WO2008091364A2 - Nanoparticles as tags for bioanalyte detection by nuclear magnetic resonance or electron spin resonance - Google Patents

Nanoparticles as tags for bioanalyte detection by nuclear magnetic resonance or electron spin resonance Download PDF

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Publication number
WO2008091364A2
WO2008091364A2 PCT/US2007/072297 US2007072297W WO2008091364A2 WO 2008091364 A2 WO2008091364 A2 WO 2008091364A2 US 2007072297 W US2007072297 W US 2007072297W WO 2008091364 A2 WO2008091364 A2 WO 2008091364A2
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Prior art keywords
nanoparticle
target analyte
probe
substrate
nuclear magnetic
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PCT/US2007/072297
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French (fr)
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WO2008091364A3 (en
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Chang-Min Park
Shriram Ramanathan
Xing Su
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Intel Corporation
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Publication of WO2008091364A3 publication Critical patent/WO2008091364A3/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/08Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/10Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using electron paramagnetic resonance
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587Nanoparticles
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01RMEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00Arrangements or instruments for measuring magnetic variables
    • G01R33/20Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/28Details of apparatus provided for in groups G01R33/44 - G01R33/64
    • G01R33/30Sample handling arrangements, e.g. sample cells, spinning mechanisms
    • G01R33/302Miniaturized sample handling arrangements for sampling small quantities, e.g. flow-through microfluidic NMR chips
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01RMEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00Arrangements or instruments for measuring magnetic variables
    • G01R33/20Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/28Details of apparatus provided for in groups G01R33/44 - G01R33/64
    • G01R33/32Excitation or detection systems, e.g. using radio frequency signals
    • G01R33/34Constructional details, e.g. resonators, specially adapted to MR
    • G01R33/341Constructional details, e.g. resonators, specially adapted to MR comprising surface coils
    • G01R33/3415Constructional details, e.g. resonators, specially adapted to MR comprising surface coils comprising arrays of sub-coils, i.e. phased-array coils with flexible receiver channels
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01RMEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00Arrangements or instruments for measuring magnetic variables
    • G01R33/20Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/46NMR spectroscopy
    • G01R33/465NMR spectroscopy applied to biological material, e.g. in vitro testing

Definitions

  • FIG. 1 shows a cross-sectional view of components for on-chip NMR detection of biomolecules utilizing nanoparticle tags.
  • a "microchannel” is a channel, groove, or conduit having at least one dimension in the micrometer ( ⁇ m), or less than 10 " meter (mm), scale.
  • microchannels are typically straight along their length, they may contain angles and curves of different degrees along their length.
  • the microchannels typically have rectangular cross-sections, they may also have other shapes of cross-sections, such as circle.
  • the microchannels are usually suitable for fluidic communications, such as carrying through a biological liquid.
  • the microchannels are often part of an integrated device, such a microfluidic device or an integrated circuit such that liquid flowing through the microchannels are in a controlled pattern and able to be analyzed as desired.
  • bi-functional linker group refers to an organic chemical compound that has at least two chemical groups or moieties, such are, carboxyl group, amine group, thiol group, aldehyde group, epoxy group, that can be covalently modified specifically; the distance between these groups is equivalent to or greater than 5 -carbon bonds.
  • capture molecule refers to a molecule that is immobilized on a surface. The capture molecule is generally, but not necessarily, binds to a target or target molecule or cell.
  • 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 linkers may be the same molecule type as that being synthesized (i.e., nascent polymers), such as polynucleotides, oligopeptides, or oligosaccharides.
  • nascent polymers such as polynucleotides, oligopeptides, or oligosaccharides.
  • a chip includes substrates made from silicon, glass, metal, polymer, or combinations and capable of functioning as a microarray, a macroarray, a microfluidic device, a MEMS, and/or an integrated circuit.
  • a chip may be a microelectronic device made of semiconductor material and having one or more integrated circuits or one or more devices.
  • 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.
  • MEMS devices are 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.
  • MEMS devices can be further integrated with microchannels, microfluidic devices, and/or magnetic tunnel junction sensors, such that, together, they perform separation and detection function for biological cells and biomolecules.
  • DNA Synthesis DNA strands are synthesized and purified according to standard procedures using an automated synthesizer (Expedite) and HPLC (1100 HPLC series, Hewlett-Packard), respectively. All of the reagents for the phosphoramidite synthesis, including 3 - and 5_-thiol modifiers, can be purchased from Glen Research (Sterling, VA). Thiol modification is carried out manually by following known procedures. Absorption and extinction spectra are recorded by using an 8452a diode array spectrophotometer (Hewlett-Packard). The concentrations of stock DNA solutions are calculated based on the extinction coefficient of each strand.
  • the microcoil is a planar spiral coil, which is a microcoil with its windings substantially remained in an actual or imaginative plane.
  • the microcoil is wound around a center, which often is also the center, or on the same axis of the center, of the associated space for holding a liquid sample and expands in a spiral like manner.
  • the winding may take many different forms, depending on the needs of the specific device and analysis. For example, the winding may take a generally circular, square, or rectangular shape.
  • an integrated on-chip NMR device includes a magnet that generates a static magnetic field, Bo, across a sample holding space, which may contain a bio-molecular sample.
  • a planar microcoil is defined, in part, by its inner-area dimension, the number of windings, and the winding separation.
  • the inner-area of a microcoil refers to the area at the center of the microcoil where there is no winding and around which the microcoil is wound.
  • the shape of the inner-area is usually not perfectly regular, although it is often similar to a circular, rectangular, or square shape. As shown in FIG. 3, the inner-area of the microcoil has a roughly rectangular shape.
  • the dimension of the inner-area is described herein by the length of the parameter of the area.
  • the number of windings as used herein refers to the number of times the microcoil is wound around the inner-area. For example, the number of windings for the microcoil shown in FIG. 3 is three (3).
  • the winding separation refers to the distance between two adjacent windings.

Abstract

Embodiments of the invention relate to detecting biological molecules with ultra-sensitivity and convenience. The embodiments are especially directed to utilizing nanoparticles as tags and identifying the tags using a nuclear magnetic resonance device. The probes containing the nanoparticles can be used in solution or attached to a substrate.

Description

NANOPARTICLES AS TAGS FOR BIOANALYTE DETECTION BY NUCLEAR MAGNETIC RESONANCE OR ELECTRON SPIN RESONANCE
Field of Invention
[0001] The embodiments of the invention relate to methods and apparatus for detecting biological molecules with ultra-sensitivity and convenience. The embodiments are especially directed to utilizing nanoparticles as tags and identifying the tags using nuclear magnetic resonance or electron spin resonance. The invention transcends several scientific disciplines such as polymer chemistry, biochemistry, molecular biology, medicine and medical diagnostics.
Background
[0002] The ability to detect and identify trace quantities of analytes has become increasingly important in virtually every scientific discipline, ranging from part per billion analyses of pollutants in sub-surface water to analysis of cancer treatment drugs in blood serum.
[0003] With the advancement of detection technologies, there are multiple techniques that promise biological detection with single molecule sensitivity. However, many of these techniques have not yet found commercial applications. The main reasons are the complexity associated with these ultra-sensitive methods. Many require multiple steps of chemical treatments, bulky and expensive instruments, and/or extreme care in sample handling and observation. These are not ideal for practical applications that require easy and reliable measurements.
[0004] Nuclear Magnetic Resonance (NMR) and Electron Spin Resonance
(ESR) are widely used in chemical analysis and medical diagnostics. NMR is a physical phenomenon that occurs when the nuclei of certain atoms that are subject to a static magnetic field are exposed to a second oscillating magnetic field. The oscillating magnetic field, often generated by an electromagnet, is also called a perturbing or excitation magnetic field. Some nuclei experience this phenomenon, and others do not, dependent upon whether they possess a property called spin. ESR, which is also called Electron Paramagnetic Resonance (EPR), is a physical phenomenon analogous to NMR, but instead of the spins of the atom's nuclei, electron spins are excited in an ESR. Because of the difference in mass between nuclei and electrons, weaker static magnetic fields and higher frequencies for the oscillating magnetic fields are used, compared to NMR.
Brief Description of the Drawings
[0005] FIG. 1 shows a cross-sectional view of components for on-chip NMR detection of biomolecules utilizing nanoparticle tags.
[0006] FIG. 2 shows a cross-sectional view of components for on-chip NMR detection of biomolecules utilizing nanoparticle tags in an array configuration. [0007] FIG. 3 shows a schematic top-down view of an integrated on-chip NMR device.
[0008] FIG. 4 shows a schematic cross-section view of an integrated on-chip
NMR device.
[0009] FIG. 5 shows another schematic cross-section view of an integrated on- chip NMR device with a microchannel for containing the sample. [0010] FIG. 6 shows another schematic cross-section view of an integrated on- chip NMR device with an integrated electromagnet having a magnetic core. [0011] FIG. 7 shows a schematic top-down view of an integrated on-chip ESR device and a comparative NMR device.
[0012] FIG. 8 shows a schematic cross-section view of a substrate with an array of NMR microcoils and sample reservoirs. Detailed Description
[0013] 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.
[0014] A "microcoil" is a coil, or one or more connected loops, having at least one dimension in the micrometer (μm), or less than 10~3 meter (mm), scale. A microcoil usually comprises a thin material wound or gathered around a center or an imaginative center into spiral, helical or other shapes. A microcoil is defined by the material itself, the shape of the windings, and the separation between each windings. Solenoid type microcoils are multiple spiral wire loops, which may or may not be wrapped around a metallic core. A Solenoid type microcoil produces a magnetic field when an electrical current is passed through it and can create controlled magnetic fields. A Solenoid type microcoil can produce a uniform magnetic field in a predetermined volume of space. A "planar" microcoil is a microcoil with its windings substantially remained in an actual or imaginative plane.
[0015] A "microchannel" is a channel, groove, or conduit having at least one dimension in the micrometer (μm), or less than 10" meter (mm), scale. Although microchannels are typically straight along their length, they may contain angles and curves of different degrees along their length. Although the microchannels typically have rectangular cross-sections, they may also have other shapes of cross-sections, such as circle. The microchannels are usually suitable for fluidic communications, such as carrying through a biological liquid. The microchannels are often part of an integrated device, such a microfluidic device or an integrated circuit such that liquid flowing through the microchannels are in a controlled pattern and able to be analyzed as desired.
[0016] As used herein, "associated with" or "in association with" means that two or more objects are so situated that the desired results or effects are achieved. For example, a microcoil is "associated" with a space for holding a liquid sample when the microcoil is so situated that it will achieve the desired effect of generating an excitation magnetic field or creating, together with the magnet, NMR or ESR within at least a portion of a sample in the space. In such situations, the magnet is also "associated" with the space and the microcoil. A number of factors will be considered when associating the microcoil or the magnet with the space, including the type and size of the microcoil and the magnet, the size and location of the associated space, the desired strengths of the excitation magnetic field and the static magnetic field and, and the volume within which the desired NMR or ESR will be effectuated. As disclosed herein, the specific locations of the magnet and microcoil on the substrate will be determined based on the specific analysis desired by a person skilled in the art. [0017] As used herein, "dimension" or "dimensions" are the parameters or measurements required to define the shape and/or size, such as height, width, and length, of an object. As used herein, the dimension of a two-dimensional object, such as a rectangle, a polygon, or a circle, is the longest straight-line distance between any two points on the object. Therefore the dimension of a circle is its diameter; a rectangle its diagonal, and a polygon its longest diagonal. The dimension of a three-dimensional object is the longest straight- line distance between any two points on the object. The dimensions used herein are usually measured by centimeters (cm), millimeters (mm), and micrometers (μm), and nanometers (nm).
[0018] A "microfluidic device" is a device that has one or more microchannels.
A microfluidic device may be part of an integrated device, such as an integrated separation or detection equipment or an integrated circuit. Fluids used in microfluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions and various buffers and saline. Microfluidic devices can be used to obtain many interesting measurements, including fluid mechanical properties, cellular and molecular diffusion coefficients, fluid viscosity, pH values, chemical and biological binding coefficients and enzyme reaction kinetics. Other applications for microfluidic devices include cell and molecule detection and separation, capillary electrophoresis, isoelectric focusing, immunoassays, flow cytometry, sample injection of proteins for analysis via mass spectrometry, DNA analysis, cell manipulation, and cell separation. The magnetic materials and technologies may be incorporated into the microfluidic devices for applications such as cell and biomolecule detection and separation. [0019] The use of microfluidic devices to conduct biomedical assays has many significant advantages. First, because the volume of fluids within these channels is very small, usually several nano-liters, the amount of reagents and analytes required for the assays is quite small. This is especially significant for expensive reagents. The fabrications techniques used to construct microfluidic devices, discussed in more details herein, are relatively inexpensive and are very amenable both to highly elaborated, multiplexed devices and also to mass production, such as in an integrated circuit die. In manners similar to that for microelectronics, microfluidic technologies also enable the fabrication of highly integrated devices for performing different functions on the same substrate chip. Embodiments of the invention helps create integrated, portable clinical diagnostic devices for home and bedside use, thereby eliminating time consuming laboratory analysis procedures.
[0020] The flow of a fluid through a microfluidic channel, or microchannel, can be characterized by the Reynolds number (Re), defined as Re = LVaVgp/μ where L is the most relevant length scale, μ is the fluid viscosity, p is the fluid density, and Vavg is the average velocity of the flow. For many microchannels, including channels with a rectangular cross-section, L is equal to 4A/P where A is the cross- sectional area of the channel and P is the wetted perimeter of the channel. Due to the small dimensions of microchannels, the Re is usually much less than 100, often less than 1.0. In this Reynolds number regime, flow is completely laminar and no turbulence occurs. The transition to turbulent flow generally occurs in the range of Reynolds number 2000. Laminar flow provides a means by which molecules can be transported in a relatively predictable manner through microchannels. [0021] As used herein, "magnetic," "magnetic effect," and "magnetism" refer to the phenomena by which one material exert an attractive or repulsive force on another material. Although theoretically all materials are influenced to one degree or another by magnetic effect, those skilled in the art understand that magnetic effect or magnetism is only recognized for its detectability under the specific circumstance. [0022] As used herein, a "permanent magnet" is a material that has a magnetic field without relying upon outside influences. Due to their unpaired electron spins, some metals are magnetic when found in their natural states, as ores. These include iron ore (magnetite or lodestone), cobalt, and nickel. A "paramagnetic material" refers to a material that attracts and repels like normal magnets when subject to a magnetic field. Paramagnetic materials include aluminum, barium, platinum, and magnesium. A "ferromagnetic material" is a material that can exhibit a spontaneous magnetization. Ferromagnetism is one of the strongest forms of magnetism and is the basis for all permanent magnets. Ferromagnetic materials include iron, nickel, and cobalt. A "superparamagnetic material" is a magnetic material that exhibits a behavior similar to that of a paramagnetic material at temperatures below the Curie or the Neel temperature.
[0023] An "electromagnet" is a type of magnet in which the magnetic field is produced by a flow of electric current. The magnetic field disappears when the current ceases. A simple type of electromagnet is a coiled piece of wire that is electrically connected. An advantage of an electromagnet is that the magnetic field can be rapidly manipulated over a wide range by controlling the electric current. Ferromagnetic or non-magnetic materials can be used to form the electromagnets. [0024] An "array," "macroarray" or "microarray" is an intentionally created collection of substances, such as molecules, openings, microcoils, detectors and/or sensors, attached to or fabricated on a solid surface, such as glass, plastic, silicon chip or other substrate forming an array. The arrays can be used to measure the expression levels of large numbers of reactions or combinations simultaneously. The substances 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 pads on the array. A macroarray generally contains pad sizes of about 300 microns or larger and can be easily imaged by gel and blot scanners. A microarray would generally contain pad sizes of less than 300 microns.
[0025] An array of microcoils is a collection of microcoils fabricated on a substrate, such as silicon, glass, or polymeric substrate. Each of the microcoils may be associated or corresponded with a sample space across which the microcoil is capable of generating an oscillating magnetic field as part of an NMR or ESR analysis. The sample space may be a space for holding a liquid sample or a spot for immobilizing certain molecules, such as DNAs and proteins. The microcoil arrays may be a microarray or a macroarray depending on the sizes or the microcoils and the associated sample spaces.
[0026] A DNA microarray is a collection of microscopic DNA spots attached to a solid surface forming an array. DNA microarrays can be used to measure the expression levels of large numbers of genes simultaneously. In a DNA microarray, the affixed DNA segments are known as probes, thousands of which can be used in a single DNA microarray. Measuring gene expression using microarrays is relevant to many areas of biology and medicine, such as studying treatments, disease and developmental stages.
[0027] A "substrate" refers to a material or a combination of materials upon and/or within which other or additional materials are formed, attached, or otherwise associated with according to a predetermined fashion. A substrate often provides physical and functional support to the other or additional materials such that, together, they form part or whole of a functional device. A substrate may be a combination of two or more other substrates, which, due to the combination, have become an identifiable new substrate. The substrate may include metal, silicon, glass, or polymeric materials. In more specific embodiments, the substrate comprises an integrated material, such as a micro fluidic device or an integrated circuit die. [0028] "Solid support" and "support" 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.
[0029] The term "molecule" generally refers to a macromolecule or polymer as described herein. However, microchannels or arrays comprising single molecules, as opposed to macromolecules or polymers, are also within the scope of the embodiments of the invention.
[0030] 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 α-, β-, or ω-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.
[0031] The term "biomolecule" refers to any organic molecule that is part of or from a living organism. Biomolecules include a nucleotide, a polynucleotide, an oligonucleotide, a peptide, a protein, a ligand, a receptor, among others. 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. [0032] As used herein, "biological cells" and "cells" are interchangeable, unless otherwise clearly indicated, and refer to the structural and functional units of all living organisms, sometimes called the "building blocks of life." Cells, as used herein include bacteria, fungi, and animal mammalian cells. Specifically included are animal blood cells, such as red blood cells, white blood cells, and platelets. [0033] The term "analyte", "target," "target molecule," or "target cell" refers to a molecule or biological cell of interest that is to be analyzed or detected, e.g., a nucleotide, an oligonucleotide, a polynucleotide, a peptide, a protein, or a blood cell. The target or target molecule could be 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, nano wires and nanoparticles. The target molecule or cell may be magnetically tagged, or labeled to facilitate their detection and separation. [0034] The term "probe" or "probe molecule" refers to a molecule that binds to a target molecule or cell for the analysis of the target. The probe or probe molecule is generally, but not necessarily, has a known molecular structure or sequence. The probe or probe molecule is generally, but not necessarily, attached to a solid support of the microfluidic device or array. The probe or probe molecule is typically a nucleotide, an oligonucleotide, a polynucleotide, a peptide, or a protein, including, for example, cDNA or pre-synthesized polynucleotide deposited on the array. Probes molecules are biomolecules capable of undergoing binding or molecular recognition events with target molecules or cells. A probe or probe molecule can be a capture molecule. [0035] The term "bi-functional linker group" refers to an organic chemical compound that has at least two chemical groups or moieties, such are, carboxyl group, amine group, thiol group, aldehyde group, epoxy group, that can be covalently modified specifically; the distance between these groups is equivalent to or greater than 5 -carbon bonds. [0036] The term "capture molecule" refers to a molecule that is immobilized on a surface. The capture molecule is generally, but not necessarily, binds to a target or target molecule or cell. The capture molecule is typically a nucleotide, an oligonucleotide, a polynucleotide, a peptide, or a protein, but could also be 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 a target molecule that is bound to a probe molecule to form a complex of the capture molecule, target molecule and the probe molecule. The capture molecule may be magnetically or fluorescently labeled DNA or RNA. In specific embodiments the capture molecule may be immobilized on the surface of a magnetic tunnel junction sensor, which itself is part of an integrated device, such as a microfluidic device or an integrated circuit. The capture molecule may or may not be capable of binding to just the target molecule or cell, or just the probe molecule.
[0037] The terms "die," "polymer array chip," "DNA array," "array chip,"
"DNA array chip," or "bio-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.
[0038] The term "molecule" generally refers to a macromolecule 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.
[0039] 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. [0040] 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."
[0041] When the biomolecule or macromolecule of interest is a peptide, the amino acids can be any amino acids, including α, β, or ω-amino acids. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer may be used. Additionally, unnatural amino acids, for example, β-alanine, phenylglycine and homoarginine are also contemplated by the embodiments of the invention. These amino acids are well-known in the art.
[0042] 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. [0043] 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. [0044] 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.
[0045] 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. [0046] 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.
[0047] 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.
[0048] 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. [0049] 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.
[0050] 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. [0051] A "receptor" is a 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. As the term "receptors" 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. [0052] A "linker" molecule refers to any of those molecules described supra and preferably should be about 4 to about 100 atoms long to provide sufficient exposure. The linker molecules may be, for example, aryl acetylene, alkane derivatives, 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. [0053] The term "chip" or "microchip" refers to a small device or substrate that comprises components for performing certain functions. A chip includes substrates made from silicon, glass, metal, polymer, or combinations and capable of functioning as a microarray, a macroarray, a microfluidic device, a MEMS, and/or an integrated circuit. A chip may be a microelectronic device made of semiconductor material and having one or more integrated circuits or one or more devices. A "chip" or "microchip" is typically a section of a wafer and made by slicing the wafer. A "chip" or "microchip" may comprise many miniature transistors and other electronic components on a single thin rectangle of silicon, sapphire, germanium, silicon nitride, silicon germanium, or of any other semiconductor material. A microchip can contain dozens, hundreds, or millions of electronic components. As discussed herein, microchannels, microfluidic devices, and magnetic tunnel junction sensors can also be integrated into a microchip.
[0054] "Predefined region" or "spot" or "pad" refers to a localized area on a solid support. The spot could be intended to be used for formation of a selected molecule and is otherwise referred to herein in the alternative as a "selected" region. The spot 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 cm2 or less than 1 mm2, and still more preferably less than 0.5 mm2. In most preferred embodiments the regions have an area less than about 10,000 μm2 or, more preferably, less than 100 μm2, and even more preferably less than 10 μm2 or less than 1 μm2. Additionally, multiple copies of the polymer will typically be synthesized within any preselected region. The number of copies can be in the hundreds to the millions. 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.
[0055] "Micro-Electro-Mechanical Systems (MEMS)" is the integration of mechanical elements, sensors, actuators, and electronics on a common silicon substrate through microfabrication technology. 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 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. As discussed herein, MEMS devices can be further integrated with microchannels, microfluidic devices, and/or magnetic tunnel junction sensors, such that, together, they perform separation and detection function for biological cells and biomolecules.
[0056] "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.
[0057] The terms "nanomaterial" and "nanoparticles" 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 - 100 nanometer range. Preferably, a nanomaterial has properties and functions because of the size and can be manipulated and controlled on the atomic level. Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub IOnm) that quantization of electronic energy levels occurs. Preferred nanoparticles as used herein are metallic nanoparticles. More preferred nanoparticles that include coinage (Au, Ag, Cu), alkalis (Li, Na, K), Al, Pd and Pt.
[0058] 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. [0059] The term "fluid" used herein means an aggregate of matter that has the tendency to assume the shape of its container, for example a liquid or gas. Analytes in fluid form can include fluid suspensions and solutions of solid particle analytes. [0060] Magnetic probes are particles whose core is made of magnetic materials, such as compounds containing iron, palladium, platinum, aluminium, barium, calcium, sodium, strontium, uranium, magnesium, cobalt, nickel, or technetium. Magnetic probes show attraction to magnets, but are not magnets by themselves. The surface of the particle can be first chemically treated (e.g. silanization) to facilitate the binding of the probe molecule. The surface of the particle is coated with the probe molecules, such as DNA or antibodies.
[0061] Embodiments of the invention relate to detecting biological molecules with ultra-sensitivity and convenience. The embodiments are especially directed to utilizing nanoparticles as tags and identifying the tags using NMR. The probes containing the nanoparticles can be used in solution or attached to a substrate. [0062] The unique NMR or ESR spectra of nanoparticles can be used as barcodes to identify specific target analytes. The NMR or ESR spectra can be identified using either a conventional NMR or ESR device or an on chip NMR or ESR device as described herein. The claimed methods and devices present higher sensitivity compared to untagged NMR or ESR identification of biomolecules. This is because the use of nanoparticles with known unique NMR or ESR spectrum enables efficient identification of biomolecules which have complex NMR or ESR spectrum. In addition, the claimed methods allow for multiplex detection.
[0063] Further, sample preparation for tagging biomolecules with nanoparticles is simpler than sample preparation for standard direct NMR or ESR identification of biomolecules. This is because the direct identification of biomolecues typically requires a complex sample preparation in order to increase the sensitivity of samples. Such sample preparation is not needed when using molecular probes that are tagged with nanoparticles that have easily identifiable unique NMR and ESR spectrums. [0064] An embodiment of the invention relates to a method of detecting an analyte with a nanoparticle probe comprising attaching a detection probe comprising a nanoparticle to a target analyte; and detecting the presence of the nanoparticle utilizing nuclear magnetic resonance or electron spin resonance. Preferably, the presence of the nanoparticle is detected utilizing nuclear magnetic resonance. Preferably, the presence of the nanoparticle is detected utilizing electron spin resonance. Preferably, the target analyte is a biomolecule, a nucleic acid or a protein. Preferably, the detection probe comprises an antibody, a nucleic acid or a metal. Preferably, the nanoparticle comprises a metal selected from the group consisting of Au, Ag, Cu, Al, Pd and Pt. In one variation, the method could further comprise attaching the target analyte to a capture molecule. Preferably, the capture molecule is bound to a substrate surface. Preferably, the capture molecule comprises an antibody or a nucleic acid. Preferably, the substrate comprises silicon, glass, a polymeric material, or a combination thereof. Preferably, the substrate comprises a plurality of different capture molecules. In one variation, the presence of the nanoparticle is detected using an on chip nuclear magnetic resonance device or electron spin resonance device or using an off chip nuclear magnetic resonance device or electron spin resonance device. Preferably, the target analyte is exposed to a plurality of different nanoparticle probes prior to detecting the presence of the nanoparticle. Preferably, the difference between the nanoparticle probes comprises a difference in the composition of a nanoparticle in the probe. Preferably, the difference between the nanoparticle probes comprises a difference in the particle size of a nanoparticle in the probe.
[0065] Another embodiment of the invention relates to a method of determining the identity of an analyte with a nanoparticle probe comprising exposing a target analyte to a plurality of different detection probes, wherein at least one detection probe comprises a nanoparticle probe; attaching a target analyte to at least one matching detection probe of the plurality of different detection probes; determining the identity of the matching nanoparticle probe attached to the target analyte utilizing nuclear magnetic resonance or electron spin resonance; and determining the identity of the target analyte that corresponds to the matching detection probe, wherein the preferred embodiments and variations could be as those described in the previous paragraphs. [0066] Yet another embodiment of the invention relates to a system comprising a substrate; a capture molecule attached to the substrate and configured to bind to a target analyte; a detection probe comprising a nanoparticle configured to bind to a target analyte; and a nuclear magnetic resonance device or electron spin resonance device configured to detect the nanoparticle, wherein the preferred embodiments and variations could be as those described in the previous paragraphs.. [0067] Nanoparticles, particularly metallic nanoparticles, are very good for
NMR and ESR detection because these particles have unique easily identifiable spectrums. Accordingly, these particles attached to a probe, can be used to "barcode" a target analyte for NMR or ESR identification. This system is also stable as metal nanoparticles do not suffer from optical bleach or decay during NMR or ESR detection. [0068] Any nanoparticles described herein may be used as an NMR or ESR tag.
Preferred nanoparticles as used herein are metallic nanoparticles. More preferred nanoparticles include coinage (Au, Ag, Cu), alkalis (Li, Na, K), Al, Pd and Pt. Even more preferred nanoparticles include 195Pt, 109Ag, 63Cu, which have easily identifiable unique NMR and ESR spectrums. Preferred nanoparticles also have an average particle size of about lnm -10 nm.
[0069] Once the nanoparticles are obtained a probe molecule for a target analyte of interest can be prepared. For example, probes made up of DNA, RNA, or proteins (antibody) may be prepared according to known methods. The nanoparticle tag can then be attached to the probe.
[0070] In one embodiment, the tagged probes are placed or flowed through an
NMR or ESR device (either conventional or on-chip) containing captured target biomolecules (analytes). The spectrum of the target analyte is then collected. If the unique spectrum of nano particle is detected, the existence of the biomolecule analyte is identified.
[0071] FIG. 1 is a cross-sectional schematic illustration of components of a nanoparticle tag based NMR detection device. This embodiment utilizes an on chip NMR device which includes an NMR detection coil, a magnet and a functionalized layer, however the functionalize surface can be used without the other on chip components for detection in a conventional NMR device as well. The functionalized layer is functionalized with a first binding partner which is a capture molecule configured to bind to the target analyte. The second binding partner is a probe molecule configured to bind to the same target analyte as the first binding partner. The second binding partner is attached to the nanoparticle tag.
[0072] For detection of a protein target analyte the first binding partner and the second binding partner will include antibodies that are configured to bind to the target analyte protein at a specific location. When the target analyte and the second binding partner are exposed to the functionalized surface that includes the first binding partner they form a complex, which is called a sandwich (the target protein is bound between the first binding partner, which is the capture antibody and the second binding partner, which is the probe antibody). The second binding partner that is unattached to the substrate surface of the functionalized layer through the target analyte can be washed out of the NMR device before detection. The bound detection probes can then be detected using the on chip NMR device.
[0073] In an alternative embodiment, the first binding partner is attached to a substrate surface. The substrate surface is then exposed to the second binding partner including the nanoparticle tag. After washing the substrate surface to remove any of the second binding partner that is unattached to the substrate surface through the target analyte, the substrate can be placed into a conventional NMR or ESR device. The bound detection probes can then be detected using the conventional NMR or ESR device.
[0074] FIG. 2 shows an on-chip array that is functionalized with a plurality of different capture molecules. A plurality of different probe molecules that correspond to the different capture molecules can also be produced. Each of the different capture molecules can then be tagged with different nanoparticle tags producing different NMR signatures. For example, a different type of nanoparticle can be attached to each different type of probe antibody. The NMR device can then be used to determine which the presence of different target analytes by detecting the signature of the nanoparticle tag attached to the target analyte. Example 1 Nucleic acid detection on a substrate surface —
[0075] When multiple analytes are to be detected in the same sample, capture antibodies or capture DNA can be immobilized on a functionalized substrate. By detecting the NMR or ESR signature of a particular nanoparticle tag, we can measure which biological molecules are present in the sample.
[0076] Following is an example of detecting a small soluble nucleic acids using capture DNA secured to a substrate surface.
[0077] DNA Synthesis — DNA strands are synthesized and purified according to standard procedures using an automated synthesizer (Expedite) and HPLC (1100 HPLC series, Hewlett-Packard), respectively. All of the reagents for the phosphoramidite synthesis, including 3 - and 5_-thiol modifiers, can be purchased from Glen Research (Sterling, VA). Thiol modification is carried out manually by following known procedures. Absorption and extinction spectra are recorded by using an 8452a diode array spectrophotometer (Hewlett-Packard). The concentrations of stock DNA solutions are calculated based on the extinction coefficient of each strand. All buffers and aqueous washes are based on Nanopure water (18 M(; Barnstead), and reagents are used as received unless indicated otherwise. The following DNA strands are synthesized for the nucleic acid assay: probe DNA, 5 -TTATAACTATTCCTAIO- (CH2)6-SH-3; capture DNA, 5 -HS- (CH2)6-A10-CTCCCTAATAACAAT-3; both are designed to bind to part of the target, 5 - TAGGAAT AGTT ATAAATTGTT ATTAGGGAG-3.
[0078] Functionalization of Detection Probe — Nanoparticles particles 195Pt,
109Ag, 63Cu can be modified with thiolated probe DNA (final concentration, 2 (M) by slow salt aging (40 h) to a final concentration of PBS (0.1 M NaCl in 0.01 M of phosphate buffer, pH 7; denoted as PBS unless indicated otherwise). Unbound probe DNA can be removed by repetitive centrifugation (15,700 x g for 30 min) of the particles, followed by rinsing and resuspension in PBS. The concentration of the detection probes can be calculated based on extinction spectra by using known values (28) of the extinction coefficients. The diameter of the detection probes can be determined by transmission electron microscopy by using a commercially available instrument (e.g. Model 8100 manufactured by HITACHI, Tokyo). [0079] Functionalization of Glass Substrate — A functionalized glass can be modified with half-complementary thiolated capture DNA strands (100 (M) using a microarrayer (AFFYMETRIX, Santa Clara, CA) according to a standard procedures. The DNA strands are covalently immobilized on the chip, the unbound strands are washed away with H2O, and the residual binding sites are passivated by immersion in 40 mM mercaptosuccinic acid for 30 min, followed by repetitive washing with H2O. [0080] Bio-Barcode Assay — In a typical assay, the glass slide is treated with the sample suspected of containing the target DNA to allow the target DNA to partially hybridize to the capture DNA. After 4 hour incubation at room temperature, the slide is washed with PBS four times to remove the sample and unbound DNA. The glass slide is then treated with the probe solution (5 mg/ml) to allow the detection probe to partially hybridize to the target DNA already partially hybridized to the capture DNA. After 4 hour incubation at room temperature, the slide is again washed with PBS four times to remove unbound detection probes. The slide can be dried with dry nitrogen gas prior to NMR detection. Alternatively, the slide can be directly scanned by NMR without drying.
[0081] As described above, the NMR or ESR detector can be a conventional
NMR or ESR device in which the nanoparticle tagged sample is inserted into the device for detection. Alternatively, the NMR or ESR device can be an on chip NMR or ESR device in which the functionalized surface is located on the NMR chip or ESR chip. [0082] The on-chip NMR or ESR device can include a functionalized substrate that includes a space for holding a liquid sample, a magnet capable of generating a static magnetic field across at least a portion of the space, and a microcoil capable of generating an excitation magnetic field across at least a portion of the functionalized substrate. According to the embodiment, the static magnetic field and the excitation magnetic field are capable of creating Nuclear Magnetic Resonance or Electron Spin Resonance on the functionalized substrate and the microcoil is capable of detecting signals from the NMR or ESR. [0083] Therefore, the embodiment of the invention encompasses a functionalized substrate that integrates a sample holding space, a magnet for generating a static magnetic field, and a microcoil for generating an excitation magnetic field so that NMR or ESR can occur on the functionalized substrate. Furthermore, the microcoil, in addition to generating the excitation magnetic field, is able to detect and collect signals from the NMR or ESR.
[0084] The substrate may include silicon, glass, a polymeric material, or a combination thereof. More specifically, the substrate includes an integrated circuit, a MEMS device, a microarray, a macroarray, a multi-well plate, a microfluidic device, or a combination thereof. In other words, the embodiment can be integrated into a wide range of materials used in a variety of existing devices. The substrate surface that comes in contact with an analyte sample is functionalized with one or more capture molecules.
[0085] Molecular probes can be immobilized on the surface of individual or individually addressable reservoirs and/or microcoils on the substrate through surface functionalization techniques. The microcoils allow the NMR or ESR signals of nanoparticle probes to be detected and/or measured. The nanoparticle probe in a DNA chip can be 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).
[0086] Silicon is a suitable material for forming micro-channels coupled with microelectronics or other microelectromechanical systems (MEMS). It also has good stiffness, allowing the formation of fairly rigid microstructures, which can be useful for dimensional stability. In a specific embodiment the substrate includes an integrated circuit (IC), a packaged integrated circuit, and/or an integrated circuit die. For example, the substrate may be a packaged integrated circuit that includes a microprocessor, a network processor, or other processing device. The substrate may be constructed using, for example, a Controlled Collapse Chip Connection (or "C4") assembly technique, wherein a plurality of leads, or bond pads are internally electrically connected by an array of connection elements (e.g., solder bumps, columns). [0087] Specific materials useful as the substrate also include, but not limited to, polystyrene, polydimethylsiloxane (PDMS), glass, chemically functionalized glass, polymer-coated glass, nitrocellulose coated glass, uncoated glass, quartz, natural hydrogel, synthetic hydrogel, plastics, metals, and ceramics.
[0088] In another embodiment, the substrate includes circuitry that is capable of amplifying or processing the NMR or ESR signals detected by the microcoil. Any suitable conventional circuits may be used and integrated into the substrate for amplifying and/or processing, including filtering, the NMR or ESR signals detected and collected by the microcoil. The integrated circuitry may be able to generate NMR or ESR spectra independently or connected to an external device for generating the device.
[0089] According to another embodiment, the space for holding a liquid sample, which includes an analyte and one ore more nanoparticle probes, includes a reservoir, a microchannel, an opening, a surface, or a combination thereof. The embodiment accommodates a variety of applications in which NMR or ESR is involved for detecting nanoparticles. For example, the sample holding space may be a reservoir, an opening void, or a surface that can hold a liquid sample. In such cases, the sample holding space may be an open reservoir or surface, or a substantially closed void with an opening for sample input. The design of the space depends not only on the specific analysis to be done, but also on how to best situate and design the sample holding space in relation to the associated magnet and microcoil, as discussed herein. [0090] According to the embodiment, the space for holding a liquid sample may also be the whole or part of a microchannel fabricated on the substrate. Depending on the specific requirement, the microchannel may be open (a trench) or closed. The microchannel typically includes an inlet and an outlet, but may also include other opening for fluidic communication. In another embodiment, the microchannel include two or more inlets and at least one outlet such that different reactants may be introduced into the channel from different inlets and mixed at a mixing section within the channel for specific chemical reaction. Furthermore, the microchannel may include more than two inlets and more than one mixing sections such that more than one reaction may occur within different sections of the mircochannel according predetermined manners.
[0091] The integrated on-chip NMR or ESR devices may accommodate a wide range of sample volume, including very small amount of samples. In one embodiment, the space for holding a liquid sample has a volume of from about 1.0 nL to about 1.0 mL. In another embodiment, the space has a volume of from about 10 nL to about 10 μL. As understood by a person skilled in the art, actual sample volumes will depend on the nature of the analysis to be conducted, in addition to the limitation of the device. [0092] In one embodiment, the magnet includes a permanent magnet or an electromagnet. As disclosed herein, the permanent magnet or electromagnet generates a static magnetic field across at least a portion of the space for holding a liquid sample. Materials suitable for use as the permanent magnet or electromagnet include permanent magnetic materials, ferromagnetic materials, paramagnetic materials, and non-magnetic metals. When a ferromagnetic material is used for the magnet, an external magnetic field is used to magnetize the material. Further, when either a ferromagnetic or nonmagnetic material is used for the magnet, an electrical current is applied to the material to create an electromagnet. In one embodiment, the magnet includes one or more of iron, nickel, cobalt, a rare-earth material such as neodymium, copper, aluminum, and mixtures thereof. More specifically, a Neodymium-Iron-Boron type magnet can be used.
[0093] In the embodiment, the static magnetic field, together with the excitation magnetic field generated by the microcoil, is capable of creating Nuclear Magnetic Resonance or Electron Spin Resonance within a liquid sample contained in the space. In this regard, the magnet is "associated" with the space for holding a liquid sample, meaning that the magnet is so situated that it will achieve the desired effect. A number of factors will be considered when associating the magnet with the space, including the size of the magnet, the sizes and locations of the associated space and microcoil, the desired strength of the static magnetic field, and the volume within which the desired NMR or ESR will be effectuated. In a specific embodiment, the magnet is placed near or adjacent to the space for holding a liquid sample. The specific type, size, strength, and location of the magnet on the substrate will be determined based on the specific analysis desired by a person skilled in the art.
[0094] In a specific embodiment, the magnet is capable of generating a static magnetic field strength of from about 0.01 Tesla (T) to about 30 T, or more specifically, from about 0.01 T to about 10 T. As disclosed herein, NMR and ESR require different static magnetic field strength. For NMR, a static magnetic field strength of 0.5 T or more is typically required. Usually, a static magnetic field strength of 1.0 T or more is used in an NMR. An ERS, on the other hand, usually requires a static magnetic field strength of less than 0.5 T. Thus, in a specific embodiment, the magnet is capable of generating a static magnetic field strength of from about 0.01 T to about 0.5 T. In another specific embodiment, the magnet is capable of generating a static magnetic field strength of from about 0.5 T to about 5 T.
[0095] Many conductive materials are suitable for the microcoil. The selection of materials for the microcoil depends on several factors including the type and size of the coil, the desired strength of excitation magnetic field, the size and location of space for holding a liquid sample, and the nature and location of the magnet. The conductivity of the material is important to the selection. In one embodiment, the microcoil comprises copper, aluminum, gold, silver, or a mixture thereof. [0096] The microcoil is "associated" with the space for holding a liquid sample, meaning that meaning that the microcoil is so situated that it will achieve the desired effect of creating NMR or ESR within at least a portion of the space. A number of factors will be considered when associating the microcoil with the space, including the type and size of the microcoil, the sizes and locations of the associated space and magnet, the desired strength of the static magnetic field and the excitation magnetic field, and the volume within which the desired NMR or ESR will be effectuated. In a specific embodiment, the microcoil is placed near or adjacent to the space for holding a liquid sample. The specific type, size, strength, and location of the microcoil on the substrate will be determined based on the specific analysis desired by a person skilled in the art.
[0097] In one embodiment, the microcoil is a Solenoid type coil. Solenoid type microcoils are multiple spiral wire loops, which may or may not be wrapped around a metallic core. A Solenoid type microcoil produces a magnetic field when an electrical current is passed through it and can create controlled magnetic fields. The Solenoid type microcoil can produce a uniform magnetic field in a predetermined volume of the space.
[0098] In another embodiment, the microcoil is a planar spiral coil, which is a microcoil with its windings substantially remained in an actual or imaginative plane. In the embodiment, the microcoil is wound around a center, which often is also the center, or on the same axis of the center, of the associated space for holding a liquid sample and expands in a spiral like manner. The winding may take many different forms, depending on the needs of the specific device and analysis. For example, the winding may take a generally circular, square, or rectangular shape. [0099] As illustrated in FIGS. 3 and 4, an integrated on-chip NMR device includes a magnet that generates a static magnetic field, Bo, across a sample holding space, which may contain a bio-molecular sample. The magnet is attached to an insulating layer and is located beside the sample holding space. A planar microcoil is located just beneath the sample holding space and wound around the space, which serves as an imaginative center of the windings. The windings of the microcoil are imbedded in the insulating layer, which is attached to a silicon substrate. As shown in the figures, the microcoil is wound in a square shaped and spiral like manner around the center. As disclosed herein, the microcoil may also be wound around the center in circular shaped manners.
[00100] A planar microcoil is defined, in part, by its inner-area dimension, the number of windings, and the winding separation. The inner-area of a microcoil refers to the area at the center of the microcoil where there is no winding and around which the microcoil is wound. The shape of the inner-area is usually not perfectly regular, although it is often similar to a circular, rectangular, or square shape. As shown in FIG. 3, the inner-area of the microcoil has a roughly rectangular shape. The dimension of the inner-area is described herein by the length of the parameter of the area. The number of windings as used herein refers to the number of times the microcoil is wound around the inner-area. For example, the number of windings for the microcoil shown in FIG. 3 is three (3). As used herein, the winding separation refers to the distance between two adjacent windings.
[00101] In one embodiment, the planar spiral microcoil includes an inner-area dimension of from about 10 μm to 10 mm, about 1 to 100 windings, and a winding separation of about lμm to 500μm. In another embodiment, the microcoil includes an inner-area dimension of from about 100 μm to 1.0 mm, about 1 to 30 windings, and a winding separation of about lOμm to lOOμm. In another specific embodiment, the microcoil has a cross-section dimension of from about 0.0 lμm to about lOOμm. [00102] FIGS. 5 and 6 illustrate further embodiments of the invention. FIG. 5 shows a cross-section view of an integrated on-chip NMR device that includes a permanent magnet, a microcoil, and a microchannel for holding a bio-molecular sample on a silicon substrate. The permanent magnet is connected to the rest of the substrate by an adhesive or seed layer. As shown in FIG. 5, the permanent magnet generates a static magnetic field, Bo, across the sample holding space, which is part of a microchannel. The microcoil, which has three windings, is located just above the sample holding space and is wound around an imaginative center above the sample holding space. In this embodiment, the microchannel may have one or more inlets and outlets (not shown) for sample input and output. The location and volume of the sample holding area where NMR occurs is determined according the specific needs of the analysis to be carried out, which will also determine the size and shape of the microcoil.
[00103] FIG. 6 illustrates another embodiment of the invention. As shown, the magnet is an electromagnet with a magnetic core and a soft-magnetic under layer. A static magnetic field, BO, is generated across a sample holding space, which is portion of a microchannel. A Solenoid microcoil is provided both above and under the sample holding space. The additional microcoil helps to effectuate the NMR or ESR and to detect and collect more information from the NMR or ESR.
[00104] Embodiments of the present invention can be adapted to perform either
NMR or ESR analysis. As disclosed herein, ESR occurs according to similar principles as NMR, except that unpaired electron spins are detected in ESR, whereas unpaired nuclear spins are detected in NMR. Therefore, ESR and NMR spectra reveal different aspects of the target's structure.
[00105] As disclosed herein, ESR has higher sensitivities as compared to NMR and it requires a lower strength for the static magnetic field. A static magnetic field strength of less than 0.5 Tesla (T) is usually required for ESR, whereas a strength of larger than 0.5 T, or more typically larger than 1.0 T, is required for NMR. On the other hand, ESR has a higher resonance frequency, usually larger than 1.0 GHz, as compared to less than 500 MHz for NMR. Also, for NMR, pulse wave measurement and technique are commonly used and that traditional continuous NMR is rare. For ESR, both continuous and pulse wave measurement and technique are used. Although this disclosure focuses on pulse wave measurement, the embodiments also encompass continuous wave techniques. As disclosed herein, the differences between ESR and NMR means that materials selections are different for an ESR or NMR device, and that different electronic circuitries are required to detect, collect, and/or process the signals from the ESR or NMR.
[00106] FIG. 7 illustrates a comparison between an integrated ESR device and a similar NMR device. As shown on the left, the integrated on-chip ESR device includes a magnet that generates a static magnetic field, B0, which is less than 0.5 T, across a sample holding space, which may contain a sample with unpaired electrons. The magnet is attached to an insulating layer and is located beside the sample holding space. A planar microcoil is located just beneath the sample holding space and wound three times around an imaginative center just above the sample holding space. As shown, the resonance frequency for the ESR is larger than 1.0 GHz. The comparative NMR device has a similar design and physical components, except that the magnet used is different with a static magnetic field strength of larger than LO T and a resonance frequency of less than 300 MHz. Therefore, designs for ESR and NMR devices are generally interchangeable with the exception of the magnet and the circuitry, including the microcoils, used for the devices.
[00107] The NMR and ESR devices 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 magnets, microcoils, and circuitries on the substrate may be formed via semiconductor manufacturing methods on a semiconductor substrate. Thin film coatings may be selectively deposited on portions of the substrate surface. Examples of suitable deposition techniques include vacuum sputtering, electron beam deposition, solution deposition, and chemical vapor deposition. The 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 the microcoils. Coatings may be used to provide a physical barrier on the surface, e.g. to retain fluid at specific sites on the surface. [00108] In one embodiment, the NMR or ESR device is made through combining two or more smaller substrates to form a larger substrate. Specifically, the fabricating of the space, the attaching of the magnet, or the fabricating of the microcoil involves combining two or more smaller substrates to form the substrate. [00109] The substrate used may include various materials including, but not limited to silicon, glass, metal, and polymeric material. According to the embodiments, the substrate includes an integrated circuit, a MEMS device, a microarray, a macroarray, a multi-well plate, a microfluidic device, or a combination thereof. [00110] In one embodiment, a permanent magnet or electromagnet is attached to a silicon substrate through an adhesive layer. The adhesive layer can also be referred to and regarded as a seed layer used to join the magnet with the substrate. Any suitable adhesive materials may be used as the adhesive layer. In one embodiment, the adhesive layer includes one or more of titanium, tantalum, platinum, and palladium. [00111] Microcoils can be fabricated on or within the substrate using a number of techniques, including etching, bonding, annealing, adhering/seeding, lithography, molding, and printing. Physical vapor deposition (PVD) and chemical vapor deposition (CVD) can also be used. In one embodiment, microcoils are fabricated on an oxidized silicon substrate by electroplating metals inside a deep photoresist mold and then passivated using an epoxy based resist.
[00112] The substrate is suitable for forming openings, voids, surfaces, or microchannels thereon for holding fluid and fluidic communications. The sample holding space may be open or closed along. Various methods may be used to form the sample holding space on the substrate. For example, a reservoir or an open microchannel can be fabricated on a silicon substrate by etching methods known to those skilled in the art. Closed microchannels can be formed by sealing the open channels at top using methods such as anodic bonding of glass plates onto the open microchannels on the silicon substrate.
[00113] The microchannels formed on the substrate may be straight or have angles or curves along their lengths. The characteristics and layout of the microchannels are determined by the specific applications the device is designed for. Although straight microchannels lining next to one another are a typical design for microfluidic devices, the microchannels in the embodiments of the invention may be designed in many different patterns to serve specific separation and detection requirements. Specifically, the design of the microchannels takes into consideration of the microcoils associated with the microchannels such that the microcoils are capable of generating excitation magnetic fields across relevant portions of the microchannels. Further, the cross-section of the micro-channel so formed may be uniform or vary along the channel's length, and may have various shapes, such as rectangle, circle, or polygon.
[00114] A method of performing an NMR or ESR detection of nanoparticles may include: (1) providing a device that includes a functionalized substrate, a magnet capable of generating a static magnetic field across at least a portion of the functionalized substrate, and a microcoil capable of generating an excitation magnetic field across at least a portion of the functionalized substrate; (2) exposing the functionalized substrate to an analyte capable of binding the to the functionalized substrate and to a nanoparticle probe that is capable of binding to the analyte; (3) using the magnet to generate a static magnetic field across at least a portion of the functionalized substrate; (4) using the microcoil to generate an excitation magnetic field across at least a portion of the functionalize substrate; and (5) using the microcoil to detect signals from nanoparticles attached to the functionalized substrate through the analyte.
[00115] FIG. 8 illustrates another embodiment of the invention in which an array of microcoils and their associated sample holding spaces are integrated in a single substrate for performing an NMR analysis. FIG. 8 shows a portion of the substrate in which an array of reservoirs for holding samples is located on the portion of the substrate. Associated with each reservoir is an NMR microcoil located beneath the reservoir. The device may be used to carry out multiple NMR analysis for single or multiple samples. For example, each of the plurality of reservoirs can be associated with a biomolecule, such as a DNA or protein. The association may be through conventional surface functionalization techniques or techniques disclosed herein. After association of DNAs to the plurality of reservoirs, a DNA microarray or macroarray is formed and can be used to perform multiple DNA analysis. In such cases, the DNA and nanoparticles are introduced into the sample holding spaces of the array. DNA hybridization can be detected through the identification of the nanoparticle signatures using the associated microcoils.
[00116] The devices and methods described herein can be used for a variety of applicants, for example, in the point of care and field devices for diagnostics, forensic, pharmaceutical, agricultural, food inspection, biodefense, environmental monitoring, and industrial process monitoring.
[00117] 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.

Claims

Claims:
1. A method of detecting an analyte with a nanoparticle probe comprising: attaching a detection probe comprising a nanoparticle to a target analyte; and detecting the presence of the nanoparticle utilizing nuclear magnetic resonance or electron spin resonance.
2. The method of claim 1, wherein the presence of the nanoparticle is detected utilizing nuclear magnetic resonance.
3. The method of claim 1, wherein the presence of the nanoparticle is detected utilizing electron spin resonance.
4. The method of claim 1, wherein the target analyte is a biomolecule.
5. The method of claim 1, wherein the target analyte is a nucleic acid.
6. The method of claim 1, wherein the target analyte is a protein.
7. The method of claim 1, wherein the detection probe comprises an antibody.
8. The method of claim 1, wherein the detection probe comprises a nucleic acid.
9. The method of claim 1, wherein the nanoparticle comprises a metal.
10. The method of claim 1, wherein the nanoparticle comprises a metal selected from the group consisting of Au, Ag, Cu, Al, Pd and Pt.
11. The method of claim 1 , wherein the nanoparticle comprises a metal selected from the group consisting of Pt, Ag and Cu.
12. The method of claim 1, further comprising attaching the target analyte to a capture molecule.
13. The method of claim 10, wherein the capture molecule is bound to a substrate surface.
14. The method of claim 10, wherein the capture molecule comprises an antibody.
15. The method of claim 10, wherein the capture molecule comprises a nucleic acid.
16. The method of claim 13, wherein the substrate comprises silicon, glass, a polymeric material, or a combination thereof.
17. The method of claim 13, wherein the substrate comprises a plurality of different capture molecules.
18. The method of claim 1, wherein the presence of the nanoparticle is detected using an on chip nuclear magnetic resonance device or electron spin resonance device.
19. The method of claim 1, wherein the presence of the nanoparticle is detected using an off chip nuclear magnetic resonance device or electron spin resonance device.
20. The method of claim 1 , wherein the target analyte is exposed to a plurality of different nanoparticle probes prior to detecting the presence of the nanoparticle.
21. The method of claim 20, wherein the difference between the nanoparticle probes comprises a difference in the composition of a nanoparticle in the probe.
22. The method of claim 20, wherein the difference between the nanoparticle probes comprises a difference in the particle size of a nanoparticle in the probe.
23. A method of determining the identity of an analyte with a nanoparticle probe comprising: exposing a target analyte to a plurality of different detection probes, wherein at least one detection probe comprises a nanoparticle probe; attaching a target analyte to at least one matching detection probe of the plurality of different detection probes; determining the identity of the matching nanoparticle probe attached to the target analyte utilizing nuclear magnetic resonance or electron spin resonance; and determining the identity of the target analyte that corresponds to the matching detection probe.
24. The method of claim 23, wherein the identity of the matching nanoparticle probe attached to the target analyte is determined utilizing nuclear magnetic resonance.
25. The method of claim 23, wherein the identity of the matching nanoparticle probe attached to the target analyte is determined utilizing electron spin resonance.
26. The method of claim 23, wherein the target analyte is a biomolecule.
27. The method of claim 23, wherein the target analyte is a nucleic acid.
28. The method of claim 23, wherein the target analyte is a protein.
29. The method of claim 23, wherein the detection probe comprises an antibody.
30. The method of claim 23, wherein the detection probe comprises a nucleic acid.
31. The method of claim 23, wherein the nanoparticle comprises a metal.
32. The method of claim 23, wherein the nanoparticle comprises a metal selected from the group consisting of Au, Ag, Cu, Al, Pd and Pt.
33. The method of claim 23, wherein the nanoparticle comprises a metal selected from the group consisting of Pt, Ag and Cu.
34. The method of claim 23, further comprising attaching the target analyte to a capture molecule.
35. The method of claim 23, wherein the capture molecule is bound to a substrate surface.
36. The method of claim 34, wherein the capture molecule comprises an antibody.
37. The method of claim 34, wherein the capture molecule comprises a nucleic acid.
38. The method of claim 35, wherein the substrate comprises silicon, glass, a polymeric material, or a combination thereof.
39. The method of claim 35, wherein the substrate comprises a plurality of different capture molecules.
40. The method of claim 23, wherein the identity of the matching nanoparticle probe attached to the target analyte is determined utilizing an on-chip nuclear magnetic resonance device or electron spin resonance device.
41. The method of claim 23 , wherein the identity of the matching nanoparticle probe attached to the target analyte is determined utilizing an off-chip nuclear magnetic resonance device or electron spin resonance device.
42. The method of claim 23, wherein the difference between the nanoparticle probes comprises a difference in the composition of a nanoparticle in the probe.
43. The method of claim 23, wherein the difference between the nanoparticle probes comprises a difference in the particle size of a nanoparticle in the probe.
44. The method of claim 35, wherein the matching detection probe is attached to the substrate prior to determining the identity of the matching detection probe.
45. A system comprising: a substrate; a capture molecule attached to the substrate and configured to bind to a target analyte; a detection probe comprising a nanoparticle configured to bind to a target analyte; and a nuclear magnetic resonance device or electron spin resonance device configured to detect the nanoparticle.
46. The system of claim 45, comprising a nuclear magnetic resonance device.
47. The system of claim 45, comprising a nuclear electron spin resonance device.
48. The system of claim 45, further comprising a target analyte.
49. The system of claim 48, wherein the target analyte is a biomolecule.
50. The system of claim 48, wherein the target analyte is a nucleic acid.
51. The system of claim 48, wherein the target analyte is a protein.
52. The system of claim 45, wherein the detection probe comprises an antibody.
53. The system of claim 45, wherein the detection probe comprises a nucleic acid.
54. The system of claim 45, wherein the nanoparticle includes a metal.
55. The system of claim 45, wherein the nanoparticle includes a metal selected from the group consisting of Au, Ag, Cu, Al, Pd and Pt.
56. The system of claim 45, wherein the nanoparticle includes a metal selected from the group consisting of Pt, Ag and Cu.
57. The system of claim 45, wherein the capture molecule comprises an antibody.
58. The system of claim 45, wherein the capture molecule comprises a nucleic acid.
59. The system of claim 45, wherein the substrate comprises silicon, glass, a polymeric material, or a combination thereof.
60. The system of claim 45, wherein the substrate comprises a plurality of different capture molecules.
61. The system of claim 45, wherein the nuclear magnetic resonance device or electron spin resonance device is attached to the substrate.
62. The system of claim 45, wherein the nuclear magnetic resonance device is not attached to the substrate.
63. The system of claim 45, comprising a plurality of magnetic resonance devices or electron spin resonance devices attached to the substrate.
64. The system of claim 45, further comprising a plurality of different nanoparticle probes.
65. The system of claim 64, wherein the difference between the nanoparticle probes comprises a difference in the composition of a nanoparticle in the probe.
66. The system of claim 64, wherein the difference between the nanoparticle probes comprises a difference in the particle size of a nanoparticle in the probe.
PCT/US2007/072297 2006-06-30 2007-06-27 Nanoparticles as tags for bioanalyte detection by nuclear magnetic resonance or electron spin resonance WO2008091364A2 (en)

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