter can be COIN
(composite-organic-inorganic nanoparticle), magnetic-COIN, quantum
dots, and other Raman or fluorescent tags, for example.
[0103] The term "COIN" refers to a composite-organic-inorganic
nanoparticle(s). The COIN could be surface-enhanced Raman
spectroscopy (SERS)-active nanoparticles incorporated into a gel
matrix and used in certain other analyte separation techniques
described herein. COINs are composite organic-inorganic
nanoparticles. These SERS-active probe constructs comprise a core
and a surface, wherein the core comprises a metallic colloid
comprising a first metal and a Raman-active organic compound. The
COINs can further comprise a second metal different from the first
metal, wherein the second metal forms a layer overlying the surface
of the nanoparticle. The COINs can further comprise an organic
layer overlying the metal layer, which organic layer comprises the
probe. Suitable probes for attachment to the surface of the
SERS-active nanoparticles include, without limitation, antibodies,
antigens, polynucleotides, oligonucleotides, receptors, ligands,
and the like.
[0104] The metal required for achieving a suitable SERS signal is
inherent in the COIN, and a wide variety of Raman-active organic
compounds can be incorporated into the particle. Indeed, a large
number of unique Raman signatures can be created by employing
nanoparticles containing Raman-active organic compounds of
different structures, mixtures, and ratios. Thus, the methods
described herein employing COINs are useful for the simultaneous
detection of many analytes in a sample, resulting in rapid
qualitative analysis of the contents of "profile" of a body fluid.
In addition, since many COINs can be incorporated into a single
nanoparticle, the SERS signal from a single COIN particle is strong
relative to SERS signals obtained from Raman-active materials that
do not contain the nanoparticles described herein as COINs. This
situation results in increased sensitivity compared to
Raman-techniques that do not utilize COINs.
[0105] COINs could be prepared using standard metal colloid
chemistry. The preparation of COINs also takes advantage of the
ability of metals to adsorb organic compounds. Indeed, since
Raman-active organic compounds are adsorbed onto the metal during
formation of the metallic colloids, many Raman-active organic
compounds can be incorporated into the COIN without requiring
special attachment chemistry.
[0106] In general, the COINs could be prepared as follows. An
aqueous solution is prepared containing suitable metal cations, a
reducing agent, and at least one suitable Raman-active organic
compound. The components of the solution are then subject to
conditions that reduce the metallic cations to form neutral,
colloidal metal particles. Since the formation of the metallic
colloids occurs in the presence of a suitable Raman-active organic
compound, the Raman-active organic compound is readily adsorbed
onto the metal during colloid formation. This COIN can typically be
isolated by membrane filtration. In addition, COINs of different
sizes can be enriched by centrifugation.
[0107] The COINs can include a second metal different from the
first metal, wherein the second metal forms a layer overlying the
surface of the nanoparticle. To prepare SERS-active nanoparticle,
COINs are placed in an aqueous solution containing suitable second
metal cations and a reducing agent. The components of the solution
are then subject to conditions that reduce the second metallic
cations so as to form a metallic layer overlying the surface of the
nanoparticle. In certain embodiments, the second metal layer
includes metals, such as, for example, silver, gold, platinum,
aluminum, and the like. These COINs can be isolated and or enriched
in the same manner as explained below. Typically, COINs are
substantially spherical and range in size from about 20 nm to 60
nm. The size of the nanoparticle is selected to be about one-half
the wavelength of light used to irradiate the COINs during
detection.
[0108] Typically, organic compounds of COINs are attached to a
layer of a second metal by covalently attaching organic compounds
to the surface of the metal layer Covalent attachment of an organic
layer to the metallic layer can be achieved in a variety ways well
known to those skilled in the art, such as for example, through
thiol-metal bonds. In alternative embodiments, the organic
molecules attached to the metal layer can be crosslinked to form a
molecular network.
[0109] The COIN(s) can include cores containing magnetic materials,
such as, for example, iron oxides, and the like such that the COIN
is a magnetic COIN. Magnetic COINs can be handled without
centrifugation using commonly available magnetic particle handling
systems. Indeed, magnetism can be used as a mechanism for
separating biological targets attached to magnetic COIN particles
tagged with particular biological probes.
[0110] As used herein, "Raman-active organic compound" refers to an
organic molecule that produces a unique SERS signature in response
to excitation by a laser. A variety of Raman-active organic
compounds are contemplated for use as components in COINs. In
certain embodiments, Raman-active organic compounds are polycyclic
aromatic or heteroaromatic compounds. Typically the Raman-active
organic compound has a molecular weight less than about 300
Daltons.
[0111] Additional, non-limiting examples of Raman-active organic
compounds useful in COINs 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, aminoacridine, and the like.
[0112] In certain embodiments, the Raman-active compound is
adenine, adenine, 4-amino-pyrazolo(3,4-d)pyrimidine,
2-fluoroadenine, N6-benzolyadenine, kinetin,
dimethyl-allyl-amino-adenine, zeatin, bromo-adenine, 8-aza-adenine,
8-azaguanine, 6-mercaptopurine,
4-amino-6-mercaptopyrazolo(3,4-d)pyrimidine, 8-mercaptoadenine, or
9-amino-acridine 4-amino-pyrazolo(3,4-d)pyrimidine, or
2-fluoroadenine. In one embodiment, the Raman-active compound is
adenine.
[0113] When "fluorescent compounds" are incorporated into COINs,
the fluorescent compounds can include, but are not limited to,
dyes, intrinsically fluorescent proteins, lanthanide phosphors, and
the like. Dyes useful for incorporation into COINs include, for
example, rhodamine and derivatives, such as Texas Red, ROX
(6-carboxy-X-rhodamine), rhodamine-NHS, and TAMRA
(5/6-carboxytetramethyl rhodamine NHS); fluorescein and
derivatives, such as 5-bromomethyl fluorescein and FAM
(5'-carboxyfluorescein NHS), Lucifer Yellow, IAEDANS, 7-Me.sub.2,
N-coumarin-4-acetate, 7-OH-4-CH.sub.3-coumarin-3-acetate,
7-NH.sub.2-4CH.sub.3-coumarin-3-acetate (AMCA), monobromobimane,
pyrene trisulfonates, such as Cascade Blue, and
monobromotrimethyl-ammoniobimane.
[0114] Multiplex testing of a complex sample would generally be
based on a coding system that possesses identifiers for a large
number of reactants in the sample. The primary variable that
determines the achievable numbers of identifiers in currently known
coding systems is, however, the physical dimension. Tagging
techniques, based on surface-enhanced Raman scattering (SERS) of
fluorescent dyes, could be used in the embodiments of this
invention for developing chemical structure-based coding systems.
The organic compound-assisted metal fusion (OCAM) method could be
used to produce composite organic-inorganic nanoparticles (COIN)
that are highly effective in generating SERS signals allows
synthesis of COIN labels from a wide range of organic compounds to
produce sufficient distinguishable COIN Raman signatures to assay
any complex biological sample. Thus COIN particles may be used as a
coding system for multiplex and amplification-free detection of
bioanalytes at near single molecule levels.
[0115] COIN particles generate intrinsic SERS signal without
additional reagents. Using the OCAMF-based COIN synthesis
chemistry, it is possible to generate a large number of different
COIN signatures by mixing a limited number of Raman labels for use
in multiplex assays in different ratios and combinations. In a
simplified scenario, the Raman spectrum of a sample labeled with
COIN particles may be characterized by three parameters: (a) peak
position (designated as L), which depends on the chemical structure
of Raman labels used and the umber of available labels, (b) peak
number (designated as Al), which depends on the number of labels
used together in a single COIN, and (c) peak height (designated as
i), which depends on the ranges of relative peak intensity.
[0116] The total number of possible distinguishable Raman
signatures (designated as 7) may be calculated from the following
equation:
T = k = 1 M L ! ( L - k ) ! k ! P ( i , k ) ##EQU00001##
where P(i, k)=i.sup.k-i+1, being the intensity multiplier which
represents the number of distinct Raman spectra that may be
generated by combining k (k=1 to M) labels for a given i value. The
multiple organic compounds may be mixed in various combinations,
numbers and ratios to make the multiple distinguishable Raman
signatures. It has been shown that spectral signatures having
closely positioned peaks (15 cm.sup.-1) may be resolved visually.
Theoretically, over a million of Raman signatures may be made
within the Raman shift range of 500-2000 cm.sup.-1 by incorporating
multiple organic molecules into COIN as Raman labels using the
OCAMF-based COIN synthesis chemistry.
[0117] Thus, OCAMF chemistry allows incorporation of a wide range
of Raman labels into metal colloids to perform parallel synthesis
of a large number of COIN labels with distinguishable Raman
signatures in a matter of hours by mixing several organic
Raman-active compounds of different structures, mixtures, and
ratios for use in the invention methods described herein.
[0118] COINs may be used to detect the presence of a particular
target analyte, for example, a nucleic acid, oligonucleotide,
protein, enzyme, antibody or antigen. The nanoparticles may also be
used to screen bioactive agents, i.e. drug candidates, for binding
to a particular target or to detect agents like pollutants. Any
analyte for which a probe moiety, such as a peptide, protein,
oligonucleotide or aptamer, may be designed can be used in
combination with the disclosed nanoparticles.
[0119] Also, SERS-active COINs that have an antibody as binding
partner could be used to detect interaction of the Raman-active
antibody labeled constructs with antigens either in solution or on
a solid support. It will be understood that such immunoassays can
be performed using known methods such as are used, for example, in
ELISA assays, Western blotting, or protein arrays, utilizing a
SERS-active COIN having an antibody as the probe and acting as
either a primary or a secondary antibody, in place of a primary or
secondary antibody labeled with an enzyme or a radioactive
compound. In another example, a SERS-active COIN is attached to an
enzyme probe for use in detecting interaction of the enzyme with a
substrate.
[0120] Another group of exemplary methods could use the SERS-active
COINs to detect a target nucleic acid. Such a method is useful, for
example, for detection of infectious agents within a clinical
sample, detection of an amplification product derived from genomic
DNA or RNA or message RNA, or detection of a gene (cDNA) insert
within a clone. For certain methods aimed at detection of a target
polynucleotide, an oligonucleotide probe is synthesized using
methods known in the art. The oligonucleotide is then used to
functionalize a SERS-active COIN. Detection of the specific Raman
label in the SERS-active COIN identifies the nucleotide sequence of
the oligonucleotide probe, which in turn provides information
regarding the nucleotide sequence of the target polynucleotide.
[0121] A "quantum dot" is a particle of matter so small that the
addition or removal of an electron changes its properties in some
useful way. All atoms are, of course, quantum dots, but
multi-molecular combinations can have this characteristic. In
biochemistry, quantum dots are called redox groups. In
nanotechnology, they are called quantum bits or qubits. Quantum
dots typically have dimensions measured in nanometers, where one
nanometer is 10.sup.-9 meter or a millionth of a millimeter. The
fields of biology, chemistry, computer science, and electronics are
all of interest to researchers in nanotechnology. An example of the
overlapping of these disciplines is a hypothetical biochip, which
might contain a sophisticated computer and be grown in a manner
similar to the way a tree evolves from a seed. In this scenario,
the terms redox group and qubit are equally applicable; it is hard
to classify such a chip as either animate or inanimate. The quantum
dots in a biochip would each account for at least one data bit, and
possibly several.
[0122] The term "complementary" refers to the topological
compatibility or matching together of interacting surfaces of a
ligand molecule and its receptor. Thus, the receptor and its ligand
can be described as complementary, and furthermore, the contact
surface characteristics are complementary to each other.
[0123] The term "oxidation" means losing electron to oxidize. The
term "reduction" means gaining electrons to reduce. The term "redox
reaction" refers to any chemical reaction which involves oxidation
and reduction.
[0124] The term "wafer" means a semiconductor substrate. A wafer
could be fashioned into various sizes and shapes. It could be used
as a substrate for a microchip. The substrate could be overlaid or
embedded with circuitry, for example, a pad, via, an interconnect
or a scribe line. The circuitry of the wafer could also serve
several purpose, for example, as microprocessors, memory storage,
and/or communication capabilities. The circuitry can be controlled
by the microprocessor on the wafer itself or controlled by a device
external to the wafer.
[0125] A "scribe line" is typically an "inactive" area between the
active dies that provide area for separating the die (usually with
a saw). Often, metrology and alignment features populate this
area.
[0126] A "via" refers to a hole etched in the interlayer of a
dielectric which is then filled with an electrically conductive
material, preferably tungsten, to provide vertical electrical
connection between stacked up interconnect metal lines that are
capable of conducting electricity.
[0127] "Metal lines" within a die are interconnect lines. Metal
interconnect lines do not typically cross the scribe line boundary
to electrically connect two dies or, as in the some embodiments of
this invention, a multitude of die to one or more wafer pads.
[0128] The term "field effect transistor" (FET) is a family of
transistors that rely on an electric field to control the
conductivity of a "channel" in a semiconductor material. FETs, like
all transistors, can be thought of as voltage-controlled resistors.
Most FETs are made using conventional bulk semiconductor processing
techniques, using the single-crystal semiconductor wafer as the
active region, or channel.
[0129] The term "CMOS" means complementary metal oxide
semiconductor.
[0130] "Micro-Electro-Mechanical Systems (MEMS)" include the
integration of mechanical elements, sensors, actuators, and
electronics on a common silicon substrate through microfabrication
technology. MEMS often combine electrical and mechanical
functionalities on a single substrate. An example of a MEMS device
could be a small mechanical chamber where two liquids (biofluids,
drugs, chemicals etc.) are mixed and a sensor interprets the
results. MEMS could also be integrated with logic functionalities
i.e. having a CMOS circuit to perform some algorithm with the data
provided by the sensor. The CMOS circuit could then have circuit
elements that transport the results of the algorithm and the sensor
input to another device (i.e. output to further devices comprising
the overall micro-system). While the electronics are fabricated
using integrated circuit (IC) process sequences (e.g., CMOS,
Bipolar, or BICMOS processes), the micromechanical components could
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 electromechanical
devices. Microelectronic integrated circuits can be thought of as
the "brains" of a system and MEMS augments this decision-making
capability with "eyes" and "arms", to allow microsystems to sense
and control the environment. Sensors gather information from the
environment through measuring mechanical, thermal, biological,
chemical, optical, and magnetic phenomena. The electronics then
process the information derived from the sensors and through some
decision making capability direct the actuators to respond by
moving, positioning, regulating, pumping, and filtering, thereby
controlling the environment for some desired outcome or purpose.
Because MEMS devices are generally manufactured using batch
fabrication techniques similar to those used for integrated
circuits, unprecedented levels of functionality, reliability, and
sophistication can be placed on a small silicon chip at a
relatively low cost.
[0131] One of the mechanical processes typically performed by MEMS
is transporting small amounts of fluids through channels, which are
called "microfluidic channels." These channels are frequently
embedded in a covering layer (hereafter called: embedding layer).
One example of a microfluidic channel used in MEMS is in an
electrokinetic pump. Electrokinetic pumps use an ionic fluid and a
current imposed at one end of the channel and collected at the
other end of the channel. This current in the ionic fluid impels
the ionic fluid towards the collection pad of the electrokinetic
pump.
[0132] The term "waveguide" refers to a device that controls the
propagation of an electromagnetic wave so that the wave is forced
to follow a path defined by the physical structure of the guide.
Generally speaking, the electric and magnetic fields of an
electromagnetic wave have a number of possible arrangements when
the wave is traveling through a waveguide. Each of these
arrangements is known as a mode of propagation. Optical waveguides
are used at optical frequencies. An "optical waveguide" is any
structure having the ability to guide optical energy. Optical
waveguides may be (a) thin-film deposits used in integrated optical
circuits (IOCs) or (b) optical fibers.
[0133] "Microprocessor" is a processor on an integrated circuit
(IC) chip. The processor may be one or more processor on one or
more IC chip. The chip is typically a silicon chip with thousands
of electronic components that serves as a central processing unit
(CPU) of a computer or a computing device.
[0134] One embodiment of the invention relates to a complex
comprising a first binding partner, a first reporter associated
with the first binding partner, an analyte, a second binding
partner, and a second reporter associated with the second binding
partner. Preferably, the complex comprises the first reporter, the
first binding partner, the analyte, the second binding partner, and
the second reporter in this order. Preferably, the first reporter
or the second reporter comprises a nanomaterial having more than
50%, 60%, 70%, 80% or 90% by weight of inorganic content. The
analyte and the binding partners preferably comprise a biomolecule
such as antibody, a protein, a carbohydrate, a lipid, an antigen, a
receptor, or a ligand, or a macromolecule. The receptor preferably
comprises a quantum dot, a Raman tag, a fluorescent tag, a
composite-organic-inorganic nanoparticle (COIN) or a magnetic COIN.
The magnetic COIN could comprise a metal particle with at least one
Raman active organic compound adsorbed on the metal particle. In
one variation, the first reporter and the second reporter are
associated to the first binding partner and the second binding
partner, respectively, through a molecule in between the first or
second reporter and the first or second binding partner.
[0135] Another embodiment of the invention relates to a device for
analysis comprising a microfluidic channel (MFC) comprising a
plurality of first binding partners immobilized on spots in the
MFC, wherein the MFC comprises an inorganic support and an
optically transparent cover and further comprises a plurality of
probes (i.e., binding partners) optionally with COINs or magnetic
COINs immobilized on spots in the MFC. The binding partner
immobilized on the spot could be attached to an analyte, which in
turn could be attached another binding partner attached to the
analyte and a reporter such as a COIN or a magnetic-COIN.
[0136] The device for fluidic separation can be made by using soft
lithography method with poly-dimethyl siloxane. With these
techniques it is possible to generate patterns with critical
dimensions as small as 30 nm. These techniques use transparent,
elastomeric polydimethylsiloxane (PDMS) "stamps" with patterned
relief on the surface to generate features. The stamps can be
prepared by casting prepolymers against masters patterned by
conventional lithographic techniques, as well as against other
masters of interest. Several different techniques are known
collectively as soft lithography. They are as described below:
[0137] Near-Field Phase Shift Lithography. A transparent PDMS phase
mask with relief on its surface is placed in conformal contact with
a layer of photoresist. Light passing through the stamp is
modulated in the near-field. If the relief on the surface of the
stamp shifts the phase of light by an odd multiple of (, a node in
the intensity is produced. Features with dimensions between 40 and
100 nm are produced in photoresist at each phase edge.
[0138] Replica Molding. A PDMS stamp is cast against a
conventionally patterned master. Polyurethane is then molded
against the secondary PDMS master. In this way, multiple copies can
be made without damaging the original master. The technique can
replicate features as small as 30 nm.
[0139] Micromolding in Capillaries (MIMIC). Continuous channels are
formed when a PDMS stamp is brought into conformal contact with a
solid substrate. Capillary action fills the channels with a polymer
precursor. The polymer is cured and the stamp is removed. MIMIC is
able to generate features down to 1 .mu.m in size.
[0140] Microtransfer Molding ((TM). A PDMS stamp is filled with a
prepolymer or ceramic precursor and placed on a substrate. The
material is cured and the stamp is removed. The technique generates
features as small as 250 nm and is able to generate multilayer
systems.
[0141] Solvent-assisted Microcontact Molding (SAMIM). A small
amount of solvent is spread on a patterned PDMS stamp and the stamp
is placed on a polymer, such as photoresist. The solvent swells the
polymer and causes it to expand to fill the surface relief of the
stamp. Features as small as 60 nm have been produced.
[0142] Microcontact Printing ((CP). An "ink" of alkanethiols is
spread on a patterned PDMS stamp. The stamp is then brought into
contact with the substrate, which can range from coinage metals to
oxide layers. The thiol ink is transferred to the substrate where
it forms a self-assembled monolayer that can act as a resist
against etching. Features as small as 300 nm have been made in this
way.
[0143] Techniques used in other groups include micromachining of
silicon for microelectricalmechanical systems (MEMS), and embossing
of thermoplastic with patterned quartz. Unlike conventional
lithography, these techniques are able to generate features on both
curved and reflective substrates and rapidly pattern large areas. A
variety of materials could be patterned using the above techniques,
including metals and polymers. The methods complement and extend
existing nanolithographic techniques and provide new routes to
high-quality patterns and structures with feature sizes of about 30
nm.
[0144] Applications of soft lithography in the near future could
include simple optical devices, such as polarizers, filters, wire
grids, and surface acoustic wave (SAW) devices. Longer term goals
include working toward optical data storage systems, flat panel
displays, and quantum devices. Soft lithographic techniques are
currently not competitive with conventional photolithography for
multilayer fabrication where there are critical requirements for
pattern regularity.
[0145] Standard lithography on silicone wafer or silica glass could
also be used to fabricate the devices of the embodiments of this
invention. Chambers or channels can be make from the devices,
fluidic flow can be controlled by pressure gradient, electrical
field gradient, gravity, heat gradient etc. The binding complexes
can also be separated by planar device with a single a plurality of
chambers, where the surfaces are modified with polymers
(polyethylene glycol (PEG)-dramatized compounds) that can minimize
non-specific binding. The substrate (solid support) can be
inorganic material (e.g., glass, ceramic) or metal (e.g.,
aluminum), biomolecules, protein, antibody, nucleic acid can be
coated on the surface for specific analyte binding.
[0146] The above embodiments could be practiced by the following
methods.
[0147] Two-reporter method as shown in FIG. 1A: The complexes are
formed when an analyte is bound to two affinity binding partners
each of which is associated with a reporter. The reporter can be
COIN (composite-organic-inorganic nanoparticles), quantum dots and
other Raman or fluorescent tags, but COINs will be particularly
useful for this purpose. Since many different types of COINs can be
made and can be used to conjugate specific antibodies, a large
collection of binding complexes can be formed (reporter1-binding
partner 1-analyte-binding partner 2-reportner 2). Preferably,
reporter I is different from reporter 2, which would allow the
binding complex formed to be distinguished over a molecule where
reporter 1 or reporter 2 is not bound to the analyte. Unbound
reporter-binding partners can be separated by size or magnetic
property if one of the reporters is paramagnetic. Binding partners
can be antibodies, antigens, receptors or ligands. Sub-set of
binding complexes is formed by controlling the concentration of the
complexes in solution. When multiple binding complexes move through
a microfluidic channel, their optical features can be recorded and
subsequently analyzed. Positive detection of an analyte is
indicated by the detection of two reporters simultaneously as
predicted by binding partner specificity.
[0148] Spatial position and reporter method as shown in FIG. 1B: A
device surface (array) containing multiple binding areas, each of
the areas contains a mixture of binding partners (antibodies). The
binding areas can be grouped into different compartments or spots,
wherein one or more of the compartments or spots could have an
organic polymer layer containing a polymeric brush or a linker
molecule. Each of the binding area is optionally surrounded by
non-binding surface and contains multiple binding partners
(different antibodies are mixed and immobilized). The binding
surfaces are fabricated to minimize non-specific binding of
analytes or reporters or binding partners. A biological sample can
be applied individually to a binding surface (area) or sub-set of
the binding surfaces of the array. Thus multiple reporters can be
located in a single binding area when sandwich binding complexes
are formed.
[0149] Another embodiment of the invention relates to a device for
data collection comprising a beam emitter, a MFC, a spectrometer,
particularly a Raman spectrometer, and a detector. The beam emitter
is to emit a beam comprising laser. The device could further
comprise an optical waveguide between the beam emitter and the MFC.
As shown in FIG. 1A, for example, the MFC could comprise a
detection site to illuminate a sample comprising a reporter
attached to an analyte by the beam. Preferably, the spectrometer is
a waveguide based spectrometer to create a phase shift in a Raman
signal emitted by the sample. The detector is to detect a
characteristic of the Raman signal emitted by the sample. The
device could further comprise a microprocessor comprising software
or a hardware to identify the Raman signal. The MFC could further
comprise a plurality of first binding partners immobilized on spots
in the MFC. The MFC could further comprise a plurality of COINs or
magnetic COINs immobilized on spots. The MFC could further comprise
a microcoil or a Micro-Electro-Mechanical System (MEMS) device.
[0150] The devices of FIGS. 3 and 5 could be fabricated in the
following way in one of the embodiments of the devices. The Fourier
transform spectrometer can be fabricated using common
semi-conductor fabrication techniques. As an example a silicon
wafer could be used as the starting material. An oxide layer could
be grown to be used as the bottom cladding of the waveguide. SiON
could then be deposited to be used as the waveguide core. This
could then have waveguides patterned onto it using wet or dry
chemical etching. Control of the index of the MZI arms could be
done using the thermo-optic effect by preferentially heating the
MZI arms with heaters deposited onto the waveguide. Filters could
be integrated onto the waveguide by etching the upper surface or
sidewall of the waveguide, or by varying the refractive-index of
the waveguide as a function of position. An integrated
photo-detector could be formed by fabricated a silicon PIN diode on
the same substrate as the spectrometer in a way similar to
obtaining planar optical devices that is known to persons of
ordinary skill in this art.
[0151] The method steps and device for the above embodiment of the
invention are shown in FIGS. 2 and 3. Even though FIGS. 2 and 3
state "Raman-on-chip," the embodiments of FIGS. 2 and 3 are equally
applicable to other analyzers. In particular, the steps are as
follows:
[0152] 1. Mix a solution containing labeled probe molecules and
labeled capture molecules with the sample to be analyzed. Both the
probe and capture molecules have a COIN label. The sample could be
a polymer, a nanomaterial, a carbon nanotube, a nucleotide, or a
biomaterial such a peptide, a protein, a ligand, a receptor, a
sequence, DNA, RNA, etc.
[0153] 2. Form a complex, which might involve hybridization, of the
COIN labeled capture molecule, a target molecule of the sample and
the COIN labeled probe molecule.
[0154] 3. Detect the complex by simultaneous detection of two COIN
labels attached to the complex, which contains the first and second
COIN labels of the probe molecule and the target molecule,
respectively.
[0155] The detection methodology is shown schematically in FIG. 3.
The laser light is focused into an optical waveguide, and is
delivered to the sample which flows through a micro-fluid channel.
The sample scatters light and emits radiation, including Raman
emission. The radiation from the sample would be collected by an
optical waveguide based spectrometer.
[0156] Another embodiment of the invention relates to a device for
data collection comprising a beam emitter, a chamber to hold a
microarray, a spectrometer and a detector. The beam emitter is to
emit a beam comprising laser. The device could further comprise an
optical waveguide between the beam emitter and the chamber. The
chamber could comprise an optical switch to detect the beam
transmitted through the microarray. The spectrometer is preferably
a waveguide based spectrometer to create a phase shift in a Raman
signal emitted by the sample. The detector is to detect a
characteristic of the Raman signal emitted by the sample. The
device could further comprise a microprocessor comprising software
or a hardware to identify the Raman signal. The device could
further comprise a microarray, wherein the microarray comprises a
plurality of first binding partners immobilized on spots on the
microarray. The microarray could further comprise a plurality of
COINs or magnetic COINs immobilized on spots.
[0157] The method steps and device of this embodiment of the
invention are shown in FIGS. 4 and 5. Even though FIGS. 4 and 5
state "Raman-on-chip," the embodiments of FIGS. 4 and 5 are equally
applicable to other analyzers. In particular, the steps are as
follows:
[0158] 1. Introduce the sample and the COIN labeled probe molecules
on a substrate of a microarray having spots containing capture
molecules (which may or may not be labeled). The sample could be a
polymer, a nanomaterial, a carbon nanotube, a nucleotide, or a
biomaterial such a peptide, a protein, a ligand, a receptor, a
sequence, DNA, RNA, etc.
[0159] 2. Form a complex, which might involve hybridization, of the
capture molecule, a target molecule of the sample and the COIN
labeled probe molecule.
[0160] 3. Detect the complex by detection of one COIN label (or
simultaneous detection of two COIN labels if the capture molecule
is a COIN labeled capture molecule) attached to the complex.
[0161] The detection methodology to detect the sample on the
microarray is shown schematically in FIG. 5. The laser light
through an optical waveguide is focused on the microarray and the
complex on microarray could be illuminated from either above or
below the microarray. The complex emits its own signature spectrum
comprising a Raman signal. The signature spectrum is collected by
an optical waveguide based spectrometer.
[0162] In the embodiments of the invention such as the two
embodiments described above with reference to FIGS. 2-5, the sample
receives the laser light, and emits a unique spectrum of light
specific to the COIN. A miniaturized spectrometer and detector
could be placed to analyze the spectrum of the emitted light by a
miniaturized spectrometer and detector system, for example.
[0163] The above embodiments relating to FIGS. 1-5 could further
include a sample collection device for collecting the sample that
has to be analyzed by the analyzer of the embodiments of the
invention. The sample collection device could include suction and
sample concentration devices. For example, a solid, liquid or
gaseous sample could be sucked into a sample collection device that
produces a known background signal. Then, the sample could be
concentrated within the sample collection device. For example, a
gas could be cooled to create condensate in the sample collection
device. By concentrating the sample in the sample collection
device, it could reduce the analysis time, particularly for a
gaseous sample.
[0164] Another embodiment of the invention relates to a device for
analyte concentration comprising a first chamber comprising an
analyte, a magnetic COIN, and a non-analyte, and a second chamber
to hold a concentrate comprising the analyte bound to the magnetic
COIN. Preferably, the first chamber is exposed to a first magnetic
field and the second chamber is exposed to a second magnetic field,
wherein the second magnetic field is different than the first
magnetic field. The first chamber could further comprise a magnet
to produce the first magnetic field and the second chamber could
further comprise a magnet to produce the second magnetic field. In
one variation of the device, the first chamber could comprise an
instrument to drain a liquid comprising the non-analyte and the
second magnetic field is the magnetic field of the earth.
[0165] In FIG. 6, the sample and the binding partner-conjugated
magnetic COINs are mixed in a reaction chambers (centrifugation
tubes), after the binding, the binding complexes are cleaned by
repeating washing with buffers (applying magnetic field to
concentrate and releasing the field to re-suspend the binding
complexes). The cleaned binding complexes are then transferred to a
binding partner array chip (antibody chip, DNA chip, microfluidic
chip etc., for example, see devices of FIG. 1).
[0166] The above embodiment could be practiced by the following
method and device of FIG. 6:
[0167] Magnetic COIN as reporter and analyte carrier as shown in
FIG. 6: The device of FIG. 6 allows concentration in magnetic field
of certain analytes that are attached to magnetic COINs while
excluding non-analytes and other analytes that are not attached to
the magnetic COINs. Magnetic COINs can be used as reporters as well
as analyte detector and carriers. Analytes in solution can be
captured by magnetic COINs that are conjugated with an affinity
probe, and concentrated in magnetic field. The concentrated sample
with most non-analyte material removed can be placed on an affinity
binding surface (with mixed affinity binding partners (Abs)), thus
different types sandwich binding complexes can be formed with
magnetic COINs as the reporters. The COIN signals can be detected
by a Raman spectroscope. Each of the Raman spectra can cover
multiple COINs; multiple spectra are needed to collect sufficient
data for analysis.
[0168] Yet another embodiment of the invention relates to an
analyte separation method comprising capturing a first analyte in a
solution by a magnetic COIN attached to a first binding partner to
form a magnetic COIN-containing complex comprising the first
analyte and the magnetic COIN attached to the first binding
partner, capturing a second analyte in the solution by a
non-magnetic COIN attached to a second binding partner to form a
non-magnetic COIN-containing complex comprising the second analyte
and the non-magnetic COIN attached to the second binding partner,
and grouping the magnetic COIN-containing complex separate from the
non-magnetic COIN-containing complex on a surface of a microcoil by
generating a local magnetic field on the surface of the microcoil.
The method could further comprise controlling the current passing
though the microcoil to control a strength of the local magnetic
field on the surface of the microcoil. The method could further
comprise releasing the magnetic COIN-containing complex from the
surface of the microcoil.
[0169] The method of manufacturing a microcoil of an embodiment of
the device shown in FIG. 7 could be as follows. Briefly, dielectric
material, which could be silicon oxide, silicon nitride, or a
polymer material, such as benzocyclobutene (BCB) dielectric layer,
could be spun on a silicon wafer and cured. This BCB layer defines
the separation distance between the coils and the substrate. By
using a sputter/lift-off process, the first Cu layer of 3 .mu.m
could be deposited and patterned. A second BCB layer of 5 .mu.m
could be spun as an inter-layer-dielectric, which also plannerized
the surface. Via holes could be opened and a second Cu layer of 3
.mu.m deposited and patterned similarly. The coils could be
passivated by using a BCB layer on the top, which could be
patterned to expose the probing pads. The width of the Cu trace
could range from 12 to 30 .mu.m, the spacing was 12 .mu.m.
[0170] The above embodiment could be practiced by the following
method.
[0171] Microcoil for concentrating magnetic-COIN sandwich complexes
shown in FIG. 7: Similar to FIG. 6, magnetic beads conjugated with
a 1.sup.st set of affinity binding partners (Abs) can be used to
capture analytes in solution; non-magnetic COINs conjugated with a
2.sup.nd set of affinity binding partners are also in contact with
the sample. Sandwich binding complexes are formed, which can be
sub-grouped on to microcoil surfaces when electricity is applied to
generate local magnetic fields. The microcoil magnetic device can
be fabricated using lithography techniques. An electronic control
board is used to control the current passing the microcoils and
thus control the magnetic fields. The binding complexes can be
released and new sample can be introduced.
[0172] Yet another embodiment of the invention relates to a
computer implemented system comprising a first algorithm to
simulate spectral features produced by a hypothetical composition
comprising a plurality of reporters mixed in different ratios, a
second algorithm to compare the simulated spectral features with
experimentally obtained spectral features produced by an actual
composition comprising a plurality of reporters in different
ratios, and a third algorithm to determine a goodness-of-fit
between the simulated spectral features and the experimentally
obtained spectral features and to iteratively adjust the simulated
spectral features by adjusting the hypothetical composition to
maximize the goodness-of-fit to meet a pre-set statistical
criteria. It is possible that the first, second and third
algorithms are bundled into one or more software programs or one or
more hardware components. Preferably, the plurality of reporters in
the actual composition is associated with a plurality of analytes
of a biological sample. In one variation, the goodness-of-fit is
maximized by minimizing the difference between the between the
simulated spectral features and the experimentally obtained
spectral features. Preferably, the difference between the between
the simulated spectral features and the experimentally obtained
spectral features is determined by a genetic algorithm that
qualitatively optimizes the genetic algorithm, by a neural network
that optimizes a set of selected parameters for a selected neural
patterns or circuits, or by a principal component analysis that
statistically decomposes components with maximum likelihood.
[0173] For the device of FIG. 8, any device with low Raman
background can be used, for example, solution on an aluminum
surface, enclosed glass chambers, or any device described above for
Raman measurement.
[0174] The above embodiment could be practiced by the following
method and device of FIG. 8:
[0175] Machine learning and analyte quantification: FIG. 8 shows a
general scheme of machine learning (software training). Machine
learning is a part of multiplex data analysis. When multiple
reporters (COINs or quantum dots) are to be used in an assay,
spectra of each of these reporters at various concentrations are
recorded. In a simplified data analysis a spectral peak (with a
unique wavelength or wavenumber) and associated combined parameters
such as ratios or polynomials can be used to quickly identify a
single reporter. Computer simulation can be used to predict the
complex spectral features when a given set of reporters are mixed
in a different ratios. The predications will be statistically
verified by comparing the computations with actual experiments
using pre-determined/known compositions of reporters. The software
algorithm and associated parameters are iteratively adjusted to
maximize the goodness-of-fit until the resulting converged
algorithm and the associated parameters meet pre-set statistical
criteria. For example, a variety of algorithms and associated
parameters can be used by statistically adjusting and minimizing
the energy of goodness-of-fit landscapes such as genetic algorithm
(focusing on qualitatively optimizing the algorithm), neural
network (preset or empirical algorithm focusing on optimizing a set
of selected parameters for a selected neural patterns/circuits),
and principal component analysis (statistical decompositions of
components with maximum likelihood) could be used. FFT
deconvolution also could be used to create a set of initial
conditions for the said methods if necessary.
[0176] Analyte quantification: specific signal activity of a
reporter needs to be known before being used for an assay (single
intensity per unit amount of reporter particles or molecules). For
surface binding, one reporter can be considered to represent one
analyte. In solution binding, the relation between reporter and
analyte needs to be determined experimentally. When a spectrum is
collected, trained software as described previously is used to
deconvolute the spectrum to determine the reporter concentration
quantitatively. Based on reporters' specific activity and sample
volume (surface area), multiple analytes can be quantified in a
single assay.
[0177] FIG. 9 shows the result of multiplexing seven COIN Tags.
From bottom to the top, each plot in different color represents
multiplexed COIN mixtures, starting from two COINs successively up
to seven COINs in a same solution. In brief, each addition of new
COIN introduces uniquely identifiable peaks on top of the existing
peaks. General "finger printing" method such as the machine
learning software with statistical software described above could
be effectively used to identify and decompose the different
signature components superimposed in the spectra of multiplex
assay.
[0178] The embodiments of the invention could use silicon
technology to fabricate interconnects for silicon chips to enable
on-die synthesis of polymers such as DNA, peptides, and
DNA-functionalized complementary nucleotide. Optionally, the
embodiments of the invention could use wafer processing cluster
tools (process instruments) for synthesis. Typically, in volume
silicon processing, a manufacturing line has a cluster of
instruments (several identical instruments). Each can support a
process step or multiple process steps. By the embodiments of the
invention, polymer synthesis can be treated as another process step
in a device manufacturing line. A cluster of instruments can be
configured within a facility to perform wafer level synthesis for
efficient high volume manufacturing.
[0179] The devices of the embodiments of the invention may be
formed by any suitable means of manufacture, including
semiconductor manufacturing methods, microforming processes,
molding methods, material deposition methods, etc., or any suitable
combination of such methods. In certain embodiments one or more of
the electrodes and/or the pad may be formed via semiconductor
manufacturing methods on a semiconductor substrate. Thin film
inorganic coatings may be selectively deposited on portions of the
substrate and/or pad surface. Examples of suitable deposition
techniques include vacuum sputtering, electron beam deposition,
solution deposition, and chemical vapor deposition. The inorganic
coatings may perform a variety of functions. For example, the
coatings may be used to increase the hydrophilicity of a surface or
to improve high temperature properties. Conductive coatings may be
used to form electrodes. Coatings may be used to provide a physical
barrier on the surface, e.g. to retain fluid at specific sites on
the surface. The devices used in the present invention may be
fabricated according to procedures well-known in the arts of
microarray and semiconductor device manufacturing.
[0180] In some embodiments the probes may be selected from
biomolecules, such as polypeptides, polynucleotides, glycoproteins,
polysaccharides, hormones, growth factors, peptidoglycans, or the
like. The probe could be natural nucleotides such as
ribonucleotides and deoxyribonucleotides and their derivatives
although unnatural nucleotide mimetics such as 2'-modified
nucleosides, peptide nucleic acids and oligomeric nucleoside
phosphonates are also used. In embodiments employing
oligonucleotide probes, the probes may be synthesized, in situ, on
the surface of the pad in either the 3' to 5' or 5' to 3' direction
using the 3'-.beta.-cyanoethyl-phosphoramidites or
5'-.beta.-cyanoethyl-phosphoramidites and related chemistries known
in the art. In situ synthesis of the oligonucleotides may also be
performed in the 5' to 3' direction using nucleotide coupling
chemistries that utilize 3'-photoremovable protecting groups.
Alternatively, the oligonucleotide probes may be synthesized on the
standard controlled pore glass (CPG) in the 3' to 5' direction
using 3'-p-cyanoethyl-phosphoramidites and related chemistries and
incorporating a primary amine or thiol functional group onto the 5'
terminus of the oligonucleotide. The oligonucleotides may then be
covalently attached to the pad surface via their 5' termini using
thiol or amine-dependent coupling chemistries known in the art. The
density of the probes on the surface can range from about 1,000 to
200,000 probe molecules per square micron. The probe density can be
controlled by adjusting the density of the reactive groups on the
surface of the pad for either the in situ synthesis or
post-synthesis deposition methods. Other suitable means for
synthesis of probe as are known in the art may be employed.
[0181] The oligonucleotide probes include, but are not limited to,
the four natural deoxyribonucleotides; deoxythymidylic acid,
deoxycytidylic acid, deoxyadenylic acid and deoxyguanylic acid. The
probes can also be ribonucleotides, uridylic acid, cytidylic acid,
adenylic acid, and guanylic acid. Modified nucleosides may also be
incorporated into the oligonucleotide probes. These include but are
not limited to; 2'-deoxy-5-methylcytidine,
2'-deoxy-5-fluorocytidine, 2'-deoxy-5-iodocytidine,
2'-deoxy-5-fluorouridine, 2'-deoxy-5-iodo-uridine,
2'-O-methyl-5-fluorouridine, 2'-deoxy-5-iodouridine,
2'-deoxy-5(1-propynyl)uridine, 2'-O-methyl-5(1-propynyl)uridine,
2-thiothymidine, 4-thiothymidine, 2'-deoxy-5(1-propynyl)cytidine,
2'-O-methyl-5(1-propynyl)cytidine, 2'-O-methyladenosine,
2'-deoxy-2,6-diaminopurine, 2'-O-methyl-2,6-diaminopurine,
2'-deoxy-7-deazadenosine, 2'-deoxy-6methyladenosine,
2'-deoxy-8-oxoadenosine, 2'-O-methylguanosine,
2'-deoxy-7-deazaguanosine, 2'-deoxy-8-oxoguanosine, 2'-deoxyinosine
or the like.
[0182] The polynucleotide probes can vary in length from a range of
about 5 to about 100 nucleotides, such as about 8 to about 80
nucleotides, such as about 10 to about 60 nucleotides, and such as
about 15 to about 50 nucleotides. Longer polynucleotide probes are
typically employed for applications where the sample contains a
high sequence-complexity target mixture. Shorter polynucleotide
probes are typically employed in applications where single
nucleotide discrimination, such as mutation detection, is
desired.
[0183] The target molecule could be a nucleic acid such as genomic
DNA, genomic RNA, messenger RNA, ribosomal RNA or transfer RNA, an
oligonucleotide or polynucleotide of DNA or RNA generated by
enzymatic process such as PCR or reverse transcription, or any
synthetic DNA, RNA, or any other desired nucleic acid or any
combination thereof. The target molecule may be double stranded or
single stranded. It is preferred that the target molecule be single
stranded in order to increase the efficiency of its interaction
with the probe sequences. The target molecule could contain
nanomaterials such a carbon nanotube, wherein the nanomaterial such
as the carbon nanotube could be functionalized at its ends to
molecules containing nucleic acid.
[0184] The architecture of the array probes may be either generic
or specific with regard to the complementary target sequences that
it may hybridize with. For example, an array of all possible 7-mer
probe sequences could be used to interrogate targets having any
sequence. The advantage of such an array is that it is not
application specific and therefore generic. Alternatively, the
probe array may contain polynucleotide sequences that are
complementary to a specific target sequence or set of target
sequences and individual or multiple mutations thereof. Such an
array is useful in the diagnosis of specific disorders, which are
characterized by the presence of a particular nucleic acid
sequence. For example, the target sequence may be that of a
particular exogenous disease causing agent, e.g. human
immunodeficiency virus, or alternatively the target sequence may be
that portion of the human genome which is known to be mutated in
instances of a particular disorder, e.g., sickle cell anemia or
cystic fibrosis, or to a portion of a genome known to be associated
with certain phenotypes, e.g., resistance to certain drugs,
over-reactivity to certain drugs, or even susceptibility to
side-effects of certain drugs.
[0185] In one embodiment of the present invention, polymers on a
plurality of dies on a wafer substrate are functionalized on the
electrodes as follows. First, a terminal end of a monomer,
nucleotide, or linker molecule (i.e., a molecule which "links," for
example, a monomer or nucleotide to a substrate) is provided with
at least one reactive functional group, which is protected with a
protecting group removable by an electrochemically generated
reagent. The protecting group(s) is exposed to reagents
electrochemically generated at the electrode and removed from the
monomer, nucleotide or linker molecule in a first selected region
to expose a reactive functional group. The substrate is then
contacted with the monomer or a pre-formed molecule (called the
first molecule) such that the surface bonds with the exposed
functional group(s) of the monomer or the pre-formed molecule. The
first molecule may also bear at least one protected chemical
functional group removable by an electrochemically generated
reagent. The monomer or pre-formed molecule can then be deprotected
in the same manner to yield a second reactive chemical functional
group. A different monomer or pre-formed molecule (called the
second molecule), which may also bear at least one protecting group
removable by an electrochemically generated reagent, is
subsequently brought in the vicinity of the substrate to bond with
the second exposed functional group of the first molecule. Any
unreacted functional group can optionally be capped at any point
during the synthesis process. The deprotection and bonding steps
can be repeated sequentially at the plurality of the predefined
regions on the substrate until polymers or oligonucleotides of a
desired sequence and length are obtained.
[0186] In another embodiment of the present invention, polymers on
a plurality of dies on a wafer substrate are functionalized on the
electrodes as follows. First, a substrate of a wafer having one or
more molecules bearing at least one protected chemical functional
group bonded on an array of electrodes on a plurality of dies is
obtained. The array of electrodes is contacted with a buffering or
scavenging solution. Following application of an electric potential
to selected electrodes in the array of electrodes sufficient to
generate electrochemical reagents capable of deprotecting the
protected chemical functional groups, molecules on the array of
electrodes are deprotected to expose reactive functional groups,
thereby preparing them for bonding. A monomer solution or a
pre-formed molecule (called the first molecule), such as proteins,
nucleic acids, polysaccharides, and porphyrins, is then contacted
with the substrate surface of the wafer and the monomers or
pre-formed molecules are bonded in parallel with a plurality of
deprotected chemical functional groups on a plurality of dies on
the wafer. Another sufficient potential is subsequently applied to
select electrodes in the array to deprotect at least one chemical
functional group on the bonded molecule or another of the molecules
bearing at least one protected chemical functional group on a
plurality of dies on the wafer. A different monomer or pre-formed
molecule (called the second molecule) having at least one protected
chemical functional group is subsequently attached to a deprotected
chemical functional group of the bonded molecule or the other
deprotected molecule located at a plurality of dies of the wafer.
The selective deprotection and bonding steps can be repeated
sequentially until polymers or oligonucleotides of a desired
sequence and length are obtained. The selective deprotection step
is repeated by applying another potential sufficient to effect
deprotection of a chemical functional group on a bonded protected
monomer or a bonded protected molecule. The subsequent bonding of
an additional monomer or pre-formed molecule to the deprotected
chemical functional group(s) until at least two separate polymers
or oligonucleotides of desired length are formed on the
substrate.
[0187] Some of the advantages of the embodiments of the invention
include: [0188] Fast data collection, e.g., in array-based assays,
one spectrum contain multiple data points; and in fluidic-based
assays, magnetic force-assisted detection concentrated the
analyte-probe complex, reducing scanning time) [0189] High
throughput (multiple tests can be performed at the same time in
multiplexed analysis). [0190] Use of small sample volume (crucial
to clinical diagnosis) [0191] Low cost (Less samples, reagents, and
labor).
[0192] This embodiment of the invention relate to generating
multiplex data and analyzing the resulting data. The embodiments of
the invention can be used to collect information from multiple
binding complexes in a single measurement (1 data integration time,
for example 0.1 second); normally a separation step is used before
any detection (for example, magnetic separation, centrifugation,
etc.). The embodiments of the invention are different from current
methods which uses beads, which are likely to be much larger than
COINs and rely on a correlation between the fluorescence of the
classification laser and the fluorescence of the reporter to detect
a single type of binding complexes, while the COIN-based assay of
some of the embodiments of the invention does not need this type of
statistical correlation method because the label/tag signatures of
COINs are directly read by Raman system.
[0193] The embodiments of the invention can be used to carry out
the electrochemical syntheses of polymers such as DNA and peptides
according to any of a variety of approaches known to person skilled
in the art. For example, any of a variety of reduction/oxidation
(redox) reactions may be employed to electrochemically control the
localization and pH of a solution on Si-based electrodes to enable
the attachment and elongation of polymers. In such methods, the
electrical current drives the oxidation of an appropriate molecule
at the anode(s) and the reduction of another molecule at the
cathode(s) to control the kinetics and stoichiometry of
acid-catalyzed organic syntheses on a Si-based circuit Such methods
can also be used to generate high pH (basic) solutions, and to
drive any other electrochemical redox reactions known to one
skilled in the art that may or may not result in pH changes (e.g.,
can also be used to generate reactive free radicals).
[0194] Another embodiment of the invention is electrochemical
detection using the array chip. Typically these methods employ
measurements of current flow across a DNA monolayer tethered to a
circuit on a silicon substrate. Current flow properties
proportionately change when the DNA monolayers are bound by an
appropriate redox molecule-tagged test DNA or untagged DNA that is
co-added with a redox-active molecule that specifically binds
double stranded DNA. Enzyme amplification methods can also be
incorporated into such assays in order to enhance the
electrochemical signal generated by binding events. Note that these
methods can also be adapted by one skilled in the art to measure
the binding between other molecular species such as between two
proteins or a protein and a small molecule.
[0195] The array chip could also be used for therapeutic materials
development, i.e., for drug development and for biomaterial
studies, as well as for biomedical research, analytical chemistry,
high throughput compound screening, and bioprocess monitoring. An
exemplary application includes applications in which various known
ligands for particular receptors can be placed on the array chip
and hybridization could be performed between the ligands and
labeled receptors.
[0196] Yet another application of the array chip of an embodiment
of this invention includes, for example, sequencing genomic DNA by
the technique of sequencing by hybridization. Non-biological
applications are also contemplated, and include the production of
organic materials with varying levels of doping for use, for
example, in semiconductor devices. Other examples of non-biological
uses include anticorrosives, antifoulants, and paints.
[0197] It is specifically contemplated that the array chip and/or
the methods of manufacturing the array chip of an embodiment of the
invention could be used for developing new materials, particularly
nanomaterials for many purposes including, but not limited to
corrosion resistance, battery energy storage, electroplating, low
voltage phosphorescence, bone graft compatibility, resisting
fouling by marine organisms, superconductivity, epitaxial lattice
matching, or chemical catalysis. Materials for these or other
utilities may be formed proximate to one or a plurality of the
electrodes in parallel on a plurality of dies of a silicon wafer,
for example. Alternatively, materials may be formed by modifying
the surface of one or a plurality of electrodes on a plurality of
dies by generating reagents electrochemically.
[0198] It is further contemplated that an array chip of the
embodiments of the invention could be used to develop screening
methods for testing materials. That is, reagents electrochemically
generated by an electrode on a die could be used to test the
physical and chemical properties of materials proximate to the
electrode. For example, the array chip could be used for testing
corrosion resistance, electroplating efficiency, chemical kinetics,
superconductivity, electro-chemiluminescence and catalyst
lifetimes.
[0199] The advantageous characteristics of some of the embodiments
of the invention are illustrated in the examples, which are
intended to be merely exemplary of the invention.
[0200] The array chips of the embodiments of the invention are
preferably silicon bio-chips built by using silicon process
technology and SRAM like architecture with circuitries including
electrode arrays, decoders, serial-peripheral interface, on chip
amplification, for example.
[0201] The embodiments of this invention have several practical
uses. For example, one embodiment of the invention allows molecules
and nanomaterials detection/analysis based on the electrical
readout of specific binding events (target to functionalized
electrodes with probes) using CMOS-based devices. Another
embodiment of the invention has potential applications for
nanomaterials study (for example, in-situ analysis of DNA-mediated
assembly of carbon nano-tubes on functionalized electrodes) to be
used in electronic devices (CNT transistors and interconnects) as
well as well as for detection of bio-species (DNA, protein, viruses
etc.) for molecular diagnostics, homeland security, drug discovery
and life science R&D work. Yet another embodiment of the
invention could be to use Nanomaterials, such as carbon-nanotubes,
in potential applications as interconnect materials.
Carbon-nanotubes have lower resistivity than Cu and higher
electromigration resistance (1000.times. higher than Cu). Yet
another application could be to develop DNA functionalized
electrodes with CMOS circuitry for immobilizing, detection,
addressing, electrical readout and amplification of the signal can
find potential application in silicon DNA chips. Silicon chips with
DNA functionalized electrodes could find potential application to
build nano-structures and in-situ assembly study of nanomaterials.
Silicon DNA chips could also find potential application in medical
diagnostics, homeland security devices, drug discovery and life
science R&D work.
[0202] This application discloses several numerical range
limitations that support any range within the disclosed numerical
ranges even though a precise range limitation is not stated
verbatim in the specification because the embodiments of the
invention could be practiced throughout the disclosed numerical
ranges. Finally, the entire disclosure of the patents and
publications referred in this application, if any, are hereby
incorporated herein in entirety by reference.
* * * * *