US20030211488A1 - Nanoparticle probs with Raman spectrocopic fingerprints for analyte detection - Google Patents
Nanoparticle probs with Raman spectrocopic fingerprints for analyte detection Download PDFInfo
- Publication number
- US20030211488A1 US20030211488A1 US10/172,428 US17242802A US2003211488A1 US 20030211488 A1 US20030211488 A1 US 20030211488A1 US 17242802 A US17242802 A US 17242802A US 2003211488 A1 US2003211488 A1 US 2003211488A1
- Authority
- US
- United States
- Prior art keywords
- raman
- substrate
- bound
- oligonucleotides
- particles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical 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
- G01N33/587—Nanoparticles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- SERS Surface Enhanced Raman Spectroscopy
- U.S. Pat. No. 5,306,403 describes a method and apparatus for DNA sequencing using SERS.
- U.S. Pat. No. 5,266,498 describes the use of SERS to detect analytes in general.
- U.S. Pat. No. 5,445,972 describes the use of a Raman label bound to a specific binding molecule.
- U.S. Pat. No. 5,376,556 describes the use of SERS in immunoassays.
- U.S. Pat. No. 6,127,120 describes the use of SERS, the detection of nucleic acid and nucleic acid subunits.
- U.S. Pat. Nos. 6,242,264 and 6,025,202 describe the use of silver to form a SERS active substrate to enhance Raman scattering of adsorbed molecules.
- a reagent comprising a particle having a Raman labeled and specific binding member bound to the particle is used for assays of analytes.
- This reagent is particularly advantageous in that it can be bound to a binding partner analyte to form a complex and directly or indirectly bound to a support.
- the Raman label in the label complex on the support can then be SERS activated by staining, for example, silver, gold or copper enhancement to achieve a SERS effect when irradiated with a laser.
- this complex is captured on a solid support and treated with silver to provide a SERS effect.
- the complex can be directly or indirectly reacted with an analyte which has already been bound to a solid support substrate.
- the SERS effect is produced near the time it is measured.
- This reagent can advantageously include multiple different Raman dyes bound to be particle carriers as a way distinguishing particular carriers with particular specific binding members as a way of indexing a vast number of reagent for multiplex application.
- Another advantageous reagent of this invention is a conjugate of several different Raman dyes bound to a specific binding substance such as DNA, RNA, polypeptide, antibody, antigen, small molecules, etc. This also serves as a reagent indexing tool.
- the invention is particularly distinguished from the prior art method in that the SERS technology is used in conjunction with nanoparticle assay techniques to provide extraordinary sensitivity and specificity of detection of analytes which is particularly amenable to multiplexed determination of analtyes.
- FIG. 1 illustrates a chip-based DNA detection method using nanoparticles functionalized with oligonucleotides and Raman labels.
- FIG. 2 illustrates a flatbed scanner image of microarrays after hybridized with nanoparticles functionalized with Cy3 labels, before (A) and after (B) silver staining.
- C A typical Raman Spectrum acquired from one of the silver stained spots.
- D A profile of Raman intensity at 1192 cm ⁇ 1 as a function of position on the chip; the laser beam from the Raman instrument is moved over the chip from left to right as defined by the line in “B”.
- FIG. 3 illustrates the unoptimized detection limit of DNA using the Raman scanning method.
- A A microarray-based sandwich detection format;
- B A flatbed scanner image of microarrays for 20 fM target concentration after hybridized with nanoparticles functionalized with Cy3.5 labels;
- C A typical Raman spectrum acquired from one of the silver-stained spots;
- D A profile of Raman intensity at 1199 cm ⁇ 1 as a function of position on the chip; the laser beam from the Raman instrument is moved over the chip from left to right as defined by the line in “B”.
- FIG. 4 illustrate Left: The Raman spectra of six dyes. Each dye correlates with a different color in our labeling scheme (see rectangular boxes). Right: six DNA target analysis systems. The information of target strand sequences were obtained from the web site of the National Center for Biological Information (http://www2.ncbi.nlm.nih.gov/Genbank/index.html).
- FIG. 5 illustrates (A) Flatbed scanner images of silver-stained microarrays and (B) corresponding Raman spectra. The colored boxes correlate with the color coded Raman spectra in FIG. 4.
- FIG. 6 illustrates the differentiation of two RNA targets (Target 1: perfect; Target 2: with one-base difference).
- FIG. 7 illustrates hybridization of pure RNA target 1 or 2, or mixture of target 1 and 2, to microarrays (A) before stringency wash, (B) after stringency wash.
- FIG. 8 illustrates (A) Typical flatbed scanner images of microarrays hybridized with nanoparticles, (1) before and (2) after stringency wash but prior to silver enhancing, and (3) after silver enhancing. Flatbed scanner image of microarrays hybridized with nanoparticles (4) before stringency wash but after silver enhancement.
- C Raman spectrum of the mixed probes (probe 1:probe 2, 1:1, after silver enhacement) compared with the spectra for probe 1 (with only TMR, blue line) or probe 2 (with only Cy3, red line).
- FIG. 9 illustrates (A) typical flatbed scanner images of nanoparticle-functionalized microarrays, (1) before and (2) after stringency wash but prior to silver staining, and (3) after silver staining.
- the full Raman spectra from 400 to 1800 cm ⁇ 1 are shown in the supporting information.
- the inset is a profile of Raman intensity ratio (I b /I 1 ) verse target ratio (T 2 /T 1 ), where I 1 is the Raman Intensity at 1650 cm ⁇ 1 (from probe 1: TMR labeled gold oligonucleotide conjugate); I 2 is the Raman Intensity at 1588 cm ⁇ 1 (from probe 2: Cy3 labeled gold oligonucleotide conjugate).
- FIG. 10 illustrates Raman spectra (400 ⁇ 1800 cm ⁇ 1 ) from the silver enhanced spots at different target 1 to target 2 ratios: (a) 1:0; (b) 5:1; (c) 3:1; (d) 1:1; (e) 1:3; (f) 1:5; and (g) 0:1.
- FIG. 11 illustrates (A) Scheme for screening protein-small molecule interactions.
- FIG. 12 illustrates the Raman-based detection format for proteins.
- FIG. 13 illustrates (A1-4) Flatbed scanner images of silver-stained microarrays associated with the protein-protein screening experiments.
- Anti-Mouse IgG was labeled with Cy3 modified-alkylthiol-capped poly adenine (A 10 ), anti-ubiquitin by Cy3.5 modified-alkylthiol-capped Poly adenine (A 10 ), and anti-human protein C by Cy5 modified-alkylthiol-capped Poly adenine (A 10 ).
- the A 10 oligonucleotide spacer was used to enhance the stability of the particle probes. 33
- FIG. 14 illustrates the examples for creating Raman-labeled nanoparticle probes with mulplexing capabilities.
- R1, R2, R3, are different Raman dyes.
- FIG. 15 illustrates the creation of massive nanoparticle probes with multiple Raman labels.
- FIG. 17 illustrates Left and Right: two Raman spectra of three-dye composite labels (black line) compared with the spectra of TMR (blue line), Cy3 (red line) and Cy3.5 (green line).
- FIG. 18 illustrates the microbead-based detection format using the scanning Raman method.
- FIG. 19 illustrates (A) and (B): The eight DNA target analysis systems. Each of the probe strands was marked by a single-dye or two-dye labels (see rectangular boxes and circles, corresponding Raman spectra. The colored boxes and circles correlate with the color coded Raman spectra in FIG. 20.
- FIG. 20 illustrates the Raman spectra of six single dyes and two mixed dyes, each spectra correlates with a different color in our labeling scheme (see rectangular boxes and circles).
- FIG. 21 illustrates microscopy image of silver-stained microspheres. The colored circles correlate with the color coded Raman spectra in FIG. 20.
- FIG. 22 illustrates optical microscope image of aligned silver-stained microspheres. The colored boxes correlate with the color coded Raman spectra in FIG. 20.
- FIG. 23 illustrates the fiberoptic-based detection format using microbeads.
- FIG. 24 illustrates the synthesis of Raman labeled oligonucleotides.
- the invention relates to reagents comprising particles having specific binding members and Raman labels bound to the particle wherein the particle can be treated with an enhancing stain such as silver, gold or copper to provide a SERS effect when irradiated.
- This reagent may be complexed with analyte which binds to the specific binding member and the resulting complex can be directly or indirectly captured on a substrate.
- the Raman label in the complex on the substrate is treated with a staining agent such as silver, gold or copper to activate the SERS effect when irradiated with a laser.
- the analyte may be captured on the solid support substrate and reacted directly or indirectly with the reagent prior to staining and SERS measurement.
- the invention also encompasses a reagent of a specific binding substance having two or more different Raman labels bound thereto.
- the invention includes methods of detecting analytes using these reagents and test kits containing reagents and other materials for carrying methods of the invention.
- the present invention relates to particles or carriers or Raman dye carriers functionalized with specific binding members and Raman labels, coupled with surface-enhanced Raman scattering (SERS) spectroscopy, to perform multiplexed detection of analytes.
- SERS surface-enhanced Raman scattering
- the Raman spectroscopic fingerprint which can be designated through choice of Raman label can be read out after silver enhancing via scanning Raman spectroscopy (FIG. 1). Because the SERS-active substrate in this strategy is generated prior to the detection event, a large and reproducible Raman scattering response can be obtained.
- a method for detecting for the presence or absence of one or more target analytes in a sample, the target analytes having at least two binding sites comprises:
- the Raman active label comprises at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- a method for detecting for the presence or absence of one or more target nucleic acids in a sample, the sequence of the nucleic acid having at least two portions comprises:
- a method for detecting for the presence or absence of a target nucleic acid in a sample, the sequence of the nucleic acid having at least two portions comprises:
- a particle comprising oligonucleotides bound thereto and a Raman label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to the particle have a sequence that is complementary to a second portion of the nucleic acid; and (ii) the Raman active label bound to particles serves as an identifier for the target nucleic acid, said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- a method for detecting for the presence or absence of a single nucleotide polymorphism in a nucleic acid in a sample, the sequence of the nucleic acid having at least two portions comprises:
- the nucleic acid is first contacted with the substrate so that the first portion of the nucleic acid sequence hybridizes with complementary oligonucleotides bound to the substrate and then the nucleic acid bound to the substrate is contacted with the particles having oligonucleotides bound thereto so that at least some of the oligonucleotides bound to the particles hybridize with the second portion of the sequence of the nucleic acid bound to the substrate.
- the nucleic acid is first contacted with the particles having oligonucleotides bound thereto so that at least some of the oligonucleotides bound to the particles hybridize with a second portion of the sequence of the nucleic acid; and then contacting the nucleic acid bound to the particles with the substrate so that the first portion of the sequence of the nucleic acid bound to the particles hybridizes with complementary oligonucleotides bound to the substrate.
- the substrate has a plurality of types of oligonucleotides attached thereto in an array to allow for the detection of multiple portions of a single type of nucleic acid, the detection of multiple types of nucleic acids, or both.
- At least two or more different Raman active labels are used.
- the ratio of the two or more types of Raman labels may be the same or different.
- a reagent comprises having at least one type of Raman active label bound thereto and a specific binding complement for binding to a specific target analyte, wherein (i) the Raman active label serves as an identifier for a specific target analyte; and (ii) the Raman active label comprises at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering.
- a reagent comprises a particle, oligonucleotides bound to the particle and at least one type of Raman label bound to a portion of the oligonucleotides, wherein at least some of the oligonucleotides bound to the particle have a sequence that is complementary to at least a portion of a target nucleic acid.
- the reagent comprises a particle, oligonucleotides bound to the particle, an oligonucleotide connector having first and second portions, an oligonucleotide having at least one type of Raman label bound thereto, wherein at least some of the oligonucleotides bound to the particles have a sequence that is complementary to the first portion of the oligonucleotide connector, the oligonucleotide having the Raman active label bound thereto has a sequence that is complementary to the second portion of the oligonucleotide connector, and at least a portion of the oligonucleotides bound to the particles have a sequence that is complementary to a target nucleic acid.
- the reagent comprises a particle, oligonucleotides bound to the particle, an oligonucleotide connector having first and second portions, an oligonucleotide having at least one type of Raman label bound thereto, and an oligonucleotide having a specific binding complement to a target analyte, wherein at least some of the oligonucleotides bound to the particles have a sequence that is complementary to the first portion of the oligonucleotide connector, the oligonucleotide having the Raman active label bound thereto has a sequence that is complementary to the second portion of the oligonucleotide connector, and the oligonucleotide having the specific binding complement bound thereto has a sequence that is complementary to the second portion of the oligonucleotide connector.
- kits for the detection of one or more target analytes in a sample.
- the kit has in one container a reagent comprising a particle having a specific binding member and at least one Raman label bound to the particle; a staining reagent; and a substrate having a capture reagent.
- a representative kit comprises:
- one or more types of conjugates comprising particles, oligonucleotides bound to the particles, a Raman label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to each type of particle have a sequence that is complementary to a second portion of the sequence of a specific target nucleic acid; and (ii) the Raman active label bound to each type of particles is different and serves as an identifier for a specific target nucleic acid, said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- kits for the detection of one or more target analytes in a sample, the sequence of the nucleic acid having at least two portions.
- the kit comprises:
- particles comprising oligonucleotides bound thereto, a Raman label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to the particle have a sequence that is complementary to a second portion of the sequence of the target nucleic acid; and (ii) the Raman active label bound to the particles serves as an identifier for the target nucleic acid, said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering; and
- kits for the detection of one or more target nucleic acids in a sample, the sequence of the nucleic acid having at least two portions.
- the kit comprises:
- a first container including oligonucleotides having Raman active labels attached thereto, wherein the oligonucleotides the Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- a second container including conjugates comprising particles and oligonucleotides bound to the particles, wherein at least some of the oligonucleotides attached to each type of particle have a sequence that is complementary to at least a portion of the sequence of the oligonucleotides having Raman active labels;
- kits for the detection of one or more target nucleic acids in a sample, the sequence of the nucleic acid having at least two portions.
- the kit comprises:
- one or more containers including oligonucleotides having one or more types of Raman active labels attached thereto, wherein the Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- a second container including conjugates comprising particles and oligonucleotides bound to the particles, wherein at least some of the oligonucleotides attached to each type of particle have a sequence that is complementary to at least a portion of the sequence of the oligonucleotides having Raman active labels;
- a method for screening one or more molecules to determine whether the molecule is a ligand to one or more specific receptors, the molecules are present in a sample comprises:
- reagents comprising particles, specific binding substance bound to the particles, a Raman active label bound to a portion of the specific binding substance, and the molecule from said sample bound to a portion of the specific binding substance, wherein said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- the invention also includes in another aspect a fiber optic analyte detection device in which a particle reagent with specific binding substance and Raman labels is associated with the ends of optical fibers in an optical cable.
- analyte is the substance to be detected in the test sample using the present invention.
- the analyte can be any substance for which there exists a naturally occurring specific binding member (e.g., an antibody, polypeptide, DNA, RNA, cell, virus, etc.) or for which a specific binding member can be prepared, and the analyte can bind to one or more specific binding members in an assay.
- a naturally occurring specific binding member e.g., an antibody, polypeptide, DNA, RNA, cell, virus, etc.
- a specific binding member e.g., an antibody, polypeptide, DNA, RNA, cell, virus, etc.
- “Analyte” also includes any antigenic substances, haptens, antibodies, and combinations thereof.
- the analyte can include a protein, a peptide, an amino acid, a carbohydrate, a hormone, asteroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances.
- analyte-analog refers to a substance which cross reacts with an analyte specific binding member although it may do so to a greater or lesser extent than does the analyte itself.
- the analyte-analog can include a modified analyte as well as a fragmented or synthetic portion of the analyte molecule so long as the analyte analog has at least one epitopic site in common with the analyte of interest.
- analyte epitope denotes that part of the analyte which contacts one member of the specific ligand binding pair during the specific binding event. That part of the specific binding pair member which contacts the epitope of the analyte during the specific binding event is termed the “paratope.”
- Analyte-mediated ligand binding event means a specific binding event between two members of a specific ligand binding pair, the extent of the binding is influenced by the presence, and the amount present, of the analyte. This influence usually occurs because the analyte contains a structure, or epitope, similar to or identical to the structure or epitode contained by one member of the specific ligand binding pair, the recognition of which by the other member of the specific ligand binding pair results in the specific binding event. As a result, the analyte specifically binds to one member of the specific ligand binding pair, thereby preventing it from binding to the other member of the specific ligand binding pair.
- an ancillary specific binding member is a specific binding member used in addition to the specific binding members of the captured reagent and the indicator reagent and becomes a part of the final binding complex.
- One or more ancillary specific binding members can be used in an assay.
- an ancillary specific binding member can be used in an assay where the indicator reagent is capable of binding the ancillary specific binding member which in turn is capable of binding the analyte.
- Associated is the state of two or more molecules and/or particulates being held in close proximity to one another.
- Capture reagent is a specific binding member, capable of binding the analyte or indicator reagent, which can be directly or indirectly attached to a substantially solid material.
- the solid phase capture reagent complex can be used to separate the bound and unbound components of the assay.
- Conjugate is a substance formed by the chemical coupling of one moiety to another.
- An example of such species include the reaction product of bovine serum albumin with chemically activated theophylline molecules and the reaction product of chemically activated Raman-active labels with a protein molecule, such as an antibody, or with a ligand, such as biotin.
- Enhancer a stain such as a silver or gold stain that provides for activating Raman labels on particles to produce a SERS effect.
- “Indicator reagent,” as used herein comprises a detectable label directly or indirectly attached to a specific binding member or metal surface.
- Intervening molecule is any substance to which both a specific binding pair member and a Raman-active label are attached.
- particles is any substance which can be dispersed in a liquid and which will support the phenomenon of a surface-enhanced Raman light scattering (SERS) or surface-enhanced resonance Raman light scattering (SERRS).
- SERS surface-enhanced Raman light scattering
- SERRS surface-enhanced resonance Raman light scattering
- particles include, but are not limited to: Colloids of gold or silver, Pt, Cu, Ag/Au, Pt/Au, Cu/Au, coreshell or alloy particles; particles or flakes of gold, silver, copper, or other substances displaying conductance band electrons.
- SERS surface-enhanced Raman light scattering
- SERRS surface-enhanced resonance Raman light scattering
- Randomtion is an energy in the form of electromagnetic radiation which, when applied to a test mixture, causes a Raman spectrum to be produced by the Raman-active label therein.
- Raman label is any substance which produces a detectable Raman spectrum, which is distinguishable from the Raman spectra of other components present, when illuminated with a radiation of the proper wavelength.
- Other terms for a Raman-active label include dye and reporter molecule. Such labels are shown on pp 25.
- SERRS Surface Enhanced Resonance Raman Scattering
- SERS Surface-Enhanced Raman Scattering
- Specific binding member is a member of a specific binding pair, i.e., two different molecules where one of the molecules, through chemical or physical means, specifically binds to the second molecule.
- other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences (including probe and captured nucleic acid sequences used in DNA hybridization assays to detect a target nucleic acid sequence), complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors a and enzymes, cells, viruses and the like.
- specific binding pairs can include members that are analogs of the original specific binding member.
- analyte-analog a derivative or fragment of the analyte, i.e., an analyte-analog, can be used so long as it has at least one epitope in common with the analyte.
- Immunoreactive specific binding members include antigens, haptens, antibodies, and complexes thereof including those formed by recombinant DNA methods or peptide synthesis.
- Test mixture means a mixture of the test sample and other substances used to apply the present invention for the detection of analyte in the test sample.
- these substances include: Specific binding members, ancillary binding members, analyte-analogs, Raman-active labels, buffers, diluents, and particulates with a surface capable of causing a surface-enhanced Raman spectroscopy, and others.
- Test sample means the sample containing the analyte to be detected and assayed using the present invention.
- the test sample can contain other components besides the analyte, can have the physical attributes of a liquid, or a solid, and can be of any size or volume, including for example, a moving stream of liquid.
- the test sample can contain any substances other than the analyte as long as the other substances do no interfere with the specific binding of the specific binding member or with the analyte or the analyte-analog.
- Examples of test samples include, but are not limited to: Serum, plasma, sputum, seminal fluid, urine, other body fluids, and environmental samples such as ground water or waste water, soil extracts, air and pesticide residues.
- the present invention contemplates the use of any suitable particle having Raman labels and specific binding substances attached thereto that are suitable for use in detection assays.
- nanoparticles are preferred.
- the size, shape and chemical composition of the particles will contribute to the properties of the resulting probe including the DNA barcode. These properties include optical properties, optoelectronic properties, electrochemical properties, electronic properties, stability in various solutions, pore and channel size variation, ability to separate bioactive molecules while acting as a filter, etc.
- the use of mixtures of particles having different sizes, shapes and/or chemical compositions, as well as the use of nanoparticles having uniform sizes, shapes and chemical composition, are contemplated.
- suitable particles include, without limitation, nano- and microsized core particles, aggregate particles, isotropic (such as spherical particles) and anisotropic particles (such as non-spherical rods, tetrahedral, prisms) and core-shell particles such as the ones described in U.S. patent application Ser. No. 10/034,451, filed Dec. 28, 2002 and International application no. PCT/US01/50825, filed Dec. 28, 2002, which are incorporated by reference in their entirety.
- Nanoparticles usefuil in the practice of the invention include metal (e.g. gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetite) colloidal materials.
- Other nanoparticles useful in the practice of the invention include ZnS, ZnO, TiO 2 , AgI, AgBr, HgI 2 , PbS, PbSe, ZnTe, CdTe, In 2 S 3 , In 2 Se 3 , Cd 3 P 2 , Cd 3 As 2 , InAs, and GaAs.
- the size of the nanoparticles is preferably from about 1.4 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm.
- the nanoparticles may also be rods, prisms, cubes, tetrahedra, or core shell particles.
- Suitable nanoparticles are also commercially available from, e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc. (gold).
- Gold nanoparticles are gold nanoparticles.
- Gold colloidal particles have high extinction coefficients for the bands that give rise to their beautiful colors. These intense colors change with particle size, concentration, interparticle distance, and extent of aggregation and shape (geometry) of the aggregates, making these materials particularly attractive for calorimetric assays. For instance, hybridization of oligonucleotides attached to gold nanoparticles with oligonucleotides and nucleic acids results in an immediate color change visible to the naked eye.
- the particles, the specific binding member or both are functionalized in order to attach to the particles.
- Such methods are well known in the art.
- oligonucleotides functionalized with alkanethiols at their 3′-termini or 5′-termini readily attach to gold nanoparticles. See Whitesides, Proceedings of the Robert A. Welch Foundation 39 th Conference On Chemical Research Nanophase Chemistry, Houston, Tex., pages 109-121 (1995).
- Mucic et al. Chem. Commun. 555-557 (describes a method of attaching 3′ thiol DNA to flat gold surfaces; this method can be used to attach oligonucleotides to nanoparticles).
- the alkanethiol method can also be used to attach oligonucleotides to other metal, semiconductor and magnetic colloids and to the other nanoparticles listed above.
- Other functional groups for attaching oligonucleotides to solid surfaces include phosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 for the binding of oligonucleotide-phosphorothioates to gold surfaces), substituted alkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4, 370-377 (1974) and Matteucci and Caruthers, J. Am. Chem.
- Oligonucleotides terminated with a 5′ thionucleoside or a 3′ thionucleoside may also be used for attaching oligonucleotides to solid surfaces.
- U.S. patent application Ser. Nos. 09/760,500 and 09/820,279 and international application nos. PCT/US01/01190 and PCT/US01/10071 describe oligonucleotides functionalized with a cyclic disulfide which are useful in practicing this invention.
- the cyclic disulfides preferably have 5 or 6 atoms in their rings, including the two sulfur atoms. Suitable cyclic disulfides are available commercially or may be synthesized by known procedures. The reduced form of the cyclic disulfides can also be used.
- any substrate can be used which allows observation of the detectable change.
- Suitable substrates include transparent solid surfaces (e.g., glass, quartz, plastics and other polymers), opaque solid surface (e.g., white solid surfaces, such as TLC silica plates, filter paper, glass fiber filters, cellulose nitrate membranes, nylon membranes), and conducting solid surfaces (e.g., indium-tin-oxide (ITO)).
- the substrate can be any shape or thickness, but generally will be flat and thin.
- transparent substrates such as glass (e.g., glass slides or glass beads) or plastics (e.g., wells of microtiter plates).
- the ends of optical fiber in a fiber optical cable serve as a substrate in one embodiment of the invention.
- oligonucleotides can be attached to the substrates as described in, e.g., Chrisey et al., Nucleic Acids Res., 24, 3031-3039 (1996); Chrisey et al., Nucleic Acids Res., 24, 3040-3047 (1996); Mucic et al., Chem. Commun., 555 (1996); Zimmermann and Cox, Nucleic Acids Res., 22, 492 (1994); Bottomley et al., J. Vac. Sci Technol. A, 10, 591 (1992); and Hegner et al., FEBS Lett., 336, 452 (1993).
- a plurality of capture probes may be attached to the substrate in an array for detecting multiple different target analytes.
- a substrate may be provided with rows of spots, each spot containing a different type of capture probes designed to bind a reagent analyte complex.
- a sample containing one or more analytes is applied to each spot, and the rest of the assay is performed in one of the ways described above using appropriate reagents of the invention.
- the Raman labels can be any one of a number of molecules with distinctive Raman scattering spectra. Unlike the enzymes used in enzyme imununoassays, these label species can be stable, simple, inexpensive molecules which can be chemically modified as required.
- the following attributes enhance the effectiveness of the label in this application: (a) A strong absorption band in the vicinity of the laser excitation wavelength (extinction coefficient near 10 4 ; (b) A functional group which will enable covalent attachment to a specific binding member; (c) Photostability; (d) Sufficient surface and resonance enhancement to allow detection of analyte in the subnanogram range; (e) Minimal interference in the binding interaction between the labeled and unlabeled specific binding members; (f) Minimal exhibition of strong fluorescence emission at the excitation-wavelength used; (g) A relatively simple scattering pattern with a few intense peaks; and/or (h) Labels with scattering patterns which do not interfere with each other so several indicator molecules may be analyzed simultaneously.
- Raman-active label 4-(4-Aminophenylazo)phenylarsonic acid monosodium salt, arsenazo I, basic fuchsin, Chicago sky blue, direct red 81, disperse orange 3, HABA (2-(4-hydroxyphenylazo)-benzoic acid), erythrosin B, trypan blue, ponceau S, ponceau SS, 1,5-difluoro-2,4-dinitrobenzene, cresyl violet and p-dimethylaminoazobenzene.
- the chosen labels may be covalently attached to the specific binding members of interest or attached or associated with.
- the invention includes a reagent which has multiple Raman dyes and a specific binding substance, such as DNA, RNA, antibody, antigen, small molecule bound to the particle.
- the multiple Raman label also need not be bound to the particle but may be complexed to the particle through specific binding reactions.
- the invention encompasses multiple SERS reagents bound to a specific binding ligand such as DNA, RNA, antibody, antigen, small molecule, cell or virus. This embodiment may be envisioned as follows:
- a laser serves as the excitation source.
- the laser may be of an inexpensive type such as a helium-neon or diode laser. An operating lifetime of such lasers may be in excess of 50,000 hours.
- a diode laser is used to excite at or at the near IR spectrum, minimizing fluorescence interference.
- the excitation sources used need not necessarily be monochromatic and they also need not necessarily have to be of high intensity. Lamps may also be used.
- the SERS effect can be excited by direct illumination of the surface or by evanescent waves from a waveguide beneath the plasmon-active surface.
- conjugates could be prepared from specific binding members having different specificities, each type with a different Raman active label having a distinctive scattering pattern. Mixing these conjugates in an assay would allow the simultaneous analysis of several different analytes in the same sample.
- the conjugate may include two or more different Raman labels.
- the ratio of Raman intensities can be extracted from a single Raman spectrum using single laser excitation.
- the number of available Raman dyes is much larger than the number of available and discernable fluorescent dyes.
- a Raman dye can be either fluorescent or non-fluorescent. A minor chemical modification of a dye molecule can lead to a new dye with different Raman spectra even though the two dyes exhibit virtually indistinguishable fluorescence spectra. 26 Therefore, this Raman fingerprinting method offers potentially greater flexibility, a larger pool of available and non-overlapping probes, and higher multiplexing capabilities than conventional fluorescence-based detection approaches. This approach has been extended to random array, bead based format where high multiplexing capabilities are essential are underway.
- the Raman-labeled probes have little or no detectable SERS activity.
- Staining material such as silver stains provide strong SERS enhancment.
- a detectable change can be produced or further enhanced by silver staining.
- Silver staining can be employed with any type of nanoparticles that catalyze the reduction of silver. Preferred are nanoparticles made of noble metals (e.g., gold and silver). See Bassell, et al., J. Cell Biol., 126, 863-876 (1994); Braun-Howland et al., Biotechniques, 13, 928-931 (1992).
- nanoparticles being employed for the detection of a nucleic acid do not catalyze the reduction of silver, then silver ions can be complexed to the nucleic acid to catalyze the reduction. See Braun et al., Nature, 391, 775 (1998). Also, silver stains are known which can react with the phosphate groups on nucleic acids.
- Silver, gold or copper staining can be used to produce or enhance a detectable change in any assay performed on a substrate, including those described above.
- silver staining has been found to provide a huge increase in sensitivity for assays employing a single type of nanoparticle so that the use of layers of nanoparticles can often be eliminated.
- Neither the angle of the incident light beam to the surface nor the position of the detector is critical using SERS. With flat surfaces positioning the surface of the laser beam at 60 degrees to the normal is commonly done and detection at either 90 degrees or 180 degrees to the beam are standard. SERS excitation can be performed in the near infrared range which would suppress intrinsic sample fluorescence. It may also be possible to perform SERS-based ligand binding assays using evanescent waves produced by optical waveguides.
- the present invention is adaptable for use as an automatic analyzer. Since the instrument would monitor discrete Stokes shifted spectral lines, the need for an elaborate monochromator system is not necessary. Recent advances in state-of-the-art optics technology, such as holographic optical elements, allow the design of a suitable spectrometer with cost and complexity below that of the laboratory grade device.
- Optical readout energies as a result of SERS are above that which require ultra-sensitive photon counting devices.
- some SERRS spectrometers now in use incorporate silicon photodiode detectors.
- the optical efficiency of a typical monochromator used in a laboratory grade spectrometer is less than 10%.
- the advances in optical materials and components mentioned above should make possible two to three-fold increases in optical efficiency for a simple spectrometer dedicated to only a few specific spectral lines. This also addresses one of the previously major concerns, blocking of the Rayleigh scattering line. With blocking capabilities of newer filters on the order of 10 ⁇ 9 , substitution of filters for one or more stages of the typical monochrometer system should be possible with significant cost savings.
- Oligonucleotide capture strands were immobilized onto the SMPB-(succinimidyl 4-(malemidophenyl)-butyrate) functionalized glass slide by spotting 5′-hexyl-thiol-capped oligoucloetides (1 mM in a 0.15 M NaCl, pH 6.5 phosphate buffer solution (PBS, 10 mM phosphate)) with a commercial arrayer (GMS 417 arrayer, Genotic MicroSystems, Inc).
- the chip After spotting the chip with the capture oligonucleotides ( ⁇ 200 ⁇ m spots), the chip was kept in a humidity chamber for 12 hours to effect the coupling reaction between SMPB and the hexylthiol-capped oligonucleotides. Then the chip was washed copiously with Nanopure water. Passivation of the areas of the chip surrounding the oligonucleotide spots was carried out by immersing the chip in a solution of hexylthiol-capped poly-adenine (A 15 ) (0.1 mM) for 4 h and then in a solution of 3-mercapto-propane sulfonic acid, sodium salt (0.2 M) for 30 minutes to cap off the remaining SMPB sites. Finally, the chip was washed with Nanopure water and dried by a microarray centrifuge (2000 g).
- This Example describes the synthesis of an oligonucleotide having a Raman label attached thereto: (3′HS-Cy3-A 10 -AAT CTC AAC GTA CCT, (SEQ ID NO 1. in FIG. 19 a ) 3′HS-Cy3-A 10 -CTC-CCT-AAT-AAC-AAT) (SEQ ID NO. 25 in FIG. 1)
- Cy3-modified, (propylthiol)-capped oligonucleotides were synthesized on a 1 ⁇ mol scale using standard phosphoramidite chemistry 5 with a Thiol-Modifier C3 S—S CPG (controlled-pore glass) solid support on a commercial synthesizer (Expedite).
- the Cy3-CE phospboramidite Indodicarbocyanine 3, 1′-O-(4-monomethoxytrityl)-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research) was used to incorporate the Cy3 unit in the oligonucleotides.
- the final dimethoxytrityl (DMT) protecting group was not removed.
- the CPG-supported oligonucleotides were placed in 1 mL of concentrated ammonium hydroxide for 8 h at 55 C o to cleave the oligonucleotide from the solid support and remove the protecting groups from the bases.
- cleavage from the solid support via the succinyl ester linkage produced a mixed disulfide composed of the (mercaptopropyl) oligonucleotide and a mercaptopropanol linker.
- TEAA triethylammonium acetate
- the DMT was cleaved by dissolving the purified oligonucleotides in an 80% acetic acid solution for 30 min, followed by evaporation; the oligonucleotides were redispersed in 500 ⁇ L of water, and the solutions were extracted with ethyl acetate (3 ⁇ 300 ⁇ L). After evaporation of the solvent, the oligonucleotides were redispersed in 400 ⁇ L of a 0.1 M dithiothreotol (DTT), 0.17 M phosphate buffer (pH 8) solution at room temperature for 2 h to cleave the 3′ disulfide. Aliquots of this solution ( ⁇ 10 ODs) were purified through a desalting NAP-5 column (Amersham Pharrnacia Biotech AB).
- DTT dithiothreotol
- pH 8 0.17 M phosphate buffer
- This Example describes the syntheses of three oligonucleotides having Raman labels bound thereto: 3′ HS-TMR-A 10 -AAC CGA AAG TCA ATA [SEQ ID NO. 2 in FIG. 19 a]; 3′ HS-Cy3.5-A 10 -CCT CAT TTA CAA CCT [SEQ ID NO. 3 in FIG. 19 a ]; and 3′HS-Cy5-A 10 -CTC CCT AAT AAC AAT [SEQ ID NO. 4 in 19 b ].
- ultramild base monomers from Glen Research were used here to allow the deprotection reaction under ultramild conditions: phenoxyacetyl (Pac) protected dA, 4-isopropyl-phenoxyacetyl (iPr-Pac) protected dG, and acetyl (Ac) protected dC.
- TAMRA-dT (TMR-dT, 5′-Dimethoxytrityloxy-5-[N-((tetramethylrhodaminyl)-aminohexyl)-3-acrylimido]-2′-deoxy
- Cy3.5-CE phosphoramidite Indodicarbocyanine 3.5, 1′-O-(4-monomethoxytrityl)-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite)
- Cy5-CE phosphoramidite (Indodicarbocyanine 5, 1′-O-(4-monomethoxytrityl)-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) were used to label the oli
- the synthesis column contents were transferred to a 2 mL reaction vial and treated with 1 mL of 0.05M potassium carbonate in anhydrous methanol for 4 h at room temperature. Then the supernatant was pipetted from the support and neutralized with 1.5 mL of 2M triethyammonium acetate. Further purification was carried out as described above for the synthesis of the Cy3-labeled-oligonucleotides. HPLC retention times are 28, 32, 30 min for TMR-, Cy3.5- and Cy5-labeled, propylthiol-capped oligonucleotides, respectively.
- This Example describes the synthesis of two olignucleotides have Raman labels attached thereto: 3′ HS-Rd-A 10 -TCA ACA TTG CCT TCT [SEQ ID NO. 5 in FIG. 19 b ] and 3′ HS-TR-A 10 -TCT TCT ATA AAC CTT ATT [SEQ ID NO. 6 in FIG. 19 a ]. See FIG. 24. Both of these oligonucleotides were prepared via two-step syntheses.
- amino-modified oligonucleotides (3′-S—S-(NH 2 )-A 10 -TCA ACA TTG CCA TCT and 3′-S—S-(NH 2 )-A 10 -TCT TCT ATA AAC CTT ATT) were synthesized via literature procedures.
- succinimide ester modified Rhod 6G (5-carboxyl-rhodamine 6G, succinimidyl ester) and Texas Red (Texas Red-X-succinimidyl ester) were coupled to the amino-modified oligonucleotides, respectively.
- Rhod 6G-(or Texas red-) labeled oligonucleotide was purified by ethanol precipitation (3 times) and further by HPLC in the conditions as described above.
- FIG. 1 In a typical experiment for DNA detection, a three-component sandwich assay is used in microarray format (FIG. 1). Gold nanoparticles (13 ⁇ 2 nm in diameter) modified with Cy3-labeled, alkylthiol-capped oligonucleotide strands (Supporting Information) were used as probes to monitor the presence of specific target DNA strands. On average, there are 110 oligonucleotide strands on each 13-nm gold nanoparticle. The Cy3 group was chosen as a Raman label due to its large Raman cross section.
- a chip spotted with the appropriate 15 mer capture strands was coated with a 0.6 M NaCl PBS buffer solution (10 mM of phosphate, pH 7) containing a 30 mer target sequence (100 pM) in a humidity chamber at room temperature. After 4 h, the chip was washed four times with 0.6 M NaCl PBS buffer solution to remove nonspecifically bound target. Then, the chip was treated with a 0.6 M NaCl PBS solution of nanoparticle probes (2 nM) for 1.5 hour to effect hybridization with the overhanging region of the target sequence (FIG. 1). The chip was then washed with 0.6 M NaNO 3 PBS buffer solution to remove chloride ions and nonspecifically bound nanoparticle probes.
- the chip was immediately treated with a silver enhancement solution (Ted Pella, Inc) for 8 minutes, subsequently rinsed with Nanopure water, and dried with a microarray centrifuge (2000 g).
- the chip which exhibits grey spots visible to the naked eye, could be imaged with a flatbed scanner (Expression 1600, Epson) via literature procedures, FIG. 2A and B. 8
- the spots also were imaged by Raman spectroscopy in a 0.3 M NaCl PBS buffer solution (Solution Raman 633 spectrometer from Detection Limit Inc., 30 mW He—Ne laser), FIG. 2C.
- the chip was scanned with a fiber-optic probe with a 0.65 N.A. adapter (25 ⁇ m laser spot), and each spot shows a consistent and strong Raman response at 1192 cm ⁇ 1 (FIG. 2D).
- the obtained spectra include both sharp (15 to 30 cm ⁇ 1 ) Raman lines and a concomitant broad underlying continuum as noted by Brus et. al. in their studies of Rhodamine 6G molecules on Ag particles. 30-31 Importantly, the Raman scattering signals arise almost exclusively from the Cy3 dye molecules immobilized on the particles; no signals were observed from other species such as the oligonucleotides, solvent molecules, and the succinimidyl 4-(maleimidophenyl)-butyrate (SMPB) on the glass surface. Moreover, the Raman scattering frequency for each Raman line remains constant from experiment to experiment, deviating by less than 2 cm ⁇ 1 . Since consistent SERS signals from the Cy3-labeled nanoparticle probes were obtained, the Raman spectrum of Cy3 can be used as a spectroscopic fingerprint to monitor the presence of a specific target oligonucleotide strand.
- a chip spotted with the appropriate capture strands (FIG. 3A) was coated with a 0.75M NaCl PBS buffer solution (10 mM of phosphate, pH 7) containing a 30-mer target sequence (20 fM) in a humidity chamber at room temperature. After 8 h, the chip was washed with 0.75M NaCl PBS buffer solution to remove nonspecifically bound target. Then, the chip was treated with a 0.75 M NaCl PBS solution of nanoparticle probes (500 pM) for 3 h to effect hybridization with the overhanging region of the target sequence (FIG. 3A).
- the chip was washed with 0.75 M NaNO 3 PBS buffer solution to remove chloride ions and nonspecifically bound nanoparticle probes.
- the chip was immediately treated with silver enhancement solution (from Ted Pella, Inc) for 15 min, subsequently rinsed with Nanopure water, and dried with a microarray centrifuge (2000 g).
- the spots can be imaged in the dry state with a flatbed scanner (FIG. 3B) or by Raman spectroscopy in the wet state (0.3 M NaCl, pH7, PBS buffer solution), FIG. 3C and D.
- the current unoptimized detection limit with this technique is 10 fM.
- This Example describes detection of multiple oligonucleotides using a plurality of Raman labeled probes.
- six commercially available dyes were selected with distinct Raman spectra that can be incorporated into oligonucleotides through standard automated DNA-syntheses.
- This Example describes the use of oligonucleotides having Raman labels in detection systems to differentiate single nucleotide polymorphisms (SNPs), and in the case of gene expression studies, one would like access to RNA detection with single spot signal ratioing capabilities. It is well known that nanoparticle probes heavily functionalized with oligonucleotides exhibit extraordinarily sharp thermally-induced denaturation transitions that lead to substantially higher selectivity than conventional molecular fluorophore probes in DNA detection. 5,8,9 However, nothing is known about the behavior of these probes in the context of RNA detection.
- RNA targets were chosen that can bind to the same capture strand DNA but have a single-base mutation in the probe binding regions (target 1:T 1 , normal; target 2:T 2 , single-base difference, FIG. 6). Therefore, two DNA-functionalized probes (probe 1: P 1 , probe 2: P 2 ), which differ in sequence and Raman label, are required to differentiate these two RNA target strands (FIG. 6). Seven separate tests were performed to demonstrate not only how the two targets (T 1 and T 2 ) can be differentiated but also how mixtures of the two targets can be analyzed in semi-quantitative fashion.
- the chip was washed with 0.3 M NaNO 3 PBS buffer solution to remove chloride ions and nonspecifically bound nanoparticle probes. If the chips were developed by silver enhancing, the Raman measurements on the grey spots at different target ratios yield similar spectra (FIG. 8), which are nearly identical to the spectrum for the sample containing probe I and probe 2 in equal amounts. This result indicates that there are equal amounts of probe 1 and probe 2 on the chip.
- the chip was washed with a 0.3 M NaCl PBS buffer to remove nonspecifically bound target.
- the chip was treated with nanoparticle probes (P 1 and P 2 at 1:1 ratio, 2nM total concentration) for 1.5 h to effect hybridization with the overhanging region of the target sequences (FIG. 6).
- the chip was washed with 0.3 M NaNO 3 PBS buffer solution to remove chloride ions and nonspecifically bound nanoparticle probes.
- a salt or temperature-based stringency wash must be applied. Accordingly, a salt stringency wash (8 mM NaCl PBS buffer) was employed to selectively denature the imperfect duplexes (T 1 :P 2 and/or T 2 :P 1 , FIG. 6C and 6D) but not the duplexes formed from the perfectly complementary oligonucleotides (T 1 :P 1 and/or T 2 :P 2 , FIG. 6A and 6B). 9 After stringency wash and subsequent silver staining, the Raman measurements on the grey spots can be used to readily identify the target composition on the spots by the obtained spectra.
- This Raman detection format also can be used in protein microarray applications for screening protein-small molecule and protein-protein interactions.
- biotin and its mouse monoclonal antibody DIG (steroid digoxigenin) and its mouse monclonal antibody
- DNP dinitrophenyl
- the three small molecules were labeled with Raman dye-functionalized gold particles: the gold particles (13 nm in diameter) were modified with a small-molecule capped, Raman dye and alkylthiol-functionalized poly-adenine(A 20 ) (FIG. 11A).
- the proteins from all three pairs were immobilized in triplet onto aldehyde-functionalized glass slides by spotting the protein solution (200 ⁇ g/ml, 5% glycerol) with a commercial arrayer (FIG. 11A).
- a commercial arrayer FIG. 11A
- BSA bovine serum albumin
- mouse immunoglobulin G IgG
- ubiquitin and its antibody mouse immunoglobulin G
- human protein C mouse immunoglobulin G
- Gold nanoparticles were first functionalized with antibodies and then with Raman-dye labeled oligonucleotides. The labeling procedure is shown in FIG.
- the supernatant containing excess oligonucleotide, BSA, and antibodies can be decanted from the particles.
- the particle probes are then be redispersed in PBS buffer.
- the probes (2 nM for gold nanoparticles, about 2 ⁇ g/ml for the antibodies) were then used to develop the protein chips.
- the protocol for screening the protein-protein interactions is similar to that for protein-small molecule interactions (described above).
- FIG. 13 A- 4 The chip in FIG. 13 A- 4 was probed with all the three Raman labeled antibodies simultaneously. After silver enhancement, all three two-by-two dots array are clearly visible after silver develioping. Raman analysis shows no detectable cross reactivity and all of the correct dyes are in the correct spots (FIG. 13).
- this new scanometric detection format provides a general approach for genomic and porteomic detection but with a higher sensitivity and a higher multiple labeling capability.
- the number of available Raman dyes is much larger than the number of available and discernable fluorescent dyes. 20,21
- a Raman dye can be a fluorescent dye and also a non-fluorescent dye.
- a small modification of a dye can lead to a new dye with different Raman spectra and even the dyes which show undistinguishable fluorescent spectra can be distinguished by Raman spectroscopy. 16
- the data readout requires multi-lasers and multiple scans.
- All the Raman labels described above are single-dye systems: one carrier and Raman dye.
- One can load two or multiple Raman dyes onto a nano-sized nanoparticle carrier.
- Massively encoded Raman labels can be generated by tailoring the ratio between the components (FIG. 14 and 15 ).
- two alkylthiol capped-oligonucleotide strands with same base sequences but different the Raman labels (Cy3 and TMR) were used to modify 13-nm gold nanoparticles simultaneously, and therefore a composite Raman label was generated.
- This two-dye labeled nanopaticle probe has similar thermodynamic and kinetic properties as the single-dye labeled nanoparticle probe (i.e.
- microbead detection shows more flexibility in hybridization-based procedures, faster analyte diffusion kinetics, and they are easier and cheaper to produce.
- the microbead detection without the positional encoding in the microarrys must rely on some sort of barcoding strategy for the particle probes.
- a major problem in the current fluorescent-dye-based encoding approach is that the number of distinguishable labels are limited due to the broad emission spectra and energy transfer between organic dyes. 11 Raman labeling, in contrast, can overcome these difficulties.
- a three-component sandwich assay format can be used.
- glass microbeads 210-250 mm in diameter
- oligonucleotide capture strands FIG. 18
- Gold nanoparticles 13 nm in diameter
- pure or mixed Raman dye-labeled and alkylthiol-capped oligonucleotides probe strands were synthesized.
- the microbeads After washing and silver staining, the microbeads are show up as dark-grey spheres and exhibit the expected Raman signatures (FIG. 21). To achieve an easy readout process, we can align these microbeads mechanically (FIG. 22 top) and read them in serial fashion via scanning Raman spectroscopy. (FIG. 22, bottom). Moreover, the Raman fingerprints of the micorbeads can also be read out by fiber optics (FIG. 23).
- Raman labeling is a nano-size labeling methodology, and has much more flexibility than those micro-size ones.
- the footprints of the probes are smaller and the specificity and sensitivity of systems based on the probes can be dramatically improved over the systems based upon larger structures.
- This new nanoparticle-based methodology is important for a variety of reasons.
- the ratio of Raman intensities can be extracted from a single Raman spectrum using single laser excitation.
- the number of available Raman dyes is much larger than the number of available and discernable fluorescent dyes. 20,21,26
- a Raman dye can be either fluorescent or non-fluorescent, but a minor chemical modification of a dye molecule can lead to a new dye with a different Raman spectrum even though the two dyes exhibit virtually indistinguishable fluorescence spectra. 26 Therefore, this fingerprinting method offers potentially greater flexibility, a larger pool of available and non-overlapping probes, and higher multiplexing capabilities than conventional fluorescence-based detection approaches.
- the method incorporates all of the previous advantages of gold-nanoparticle based detection, including several orders of magnitude higher sensitivity and many orders of magnitude higher selectivity than the analogous molecular fluorescence based approach. 8,9
Abstract
Description
- This application claims priority based on U.S. provisional applications Nos. 60/378,538, filed May 7, 2002 (case no. 02-338); and 60/383,630, filed May 28, 2002 (case no. 02-338-A) which are hereby incorporated by reference in their entirety. The work described in this application has been supported in part from grants from the Air Force Office of Scientific Research, DARPA, and the NSF. Accordingly, the United States government may have some rights to the invention.
- The development of high-sensitivity, high-selectivity detection formats for chemical and biological molecules is of paramount importance for realizing the full potential of genomics and proteomics advances made over the past decade.1-4 High density gene chips have made it possible to monitor the levels of expression of thousands of genes simultaneously. Lower density chips have shown promise for both laboratory and clinical identification of many potential biohazards in one sample. Although the core accepted and utilized labeling technology is currently based upon molecular fluorophore markers, recent advances in nanoparticle technology have pointed toward systems with significantly higher sensitivities and selectivities and potentially more straightforward and versatile readout hardware than conventional fluorescence-based approaches.5-17 A strong argument is being made for nanoparticles as the next generation labeling technology for biodiagnostic research.
- One of the most sensitive and selective detection formats for DNA relies on oligonucleotide-functionalized nanoparticles as probes, a particle-initiated silver developing technique for signal enhancement, and a flatbed scanner for optical readout.8 The current demonstrated detection limit for this “scanometric DNA detection” format is 100 μM, and the utility of the system has been demonstrated with short synthetic strands, PCR products, and genomic DNA targets.17,18 A limitation of this approach is that it is inherently a one color system based upon grey scale. The flexibility and applicability of all DNA detection systems benefit from access to multiple types of labels with addressable and individually discernable labeling information. In the case of fluorescence, others have demonstrated that one can use multiple fluorophores, including quantum dots, to prepare encoded structures with optical signatures that depend upon the types of fluorophores used and their signal ratio within the probes.11,19 These approaches typically use micron size probes so that they can obtain encoded structures with the appropriate signal intensities and uniformities. Moreover, in the case of molecular fluorophores, due to overlapping spectral features and non-uniform fluorophore photobleaching rates,1,11 this approach has several potential complications.
- The art describes the use of Surface Enhanced Raman Spectroscopy (SERS) to detect various analytes. For example, U.S. Pat. No. 5,306,403 describes a method and apparatus for DNA sequencing using SERS. U.S. Pat. No. 5,266,498 describes the use of SERS to detect analytes in general. U.S. Pat. No. 5,445,972 describes the use of a Raman label bound to a specific binding molecule. U.S. Pat. No. 5,376,556 describes the use of SERS in immunoassays. U.S. Pat. No. 6,127,120 describes the use of SERS, the detection of nucleic acid and nucleic acid subunits. U.S. Pat. Nos. 6,242,264 and 6,025,202 describe the use of silver to form a SERS active substrate to enhance Raman scattering of adsorbed molecules.
- In the present invention, a reagent comprising a particle having a Raman labeled and specific binding member bound to the particle is used for assays of analytes. This reagent is particularly advantageous in that it can be bound to a binding partner analyte to form a complex and directly or indirectly bound to a support. The Raman label in the label complex on the support can then be SERS activated by staining, for example, silver, gold or copper enhancement to achieve a SERS effect when irradiated with a laser. Generally this complex is captured on a solid support and treated with silver to provide a SERS effect. Alternatively, the complex can be directly or indirectly reacted with an analyte which has already been bound to a solid support substrate. In the present invention, the SERS effect is produced near the time it is measured. This reagent can advantageously include multiple different Raman dyes bound to be particle carriers as a way distinguishing particular carriers with particular specific binding members as a way of indexing a vast number of reagent for multiplex application.
- Another advantageous reagent of this invention is a conjugate of several different Raman dyes bound to a specific binding substance such as DNA, RNA, polypeptide, antibody, antigen, small molecules, etc. This also serves as a reagent indexing tool.
- The invention is particularly distinguished from the prior art method in that the SERS technology is used in conjunction with nanoparticle assay techniques to provide extraordinary sensitivity and specificity of detection of analytes which is particularly amenable to multiplexed determination of analtyes.
- FIG. 1 illustrates a chip-based DNA detection method using nanoparticles functionalized with oligonucleotides and Raman labels.
- FIG. 2 illustrates a flatbed scanner image of microarrays after hybridized with nanoparticles functionalized with Cy3 labels, before (A) and after (B) silver staining. (C) A typical Raman Spectrum acquired from one of the silver stained spots. (D) A profile of Raman intensity at 1192 cm−1 as a function of position on the chip; the laser beam from the Raman instrument is moved over the chip from left to right as defined by the line in “B”.
- FIG. 3 illustrates the unoptimized detection limit of DNA using the Raman scanning method. (A) A microarray-based sandwich detection format; (B) A flatbed scanner image of microarrays for 20 fM target concentration after hybridized with nanoparticles functionalized with Cy3.5 labels; (C) A typical Raman spectrum acquired from one of the silver-stained spots; (D) A profile of Raman intensity at 1199 cm−1 as a function of position on the chip; the laser beam from the Raman instrument is moved over the chip from left to right as defined by the line in “B”.
- FIG. 4 illustrate Left: The Raman spectra of six dyes. Each dye correlates with a different color in our labeling scheme (see rectangular boxes). Right: six DNA target analysis systems. The information of target strand sequences were obtained from the web site of the National Center for Biological Information (http://www2.ncbi.nlm.nih.gov/Genbank/index.html).
- FIG. 5 illustrates (A) Flatbed scanner images of silver-stained microarrays and (B) corresponding Raman spectra. The colored boxes correlate with the color coded Raman spectra in FIG. 4.
- FIG. 6 illustrates the differentiation of two RNA targets (Target 1: perfect; Target 2: with one-base difference).
- FIG. 7 illustrates hybridization of
pure RNA target target - FIG. 8 illustrates (A) Typical flatbed scanner images of microarrays hybridized with nanoparticles, (1) before and (2) after stringency wash but prior to silver enhancing, and (3) after silver enhancing. Flatbed scanner image of microarrays hybridized with nanoparticles (4) before stringency wash but after silver enhancement. (B) A typical Raman spectrum (purple line) of the silver enhanced spots in (4), compared with the spectrum (black line) for mixed probes (1:1, probe 1:
probe 2, after silver enhacement). (C) Raman spectrum of the mixed probes (probe 1:probe 2, 1:1, after silver enhacement) compared with the spectra for probe 1 (with only TMR, blue line) or probe 2 (with only Cy3, red line). - FIG. 9 illustrates (A) typical flatbed scanner images of nanoparticle-functionalized microarrays, (1) before and (2) after stringency wash but prior to silver staining, and (3) after silver staining. (B) Raman spectra (1550˜1750 cm−1) from the stained spots at different ratios of
target 1 and target 2: (a) 1:0; (b) 5:1; (c) 3:1; (d) 1:1; (e) 1:2; (f) 1:3; (g) 1:5; (h) 0:1. The full Raman spectra from 400 to 1800 cm−1 are shown in the supporting information. The inset is a profile of Raman intensity ratio (Ib/I1) verse target ratio (T2/T1), where I1 is the Raman Intensity at 1650 cm−1 (from probe 1: TMR labeled gold oligonucleotide conjugate); I2 is the Raman Intensity at 1588 cm−1 (from probe 2: Cy3 labeled gold oligonucleotide conjugate). - FIG. 10 illustrates Raman spectra (400˜1800 cm−1) from the silver enhanced spots at
different target 1 to target 2 ratios: (a) 1:0; (b) 5:1; (c) 3:1; (d) 1:1; (e) 1:3; (f) 1:5; and (g) 0:1. - FIG. 11 illustrates (A) Scheme for screening protein-small molecule interactions. (B) Flatbed scanner images of silver-stained microarrays and (C) corresponding Raman spectra according to the color coded scheme in FIG. 4. Biotin was labeled with Cy3, DIG with Cy3.5 and DNP with Cy5. See supporting information for probe preparation details.
- FIG. 12 illustrates the Raman-based detection format for proteins.
- FIG. 13 illustrates (A1-4) Flatbed scanner images of silver-stained microarrays associated with the protein-protein screening experiments. (B) Color code for the Raman identification of the probes in the silver stained spots; no cross reactivity is observed. Anti-Mouse IgG was labeled with Cy3 modified-alkylthiol-capped poly adenine (A10), anti-ubiquitin by Cy3.5 modified-alkylthiol-capped Poly adenine (A10), and anti-human protein C by Cy5 modified-alkylthiol-capped Poly adenine (A10). The A10 oligonucleotide spacer was used to enhance the stability of the particle probes.33
- FIG. 14 illustrates the examples for creating Raman-labeled nanoparticle probes with mulplexing capabilities. R1, R2, R3, are different Raman dyes.
- FIG. 15 illustrates the creation of massive nanoparticle probes with multiple Raman labels.
- FIG. 16 illustrates Left: Raman spectrum of a probe with two Raman labels (Cy3:TMR=1:1, black line) after Ag staining in microarray form compared with the spectra for probes with only TMR (blue line) or Cy3 (red line). Right: Raman spectra of two-dye functionalized nanoparticle probes as a function of Cy3 to TMR ratio.
- FIG. 17 illustrates Left and Right: two Raman spectra of three-dye composite labels (black line) compared with the spectra of TMR (blue line), Cy3 (red line) and Cy3.5 (green line).
- FIG. 18 illustrates the microbead-based detection format using the scanning Raman method.
- FIG. 19 illustrates (A) and (B): The eight DNA target analysis systems. Each of the probe strands was marked by a single-dye or two-dye labels (see rectangular boxes and circles, corresponding Raman spectra. The colored boxes and circles correlate with the color coded Raman spectra in FIG. 20.
- FIG. 20 illustrates the Raman spectra of six single dyes and two mixed dyes, each spectra correlates with a different color in our labeling scheme (see rectangular boxes and circles).
- FIG. 21 illustrates microscopy image of silver-stained microspheres. The colored circles correlate with the color coded Raman spectra in FIG. 20.
- FIG. 22 illustrates optical microscope image of aligned silver-stained microspheres. The colored boxes correlate with the color coded Raman spectra in FIG. 20.
- FIG. 23 illustrates the fiberoptic-based detection format using microbeads.
- FIG. 24 illustrates the synthesis of Raman labeled oligonucleotides.
- The invention relates to reagents comprising particles having specific binding members and Raman labels bound to the particle wherein the particle can be treated with an enhancing stain such as silver, gold or copper to provide a SERS effect when irradiated.
- This reagent may be complexed with analyte which binds to the specific binding member and the resulting complex can be directly or indirectly captured on a substrate. The Raman label in the complex on the substrate is treated with a staining agent such as silver, gold or copper to activate the SERS effect when irradiated with a laser. Alternatively the analyte may be captured on the solid support substrate and reacted directly or indirectly with the reagent prior to staining and SERS measurement.
- The invention also encompasses a reagent of a specific binding substance having two or more different Raman labels bound thereto.
- The use of two or more different Raman labels on a reagent particle or a specific binding substance provides a way of indexing vast numbers of different particles and reagents for multiplexing applications.
- The invention includes methods of detecting analytes using these reagents and test kits containing reagents and other materials for carrying methods of the invention.
- More particularly the present invention relates to particles or carriers or Raman dye carriers functionalized with specific binding members and Raman labels, coupled with surface-enhanced Raman scattering (SERS) spectroscopy, to perform multiplexed detection of analytes. This is exemplified for DNA and RNA targets in FIG. 1. Although oligonucleotides can be directly detected by SERS on aggregated particles,26 the structural similarities of oligonucleotides with different sequences results in spectra that are difficult to distinguish. Therefore, one must use different Raman dyes to label different oligonucleotides to distinguish oligonucleotide sequences.20,21 Previously a SERS-based detection methodology that allows for single or multiplexed sandwich hybridization assay formats had not been demonstrated. In part, this conspicuous technological absence is due to the difficulty in reproducibly generating and functionalizing stable SERS-active substrates23 as well as a lack of an appropriate probe design strategy to enable multiplexed detection. To get the benefits of high sensitivity and high selectivity detection coupled with multiple labeling capabilities, a new type of particle probe has been designed that can be used, for example, for DNA (or RNA) detection (FIG. 1), but is equally applicable to other specific binding substances. These probes consist of 13-nm gold particles functionalized with Raman-dye labeled oligonucleotides. Particles of various size, shape and materials may be used. The Raman spectroscopic fingerprint, which can be designated through choice of Raman label can be read out after silver enhancing via scanning Raman spectroscopy (FIG. 1). Because the SERS-active substrate in this strategy is generated prior to the detection event, a large and reproducible Raman scattering response can be obtained.
- Accordingly, in one embodiment of the invention, a method for detecting for the presence or absence of one or more target analytes in a sample, the target analytes having at least two binding sites, is provided. The method comprises:
- providing a substrate having bound thereto one or more types of a first specific binding complements for immobilizing the target analyte onto said substrate;
- providing one or more types of particles having bound thereto (a) one or more Raman active labels; and (b) a second specific binding complement for binding to a specific target analyte, wherein (i) the Raman active label bound to each type of particle is different and serves as an identifier for a specific target analyte; (ii) the second specific binding complement bound to each type of particle is different and is targeted to a specific target analyte; and (iii) the Raman active label comprises at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- contacting the particles, the sample and the substrate under conditions effective for specific binding interactions between the target analyte and first and second specific binding complements so as to form a test substrate having particles complexed thereto in the presence of one or more target analytes in the sample;
- contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and
- determining for the presence of said particle complexes on said detection substrate as an indication of the presence of one or more target analytes in the sample by obtaining and analyzing a SERS spectrum.
- In another embodiment of the invention, a method for detecting for the presence or absence of one or more target nucleic acids in a sample, the sequence of the nucleic acid having at least two portions, is provided. The method comprises:
- providing a substrate having a oligonucleotides bound thereto, the oligonucleotides bound to the substrate having a sequence that is complementary to a first portion of the nucleic acid;
- providing one or more types of particles comprising oligonucleotides bound thereto and a Raman active label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to each type of particle have a sequence that is complementary to a second portion of the sequence of a specific target nucleic acid; and (ii) the Raman active label bound to each type of particles is different and serves as an identifier for a specific target nucleic acid, said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing Raman scattering;
- contacting the particles, the substrate, and the sample under conditions effective for hybridization of the oligonucleotides bound to the substrate with the first portion of the nucleic acid and for hybridization of the oligonucleotides attached to the particle with the second portion of the nucleic acid so as to form a test substrate having one or more particle complexes bound thereto when one or more target nucleic acids are present in said sample;
- contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and
- determining for the presence of said particle complexes on said detection substrate as an indication of the presence of one or more target nucleic acids in the sample by obtaining and analyzing a SERS spectrum.
- In yet another embodiment of the invention, a method for detecting for the presence or absence of a target nucleic acid in a sample, the sequence of the nucleic acid having at least two portions, is provided. The method comprises:
- providing a substrate having oligonucleotides bound thereto, the oligonucleotides bound to the substrate having a sequence that is complementary to a first portion of the nucleic acid;
- providing a particle comprising oligonucleotides bound thereto and a Raman label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to the particle have a sequence that is complementary to a second portion of the nucleic acid; and (ii) the Raman active label bound to particles serves as an identifier for the target nucleic acid, said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- contacting the particles, the substrate, and the sample under conditions effective for hybridization of the oligonucleotides bound to the substrate with the first portion of the nucleic acid and for hybridization of the oligonucleotides attached to the particle with the second portion of the nucleic acid so as to form a test substrate having a particle complex bound thereto when said target nucleic acid is present in said sample;
- contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and
- determining for the presence of said particle complex on said detection substrate as an indication of the presence of the target nucleic acid in the sample by obtaining and analyzing a SERS spectrum.
- In yet another embodiment of the invention, a method for detecting for the presence or absence of a single nucleotide polymorphism in a nucleic acid in a sample, the sequence of the nucleic acid having at least two portions, is provided. The method comprises:
- providing a substrate having a oligonucleotides bound thereto, the oligonucleotides bound to the substrate having a sequence that is complementary to a first portion of the nucleic acid;
- providing one or more types of particles comprising oligonucleotides bound thereto and a Raman active label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to each type of particle have a sequence that is believed to be complementary to a second portion of the sequence of the nucleic acid, said second portion of the sequence of the nucleic acid is suspected of having a single nucleotide substitution when compared to a wild type sequence of the nucleic acid; and (ii) the Raman active label bound to each type of particles is different and serves as an identifier for a specific sequence having a single nucleotide substitution, said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- contacting the particles, the substrate, and the sample under conditions effective for hybridization of the oligonucleotides bound to the substrate with the first portion of the nucleic acid and for hybridization of the oligonucleotides attached to the particle with the second portion of the nucleic acid so as to form a test substrate having one or more particle complexes bound thereto;
- applying a stringency wash to the substrate to substantially remove any non-specifically bound particles and any particle complexes having oligonucleotides that are not complementary to the second portion of the nucleic acid sequence;
- contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and
- determining for the presence of any particle complexes on said detection substrate as an indication of the existence of a single nucleotide morphism in said nucleic acid in the sample by obtaining and analyzing a SERS spectrum.
- In the foregoing methods, the nucleic acid is first contacted with the substrate so that the first portion of the nucleic acid sequence hybridizes with complementary oligonucleotides bound to the substrate and then the nucleic acid bound to the substrate is contacted with the particles having oligonucleotides bound thereto so that at least some of the oligonucleotides bound to the particles hybridize with the second portion of the sequence of the nucleic acid bound to the substrate.
- In another aspect of the invention, the nucleic acid is first contacted with the particles having oligonucleotides bound thereto so that at least some of the oligonucleotides bound to the particles hybridize with a second portion of the sequence of the nucleic acid; and then contacting the nucleic acid bound to the particles with the substrate so that the first portion of the sequence of the nucleic acid bound to the particles hybridizes with complementary oligonucleotides bound to the substrate. In another embodiment, the substrate has a plurality of types of oligonucleotides attached thereto in an array to allow for the detection of multiple portions of a single type of nucleic acid, the detection of multiple types of nucleic acids, or both.
- In another aspect of the invention, at least two or more different Raman active labels are used. The ratio of the two or more types of Raman labels may be the same or different.
- In yet another embodiment of the invention, a reagent is provided. The reagent comprises having at least one type of Raman active label bound thereto and a specific binding complement for binding to a specific target analyte, wherein (i) the Raman active label serves as an identifier for a specific target analyte; and (ii) the Raman active label comprises at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering.
- In yet another embodiment, a reagent is provided. The reagent comprises a particle, oligonucleotides bound to the particle and at least one type of Raman label bound to a portion of the oligonucleotides, wherein at least some of the oligonucleotides bound to the particle have a sequence that is complementary to at least a portion of a target nucleic acid.
- In another aspect of the invention, the reagent comprises a particle, oligonucleotides bound to the particle, an oligonucleotide connector having first and second portions, an oligonucleotide having at least one type of Raman label bound thereto, wherein at least some of the oligonucleotides bound to the particles have a sequence that is complementary to the first portion of the oligonucleotide connector, the oligonucleotide having the Raman active label bound thereto has a sequence that is complementary to the second portion of the oligonucleotide connector, and at least a portion of the oligonucleotides bound to the particles have a sequence that is complementary to a target nucleic acid.
- In yet another aspect of the invention, the reagent comprises a particle, oligonucleotides bound to the particle, an oligonucleotide connector having first and second portions, an oligonucleotide having at least one type of Raman label bound thereto, and an oligonucleotide having a specific binding complement to a target analyte, wherein at least some of the oligonucleotides bound to the particles have a sequence that is complementary to the first portion of the oligonucleotide connector, the oligonucleotide having the Raman active label bound thereto has a sequence that is complementary to the second portion of the oligonucleotide connector, and the oligonucleotide having the specific binding complement bound thereto has a sequence that is complementary to the second portion of the oligonucleotide connector.
- In another embodiment of the invention, a kit is provided for the detection of one or more target analytes in a sample. The kit has in one container a reagent comprising a particle having a specific binding member and at least one Raman label bound to the particle; a staining reagent; and a substrate having a capture reagent. A representative kit comprises:
- one or more types of conjugates comprising particles, oligonucleotides bound to the particles, a Raman label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to each type of particle have a sequence that is complementary to a second portion of the sequence of a specific target nucleic acid; and (ii) the Raman active label bound to each type of particles is different and serves as an identifier for a specific target nucleic acid, said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- an optional substrate having oligonucleotides bound there, the oligonucleotides bound to the substrate have a sequence that is complementary to a first portion of a sequence of the target nucleic acid; and
- optional stain reagents for creating a substrate surface capable of causing surface-enhanced Raman scattering (SERS).
- In another embodiment of the invention, a kit is provided for the detection of one or more target analytes in a sample, the sequence of the nucleic acid having at least two portions. The kit comprises:
- particles comprising oligonucleotides bound thereto, a Raman label bound to a portion of the oligonucleotides, wherein (i) at least some of the oligonucleotides attached to the particle have a sequence that is complementary to a second portion of the sequence of the target nucleic acid; and (ii) the Raman active label bound to the particles serves as an identifier for the target nucleic acid, said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering; and
- an optional substrate having oligonucleotides bound there, the oligonucleotides bound to the substrate have a sequence that is complementary to a first portion of a sequence of the target nucleic acid; and
- In another embodiment of the invention, a kit is provided for the detection of one or more target nucleic acids in a sample, the sequence of the nucleic acid having at least two portions. The kit comprises:
- a first container including oligonucleotides having Raman active labels attached thereto, wherein the oligonucleotides the Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- a second container including conjugates comprising particles and oligonucleotides bound to the particles, wherein at least some of the oligonucleotides attached to each type of particle have a sequence that is complementary to at least a portion of the sequence of the oligonucleotides having Raman active labels; and
- an optional substrate having oligonucleotides bound there, the oligonucleotides bound to the substrate have a sequence that is complementary to a first portion of a sequence of the target nucleic acid; and
- optional stain reagents for creating a substrate surface capable of causing surface-enhanced Raman scattering (SERS).
- In another embodiment of the invention, a kit is provided for the detection of one or more target nucleic acids in a sample, the sequence of the nucleic acid having at least two portions. The kit comprises:
- one or more containers including oligonucleotides having one or more types of Raman active labels attached thereto, wherein the Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- a second container including conjugates comprising particles and oligonucleotides bound to the particles, wherein at least some of the oligonucleotides attached to each type of particle have a sequence that is complementary to at least a portion of the sequence of the oligonucleotides having Raman active labels; and
- an optional substrate having oligonucleotides bound there, the oligonucleotides bound to the substrate have a sequence that is complementary to a first portion of a sequence of the target nucleic acid and optional staining material reagents.
- In another embodiment of the invention, a method for screening one or more molecules to determine whether the molecule is a ligand to one or more specific receptors, the molecules are present in a sample, is provided. The method comprises:
- providing a substrate having bound thereto one or more specific receptors;
- providing reagents comprising particles, specific binding substance bound to the particles, a Raman active label bound to a portion of the specific binding substance, and the molecule from said sample bound to a portion of the specific binding substance, wherein said Raman active label comprising at least one Raman active molecule providing a detectable or measurable Raman scattering signal when illuminated by radiation capable of inducing a Raman scattering;
- contacting the particles, sample and substrate under conditions effective for specific binding interactions between the molecule bound to the particles with the specific receptor bound to the substrate so as to form a test substrate having particles complexed thereto when the molecule is a ligand to a specific receptor;
- contacting the test substrate with a staining material to produce a detection substrate having a surface capable of causing surface-enhanced Raman scattering (SERS); and
- determining for the presence of said particle complexes on said detection substrate as a confirmation of a ligand to a specific receptor by obtaining and analyzing a SERS spectrum.
- The invention also includes in another aspect a fiber optic analyte detection device in which a particle reagent with specific binding substance and Raman labels is associated with the ends of optical fibers in an optical cable.
- These and other embodiments of the invention will be apparent in light of the detailed description below.
- (A) Definitions
- “Analyte,” as used herein, is the substance to be detected in the test sample using the present invention. The analyte can be any substance for which there exists a naturally occurring specific binding member (e.g., an antibody, polypeptide, DNA, RNA, cell, virus, etc.) or for which a specific binding member can be prepared, and the analyte can bind to one or more specific binding members in an assay. “Analyte” also includes any antigenic substances, haptens, antibodies, and combinations thereof. The analyte can include a protein, a peptide, an amino acid, a carbohydrate, a hormone, asteroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances.
- “Analyte-analog”, as used herein, refers to a substance which cross reacts with an analyte specific binding member although it may do so to a greater or lesser extent than does the analyte itself. The analyte-analog can include a modified analyte as well as a fragmented or synthetic portion of the analyte molecule so long as the analyte analog has at least one epitopic site in common with the analyte of interest.
- “Analyte epitope,” as used herein, denotes that part of the analyte which contacts one member of the specific ligand binding pair during the specific binding event. That part of the specific binding pair member which contacts the epitope of the analyte during the specific binding event is termed the “paratope.”
- “Analyte-mediated ligand binding event,” as used herein, means a specific binding event between two members of a specific ligand binding pair, the extent of the binding is influenced by the presence, and the amount present, of the analyte. This influence usually occurs because the analyte contains a structure, or epitope, similar to or identical to the structure or epitode contained by one member of the specific ligand binding pair, the recognition of which by the other member of the specific ligand binding pair results in the specific binding event. As a result, the analyte specifically binds to one member of the specific ligand binding pair, thereby preventing it from binding to the other member of the specific ligand binding pair.
- “Ancillary Specific binding member,” as used herein, is a specific binding member used in addition to the specific binding members of the captured reagent and the indicator reagent and becomes a part of the final binding complex. One or more ancillary specific binding members can be used in an assay. For example, an ancillary specific binding member can be used in an assay where the indicator reagent is capable of binding the ancillary specific binding member which in turn is capable of binding the analyte.
- “Associated,” as used herein, is the state of two or more molecules and/or particulates being held in close proximity to one another.
- “Capture reagent,” as used herein, is a specific binding member, capable of binding the analyte or indicator reagent, which can be directly or indirectly attached to a substantially solid material. The solid phase capture reagent complex can be used to separate the bound and unbound components of the assay.
- “Conjugate,” as used herein, is a substance formed by the chemical coupling of one moiety to another. An example of such species include the reaction product of bovine serum albumin with chemically activated theophylline molecules and the reaction product of chemically activated Raman-active labels with a protein molecule, such as an antibody, or with a ligand, such as biotin.
- “Enhancer,” a stain such as a silver or gold stain that provides for activating Raman labels on particles to produce a SERS effect.
- “Indicator reagent,” as used herein comprises a detectable label directly or indirectly attached to a specific binding member or metal surface.
- “Intervening molecule,” as used herein, is any substance to which both a specific binding pair member and a Raman-active label are attached.
- “Particles,” as used herein, is any substance which can be dispersed in a liquid and which will support the phenomenon of a surface-enhanced Raman light scattering (SERS) or surface-enhanced resonance Raman light scattering (SERRS). Examples of particles include, but are not limited to: Colloids of gold or silver, Pt, Cu, Ag/Au, Pt/Au, Cu/Au, coreshell or alloy particles; particles or flakes of gold, silver, copper, or other substances displaying conductance band electrons. As the particle surface participates in the SERS and SERRS effect, flakes or particles of substances not displaying conductance band electrons, which have been coated with a substance which does, also become suitable particulates.
- “Radiation,” as used herein, is an energy in the form of electromagnetic radiation which, when applied to a test mixture, causes a Raman spectrum to be produced by the Raman-active label therein.
- “Raman label,” as used herein, is any substance which produces a detectable Raman spectrum, which is distinguishable from the Raman spectra of other components present, when illuminated with a radiation of the proper wavelength. Other terms for a Raman-active label include dye and reporter molecule. Such labels are shown on
pp 25. - “SERRS (Surface Enhanced Resonance Raman Scattering)” results when the adsorbate at a SERS active surface is in resonance with the laser excitation wavelength. The resultant enhancement is the product of the resonance and surface enhancement.
- “SERS (Surface-Enhanced Raman Scattering)” means the increase in Raman scattering exhibited by certain molecules in proximity to certain metal surfaces.
- “Specific binding member,” as used herein, is a member of a specific binding pair, i.e., two different molecules where one of the molecules, through chemical or physical means, specifically binds to the second molecule. In addition to antigen and antibody-specific binding pairs, other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences (including probe and captured nucleic acid sequences used in DNA hybridization assays to detect a target nucleic acid sequence), complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors a and enzymes, cells, viruses and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member. For example a derivative or fragment of the analyte, i.e., an analyte-analog, can be used so long as it has at least one epitope in common with the analyte. Immunoreactive specific binding members include antigens, haptens, antibodies, and complexes thereof including those formed by recombinant DNA methods or peptide synthesis.
- “Test mixture,” as used herein, means a mixture of the test sample and other substances used to apply the present invention for the detection of analyte in the test sample. Examples of these substances include: Specific binding members, ancillary binding members, analyte-analogs, Raman-active labels, buffers, diluents, and particulates with a surface capable of causing a surface-enhanced Raman spectroscopy, and others.
- “Test sample,” as used herein, means the sample containing the analyte to be detected and assayed using the present invention. The test sample can contain other components besides the analyte, can have the physical attributes of a liquid, or a solid, and can be of any size or volume, including for example, a moving stream of liquid. The test sample can contain any substances other than the analyte as long as the other substances do no interfere with the specific binding of the specific binding member or with the analyte or the analyte-analog. Examples of test samples include, but are not limited to: Serum, plasma, sputum, seminal fluid, urine, other body fluids, and environmental samples such as ground water or waste water, soil extracts, air and pesticide residues.
- (B) Reagents
- The present invention contemplates the use of any suitable particle having Raman labels and specific binding substances attached thereto that are suitable for use in detection assays. In practicing this invention, however, nanoparticles are preferred. The size, shape and chemical composition of the particles will contribute to the properties of the resulting probe including the DNA barcode. These properties include optical properties, optoelectronic properties, electrochemical properties, electronic properties, stability in various solutions, pore and channel size variation, ability to separate bioactive molecules while acting as a filter, etc. The use of mixtures of particles having different sizes, shapes and/or chemical compositions, as well as the use of nanoparticles having uniform sizes, shapes and chemical composition, are contemplated. Examples of suitable particles include, without limitation, nano- and microsized core particles, aggregate particles, isotropic (such as spherical particles) and anisotropic particles (such as non-spherical rods, tetrahedral, prisms) and core-shell particles such as the ones described in U.S. patent application Ser. No. 10/034,451, filed Dec. 28, 2002 and International application no. PCT/US01/50825, filed Dec. 28, 2002, which are incorporated by reference in their entirety.
- Nanoparticles usefuil in the practice of the invention include metal (e.g. gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetite) colloidal materials. Other nanoparticles useful in the practice of the invention include ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, and GaAs. The size of the nanoparticles is preferably from about 1.4 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm. The nanoparticles may also be rods, prisms, cubes, tetrahedra, or core shell particles.
- Methods of making metal, semiconductor and magnetic nanoparticles are well-known in the art. See, e.g., Schmid, G. (ed.)Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold: Principles, Methods, and Applications (Academic Press, San Diego, 1991); Massart, R., IEEE Taransactions On Magnetics, 17, 1247 (1981); Ahmadi, T. S. et al., Science, 272, 1924 (1996); Henglein, A. et al., J. Phys. Chem., 99, 14129 (1995); Curtis, A. C., et al., Angew. Chem. Int. Ed. Engl., 27, 1530 (1988).
- Methods of making ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, and GaAs nanoparticles are also known in the art. See, e.g., Weller, Angew. Chem. Int. Ed. Engl., 32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988); Henglein, Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A., 53, 465 (1991); Bahncmann, in Photochemical Conversion and Storage of Solar Energy (eds. Pelizetti and Schiavello 1991), page 251; Wang and Herron, J. Phys. Chem., 95, 525 (1991); Olshavsky et al., J. Am. Chem. Soc., 112, 9438 (1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992).
- Suitable nanoparticles are also commercially available from, e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc. (gold).
- Presently preferred for use in detecting analytes are gold nanoparticles. Gold colloidal particles have high extinction coefficients for the bands that give rise to their beautiful colors. These intense colors change with particle size, concentration, interparticle distance, and extent of aggregation and shape (geometry) of the aggregates, making these materials particularly attractive for calorimetric assays. For instance, hybridization of oligonucleotides attached to gold nanoparticles with oligonucleotides and nucleic acids results in an immediate color change visible to the naked eye.
- (C) Attachment of Specific Binding Members
- The particles, the specific binding member or both are functionalized in order to attach to the particles. Such methods are well known in the art. For instance, oligonucleotides functionalized with alkanethiols at their 3′-termini or 5′-termini readily attach to gold nanoparticles. See Whitesides,Proceedings of the Robert A. Welch Foundation 39th Conference On Chemical Research Nanophase Chemistry, Houston, Tex., pages 109-121 (1995). See also, Mucic et al. Chem. Commun. 555-557 (1996) (describes a method of attaching 3′ thiol DNA to flat gold surfaces; this method can be used to attach oligonucleotides to nanoparticles). The alkanethiol method can also be used to attach oligonucleotides to other metal, semiconductor and magnetic colloids and to the other nanoparticles listed above. Other functional groups for attaching oligonucleotides to solid surfaces include phosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 for the binding of oligonucleotide-phosphorothioates to gold surfaces), substituted alkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4, 370-377 (1974) and Matteucci and Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981) for binding of oligonucleotides to silica and glass surfaces, and Grabar et al., Anal. Chem., 67, 735-743 for binding of aminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes). Oligonucleotides terminated with a 5′ thionucleoside or a 3′ thionucleoside may also be used for attaching oligonucleotides to solid surfaces. The following references describe other methods which may be employed to attached oligonucleotides to nanoparticles: Nuzzo et al., J. Am. Chem. Soc., 109, 2358 (1987) (disulfides on gold); Allara and Nuzzo, Langmuir, 1, 45 (1985) (carboxylic acids on aluminum); Allara and Tompkins, J. Colloid Interface Sci., 49, 410-421 (1974) (carboxylic acids on copper); Iler, The Chemistry Of Silica,
Chapter 6, (Wiley 1979) (carboxylic acids on silica); Timmons and Zisman, J. Phys. Chem., 69, 984-990 (1965) (carboxylic acids on platinum); Soriaga and Hubbard, J. Am. Chem. Soc., 104, 3937 (1982) (aromatic ring compounds on platinum); Hubbard, Acc. Chem. Res., 13, 177 (1980) (sulfolanes, sulfoxides and other functionalized solvents on platinum); Hickman et al., J. Am. Chem. Soc., 111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv, Langmuir, 3, 1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir, 3, 1034 (1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074 (1989) (silanes on silica); Eltekova and Eltekov, Langmuir, 3, 951 (1987) (aromatic carboxylic acids, aldehydes, alcohols and methoxy groups on titanium dioxide and silica); Lec et al., J. Phys. Chem., 92, 2597 (1988) (rigid phosphates on metals). - U.S. patent application Ser. Nos. 09/760,500 and 09/820,279 and international application nos. PCT/US01/01190 and PCT/US01/10071 describe oligonucleotides functionalized with a cyclic disulfide which are useful in practicing this invention. The cyclic disulfides preferably have 5 or 6 atoms in their rings, including the two sulfur atoms. Suitable cyclic disulfides are available commercially or may be synthesized by known procedures. The reduced form of the cyclic disulfides can also be used.
- Those skilled in the art recognize a large variety of methods by which antigen, antibodies, small molecules or carbohydrates can be bound to particles.
- (D) Substrates
- Any substrate can be used which allows observation of the detectable change. Suitable substrates include transparent solid surfaces (e.g., glass, quartz, plastics and other polymers), opaque solid surface (e.g., white solid surfaces, such as TLC silica plates, filter paper, glass fiber filters, cellulose nitrate membranes, nylon membranes), and conducting solid surfaces (e.g., indium-tin-oxide (ITO)). The substrate can be any shape or thickness, but generally will be flat and thin. Preferred are transparent substrates such as glass (e.g., glass slides or glass beads) or plastics (e.g., wells of microtiter plates). The ends of optical fiber in a fiber optical cable serve as a substrate in one embodiment of the invention.
- (E) Attachment of Capture Probes to a Substrate
- Any suitable method for attaching an analyte to a substrate may be used. For instance, oligonucleotides can be attached to the substrates as described in, e.g., Chrisey et al.,Nucleic Acids Res., 24, 3031-3039 (1996); Chrisey et al., Nucleic Acids Res., 24, 3040-3047 (1996); Mucic et al., Chem. Commun., 555 (1996); Zimmermann and Cox, Nucleic Acids Res., 22, 492 (1994); Bottomley et al., J. Vac. Sci Technol. A, 10, 591 (1992); and Hegner et al., FEBS Lett., 336, 452 (1993).
- When a substrate is employed, a plurality of capture probes may be attached to the substrate in an array for detecting multiple different target analytes. For instance, a substrate may be provided with rows of spots, each spot containing a different type of capture probes designed to bind a reagent analyte complex. A sample containing one or more analytes is applied to each spot, and the rest of the assay is performed in one of the ways described above using appropriate reagents of the invention.
- (F) Raman Labels
- The Raman labels, can be any one of a number of molecules with distinctive Raman scattering spectra. Unlike the enzymes used in enzyme imununoassays, these label species can be stable, simple, inexpensive molecules which can be chemically modified as required.
- The following attributes enhance the effectiveness of the label in this application: (a) A strong absorption band in the vicinity of the laser excitation wavelength (extinction coefficient near 104; (b) A functional group which will enable covalent attachment to a specific binding member; (c) Photostability; (d) Sufficient surface and resonance enhancement to allow detection of analyte in the subnanogram range; (e) Minimal interference in the binding interaction between the labeled and unlabeled specific binding members; (f) Minimal exhibition of strong fluorescence emission at the excitation-wavelength used; (g) A relatively simple scattering pattern with a few intense peaks; and/or (h) Labels with scattering patterns which do not interfere with each other so several indicator molecules may be analyzed simultaneously.
- The following is a listing of some, but not all potential candidates for these Raman-active label: 4-(4-Aminophenylazo)phenylarsonic acid monosodium salt, arsenazo I, basic fuchsin, Chicago sky blue, direct red 81, disperse
orange 3, HABA (2-(4-hydroxyphenylazo)-benzoic acid), erythrosin B, trypan blue, ponceau S, ponceau SS, 1,5-difluoro-2,4-dinitrobenzene, cresyl violet and p-dimethylaminoazobenzene. The chosen labels may be covalently attached to the specific binding members of interest or attached or associated with. - An important aspect of the invention is that multiple Raman or Raman labels may be bound to the particle to provide a multicoding Rarnan labels for indexing different particles. Thus, the invention includes a reagent which has multiple Raman dyes and a specific binding substance, such as DNA, RNA, antibody, antigen, small molecule bound to the particle.
- The multiple Raman label also need not be bound to the particle but may be complexed to the particle through specific binding reactions. Thus, the invention encompasses multiple SERS reagents bound to a specific binding ligand such as DNA, RNA, antibody, antigen, small molecule, cell or virus. This embodiment may be envisioned as follows:
- Raman1-Raman2-Raman3-(specific binding ligand)
- (G). Excitation Sources
- In the preferred embodiment, a laser serves as the excitation source. The laser may be of an inexpensive type such as a helium-neon or diode laser. An operating lifetime of such lasers may be in excess of 50,000 hours.
- In one embodiment, a diode laser is used to excite at or at the near IR spectrum, minimizing fluorescence interference. The excitation sources used need not necessarily be monochromatic and they also need not necessarily have to be of high intensity. Lamps may also be used.
- The SERS effect can be excited by direct illumination of the surface or by evanescent waves from a waveguide beneath the plasmon-active surface.
- (H.) Raman Labeled Probes
- Several different conjugates could be prepared from specific binding members having different specificities, each type with a different Raman active label having a distinctive scattering pattern. Mixing these conjugates in an assay would allow the simultaneous analysis of several different analytes in the same sample. In another aspect of the invention, the conjugate may include two or more different Raman labels.
- It is important to note that in contrast with conventional fluorescence-based chip detection, the ratio of Raman intensities can be extracted from a single Raman spectrum using single laser excitation. Moreover, the number of available Raman dyes is much larger than the number of available and discernable fluorescent dyes.20,21,26 A Raman dye can be either fluorescent or non-fluorescent. A minor chemical modification of a dye molecule can lead to a new dye with different Raman spectra even though the two dyes exhibit virtually indistinguishable fluorescence spectra.26 Therefore, this Raman fingerprinting method offers potentially greater flexibility, a larger pool of available and non-overlapping probes, and higher multiplexing capabilities than conventional fluorescence-based detection approaches. This approach has been extended to random array, bead based format where high multiplexing capabilities are essential are underway.
- (I). SERS Enhancement (Enhancer)
- Initially, the Raman-labeled probes have little or no detectable SERS activity. Staining material such as silver stains provide strong SERS enhancment. When a substrate is employed, a detectable change can be produced or further enhanced by silver staining. Silver staining can be employed with any type of nanoparticles that catalyze the reduction of silver. Preferred are nanoparticles made of noble metals (e.g., gold and silver). See Bassell, et al.,J. Cell Biol., 126, 863-876 (1994); Braun-Howland et al., Biotechniques, 13, 928-931 (1992). If the nanoparticles being employed for the detection of a nucleic acid do not catalyze the reduction of silver, then silver ions can be complexed to the nucleic acid to catalyze the reduction. See Braun et al., Nature, 391, 775 (1998). Also, silver stains are known which can react with the phosphate groups on nucleic acids.
- Silver, gold or copper staining can be used to produce or enhance a detectable change in any assay performed on a substrate, including those described above. In particular, silver staining has been found to provide a huge increase in sensitivity for assays employing a single type of nanoparticle so that the use of layers of nanoparticles can often be eliminated.
- (J). Detection of Raman Scattering
- Several methods are available for detecting Raman scattering. These generally can be used with different types of spectrometers. In SERS, the primary measurement is one of light scattering intensity at particular wavelengths. SERS requires measuring wavelength-shifted scattering intensity in the presence of an intense background from the excitation beam. The use of a Raman-active substance having a large Stokes shift simplifies this measurement.
- Several concepts for further simplifying the readout instrument have been proposed. These include the use of wavelength selective mirrors, filters or holographic optical elements for scattered light collection.
- Neither the angle of the incident light beam to the surface nor the position of the detector is critical using SERS. With flat surfaces positioning the surface of the laser beam at 60 degrees to the normal is commonly done and detection at either 90 degrees or 180 degrees to the beam are standard. SERS excitation can be performed in the near infrared range which would suppress intrinsic sample fluorescence. It may also be possible to perform SERS-based ligand binding assays using evanescent waves produced by optical waveguides.
- No signal development time is required as readout begins immediately upon illumination and data can be collected for as long as desired without decay of signal unless the excitation light is extremely intense and chemical changes occur. The signal cannot overdevelop as in systems dependent on optical absorbance. Unlike fluorescent readout systems. SERS reporter groups will not self-quench so the signal can be enhanced by increasing the number of Raman reporter groups on the probe molecule. Fluorescent molecules near the SERS-active surface will also be surface-quenched.
- (K.) Instrumentation
- The present invention is adaptable for use as an automatic analyzer. Since the instrument would monitor discrete Stokes shifted spectral lines, the need for an elaborate monochromator system is not necessary. Recent advances in state-of-the-art optics technology, such as holographic optical elements, allow the design of a suitable spectrometer with cost and complexity below that of the laboratory grade device.
- Optical readout energies as a result of SERS are above that which require ultra-sensitive photon counting devices. In fact, some SERRS spectrometers now in use incorporate silicon photodiode detectors. The optical efficiency of a typical monochromator used in a laboratory grade spectrometer is less than 10%. The advances in optical materials and components mentioned above should make possible two to three-fold increases in optical efficiency for a simple spectrometer dedicated to only a few specific spectral lines. This also addresses one of the previously major concerns, blocking of the Rayleigh scattering line. With blocking capabilities of newer filters on the order of 10−9, substitution of filters for one or more stages of the typical monochrometer system should be possible with significant cost savings.
- Microarray Fabrication.
- Oligonucleotide capture strands were immobilized onto the SMPB-(succinimidyl 4-(malemidophenyl)-butyrate) functionalized glass slide by spotting 5′-hexyl-thiol-capped oligoucloetides (1 mM in a 0.15 M NaCl, pH 6.5 phosphate buffer solution (PBS, 10 mM phosphate)) with a commercial arrayer (GMS 417 arrayer, Genotic MicroSystems, Inc). After spotting the chip with the capture oligonucleotides (˜200 μm spots), the chip was kept in a humidity chamber for 12 hours to effect the coupling reaction between SMPB and the hexylthiol-capped oligonucleotides. Then the chip was washed copiously with Nanopure water. Passivation of the areas of the chip surrounding the oligonucleotide spots was carried out by immersing the chip in a solution of hexylthiol-capped poly-adenine (A15) (0.1 mM) for 4 h and then in a solution of 3-mercapto-propane sulfonic acid, sodium salt (0.2 M) for 30 minutes to cap off the remaining SMPB sites. Finally, the chip was washed with Nanopure water and dried by a microarray centrifuge (2000 g).
- Synthesis and Purification of Cy3-labeled-(propylthiol)-capped Oligonucleotides.
- This Example describes the synthesis of an oligonucleotide having a Raman label attached thereto: (3′HS-Cy3-A10-AAT CTC AAC GTA CCT, (
SEQ ID NO 1. in FIG. 19a) 3′HS-Cy3-A10-CTC-CCT-AAT-AAC-AAT) (SEQ ID NO. 25 in FIG. 1) - The Cy3-modified, (propylthiol)-capped oligonucleotides were synthesized on a 1 μmol scale using
standard phosphoramidite chemistry 5 with a Thiol-Modifier C3 S—S CPG (controlled-pore glass) solid support on a commercial synthesizer (Expedite). The Cy3-CE phospboramidite (Indodicarbocyanine pH 7 and a 1%/min gradient of 95% CH3CN/5% 0.03 M TEAA at a flow rate of 3 mL/min, while monitoring the UV signal of DNA at 254 nm and 550 nm. The DMT was cleaved by dissolving the purified oligonucleotides in an 80% acetic acid solution for 30 min, followed by evaporation; the oligonucleotides were redispersed in 500 μL of water, and the solutions were extracted with ethyl acetate (3×300 μL). After evaporation of the solvent, the oligonucleotides were redispersed in 400 μL of a 0.1 M dithiothreotol (DTT), 0.17 M phosphate buffer (pH 8) solution at room temperature for 2 h to cleave the 3′ disulfide. Aliquots of this solution (<10 ODs) were purified through a desalting NAP-5 column (Amersham Pharrnacia Biotech AB). - Synthesis and Purification of TMR-, Cy3.5- and Cy5-Labeled-(propylthiol)-Capped Oligonucleotides
- This Example describes the syntheses of three oligonucleotides having Raman labels bound thereto: 3′ HS-TMR-A10-AAC CGA AAG TCA ATA [SEQ ID NO. 2 in FIG. 19a]; 3′ HS-Cy3.5-A10-CCT CAT TTA CAA CCT [SEQ ID NO. 3 in FIG. 19a]; and 3′HS-Cy5-A10-CTC CCT AAT AAC AAT [SEQ ID NO. 4 in 19b]. Because the dyes are sensitive to standard cleavage reagent (ammonia), ultramild base monomers (from Glen Research) were used here to allow the deprotection reaction under ultramild conditions: phenoxyacetyl (Pac) protected dA, 4-isopropyl-phenoxyacetyl (iPr-Pac) protected dG, and acetyl (Ac) protected dC. TAMRA-dT (TMR-dT, 5′-Dimethoxytrityloxy-5-[N-((tetramethylrhodaminyl)-aminohexyl)-3-acrylimido]-2′-deoxy Uridine-3′-[2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite),Cy3.5-CE phosphoramidite (Indodicarbocyanine 3.5, 1′-O-(4-monomethoxytrityl)-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), and Cy5-CE phosphoramidite (
Indodicarbocyanine - Synthesis and Purification of
Rhodamine 6G-, and Texas Red-Labeled-(propylthiol)-Capped Oligonucleotides - This Example describes the synthesis of two olignucleotides have Raman labels attached thereto: 3′ HS-Rd-A10-TCA ACA TTG CCT TCT [SEQ ID NO. 5 in FIG. 19b] and 3′ HS-TR-A10-TCT TCT ATA AAC CTT ATT [SEQ ID NO. 6 in FIG. 19a]. See FIG. 24. Both of these oligonucleotides were prepared via two-step syntheses. In the first step, amino-modified oligonucleotides (3′-S—S-(NH2)-A10-TCA ACA TTG CCA TCT and 3′-S—S-(NH2)-A10-TCT TCT ATA AAC CTT ATT)were synthesized via literature procedures.5 The amino-modifier C6 dT(5′-dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2′-deoxyUridine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) was placed in
position 5 in the synthesizer (Expedite), and amino-modified oligonucleotides were obtained by conventional automated syntheses. The cleavage, deprotection, and purification of the oligonucleotides were carried out by the procedures described for the synthesis of the Cy3-modified oligonucleotide (above), retention time=26 min. In the second step, succinimide ester modifiedRhod 6G (5-carboxyl-rhodamine 6G, succinimidyl ester) and Texas Red (Texas Red-X-succinimidyl ester) were coupled to the amino-modified oligonucleotides, respectively. In a typical experiment, an amino-modified, alkylthiol-capped-oligonucleotide (0.15 μmol) was dissolved in a sodium borate buffer (0.1M, pH=8.5, 0.5 ml), and a DMSO solution (150 μl) containing 2.5 mg of the succiuimide ester modifiedRhod 6G (or Texas Red) was added to the oligonucleotide buffer solution, FIG. 24. The solution was stirred at room temperature for 12 hr. Then, theRhod 6G-(or Texas red-) labeled oligonucleotide was purified by ethanol precipitation (3 times) and further by HPLC in the conditions as described above. - DNA Detection Assay
- In a typical experiment for DNA detection, a three-component sandwich assay is used in microarray format (FIG. 1). Gold nanoparticles (13±2 nm in diameter) modified with Cy3-labeled, alkylthiol-capped oligonucleotide strands (Supporting Information) were used as probes to monitor the presence of specific target DNA strands. On average, there are 110 oligonucleotide strands on each 13-nm gold nanoparticle. The Cy3 group was chosen as a Raman label due to its large Raman cross section.23 A chip spotted with the appropriate 15 mer capture strands was coated with a 0.6 M NaCl PBS buffer solution (10 mM of phosphate, pH 7) containing a 30 mer target sequence (100 pM) in a humidity chamber at room temperature. After 4 h, the chip was washed four times with 0.6 M NaCl PBS buffer solution to remove nonspecifically bound target. Then, the chip was treated with a 0.6 M NaCl PBS solution of nanoparticle probes (2 nM) for 1.5 hour to effect hybridization with the overhanging region of the target sequence (FIG. 1). The chip was then washed with 0.6 M NaNO3 PBS buffer solution to remove chloride ions and nonspecifically bound nanoparticle probes. The chip was immediately treated with a silver enhancement solution (Ted Pella, Inc) for 8 minutes, subsequently rinsed with Nanopure water, and dried with a microarray centrifuge (2000 g). The chip, which exhibits grey spots visible to the naked eye, could be imaged with a flatbed scanner (
Expression 1600, Epson) via literature procedures, FIG. 2A and B.8 The spots also were imaged by Raman spectroscopy in a 0.3 M NaCl PBS buffer solution (Solution Raman 633 spectrometer from Detection Limit Inc., 30 mW He—Ne laser), FIG. 2C. The chip was scanned with a fiber-optic probe with a 0.65 N.A. adapter (25 μm laser spot), and each spot shows a consistent and strong Raman response at 1192 cm−1 (FIG. 2D). - Prior to silver enhancing, the nanoparticle probes were invisible to the naked eye, and no Raman scattering signal was detectable (FIG. 2A). This is due to a lack of electromagnetic field enhancement for the undeveloped nanoparticles (13 nm in diameter) in this state.24-26 Others have shown that closely spaced gold nanoparticles in such sizes can give surface-enhanced Raman scattering enhancement,27-30 but for DNA detection at technologically relevant target concentrations (<1 nM), nanoparticle spacings are too large to yield such effects. After silver enhancing, the Ag particles can grow around the Cy3-labeled nanoparticle probes leading to large Raman scattering enhancements. Typically, the obtained spectra include both sharp (15 to 30 cm−1) Raman lines and a concomitant broad underlying continuum as noted by Brus et. al. in their studies of
Rhodamine 6G molecules on Ag particles.30-31 Importantly, the Raman scattering signals arise almost exclusively from the Cy3 dye molecules immobilized on the particles; no signals were observed from other species such as the oligonucleotides, solvent molecules, and the succinimidyl 4-(maleimidophenyl)-butyrate (SMPB) on the glass surface. Moreover, the Raman scattering frequency for each Raman line remains constant from experiment to experiment, deviating by less than 2 cm−1. Since consistent SERS signals from the Cy3-labeled nanoparticle probes were obtained, the Raman spectrum of Cy3 can be used as a spectroscopic fingerprint to monitor the presence of a specific target oligonucleotide strand. - Detection of DNA at Low Target Concentration (Example: 20 fM)
- In a typical experiment, a chip spotted with the appropriate capture strands (FIG. 3A) was coated with a 0.75M NaCl PBS buffer solution (10 mM of phosphate, pH 7) containing a 30-mer target sequence (20 fM) in a humidity chamber at room temperature. After 8 h, the chip was washed with 0.75M NaCl PBS buffer solution to remove nonspecifically bound target. Then, the chip was treated with a 0.75 M NaCl PBS solution of nanoparticle probes (500 pM) for 3 h to effect hybridization with the overhanging region of the target sequence (FIG. 3A). The chip was washed with 0.75 M NaNO3 PBS buffer solution to remove chloride ions and nonspecifically bound nanoparticle probes. The chip was immediately treated with silver enhancement solution (from Ted Pella, Inc) for 15 min, subsequently rinsed with Nanopure water, and dried with a microarray centrifuge (2000 g). The spots can be imaged in the dry state with a flatbed scanner (FIG. 3B) or by Raman spectroscopy in the wet state (0.3 M NaCl, pH7, PBS buffer solution), FIG. 3C and D. The current unoptimized detection limit with this technique is 10 fM.
- Detection of Multiple Oligonucleotide Targets
- This Example describes detection of multiple oligonucleotides using a plurality of Raman labeled probes. One can utilize the approach described in Example 5 and nanoparticle probes functionalized with dyes other than Cy3 to create a large number of probes with distinct and measurable SERS signals. This allows multiplexed detection of a large number of oligonucleotide targets simultaneously. To demonstrate this point, six commercially available dyes were selected with distinct Raman spectra that can be incorporated into oligonucleotides through standard automated DNA-syntheses. Six types of Raman labeled and oligonucleotide-modified gold nanoparticle probes were prepared with sequences that were respectively complementary to statistically unique 30-36 mer sequences for: (A) Hepatitis A virus Vall7 polyprotein gene (HVA), (B) Hepatitis B virus surface antigen gene (HVB), (C) HIV, (D) Ebola Virus (EV), (E) Variola virus (smallpox, VV), and (F) Bacillus anthracis (BA) protective antigen gene (FIG. 4).32 With these probes, the multiplexing capabilities of the novel scanning Raman technique for the six target analytes can be demonstrated.
- Eight separate tests were carried out to evaluate the selectivity of the system and our ability to determine the number and types of strands in solutions containing mixtures of the different targets (FIG. 4 and5). The concentrations of the target strands were kept constant for all of these experiments (100 pM each), and the hybridization conditions were as described above. In the first test (FIG. 5, row 1), all spots show the same intense grey color associated with silver deposition. However, they can be differentiated simply by using the Raman scanning method, and once the spectroscopic fingerprint of the Ag-containing spot has been determined the correct Raman label and, therefore, target sequence can be identified. To simplify the analysis, a color (rectangular box) to each Raman labeled probe (FIG. 4 and FIG. 5B) was assigned. In the first test (FIG. 5A), all six targets were present, and all show strong grey scale values when measured via the flatbed scanner and the expected Raman fingerprints. In the next seven tests, one or more of the targets to evaluate the suitability of this method for multiplexing were systematically removed. Note that with the single color grey scale method one cannot determine if any cross hybridization has occurred. However, with this “multiple color” scanning Raman method, one can carefully study the SERS spectra of each spot to determine which labels make up each spot. For the experiments described in FIG. 5, where the sequences are very dissimilar, it was found that other than the expected spectroscopic probe signature for each target, there are virtually no other detectable Raman lines, which means that there is no cross-hybridization between different targets and probes.
- It should be mentioned that the obtained SERS signal only comes from areas of the substrate where the Raman dye-labeled gold particles have initiated Ag formation. Therefore, this “multiple color” scanning Raman detection method does not record background signal due to silver deposition where Au particles do not exist. This is not the case for the previous grey-scale scanometric approach, especially at ultra-low target concentrations (<50 fM).8
- Discrimination and Ratioing of Single Nucleotide Polymorphisms (SNPs) in Oligo-Ribonucleic Acid (RNA) Targets.
- This Example describes the use of oligonucleotides having Raman labels in detection systems to differentiate single nucleotide polymorphisms (SNPs), and in the case of gene expression studies, one would like access to RNA detection with single spot signal ratioing capabilities. It is well known that nanoparticle probes heavily functionalized with oligonucleotides exhibit extraordinarily sharp thermally-induced denaturation transitions that lead to substantially higher selectivity than conventional molecular fluorophore probes in DNA detection.5,8,9 However, nothing is known about the behavior of these probes in the context of RNA detection. To further test the selectivity of this Raman based system and its ability to identify SNP targets, two RNA targets were chosen that can bind to the same capture strand DNA but have a single-base mutation in the probe binding regions (target 1:T1, normal; target 2:T2, single-base difference, FIG. 6). Therefore, two DNA-functionalized probes (probe 1: P1, probe 2: P2), which differ in sequence and Raman label, are required to differentiate these two RNA target strands (FIG. 6). Seven separate tests were performed to demonstrate not only how the two targets (T1 and T2) can be differentiated but also how mixtures of the two targets can be analyzed in semi-quantitative fashion.
- In a typical experiment, the appropriate capture strands (FIG. 6) were spotted in quadruplicate on SMPB functionalized glass slides. These slides were coated with 0.3M NaCl PBS buffer solutions (10 mM of phosphate, pH 7) containing
pure RNA target 1 ortarget 2, or mixtures of 1 and 2 (1 nM total oligonucleotide concentration) in a humidity chamber at room temperature. After 2 h, the chip was washed four times with 0.3M NaCl PBS buffer solution to remove nonspecifically bound target. Then, the chip was treated with a 0.3 M NaCl PBS solution of nanoparticle probes (2 nM, probel: probe2=1:1) for 1.5 h to effect hybridization with the overhanging region of the target sequences (FIG. 7). The chip was washed with 0.3 M NaNO3 PBS buffer solution to remove chloride ions and nonspecifically bound nanoparticle probes. If the chips were developed by silver enhancing, the Raman measurements on the grey spots at different target ratios yield similar spectra (FIG. 8), which are nearly identical to the spectrum for the sample containing probe I andprobe 2 in equal amounts. This result indicates that there are equal amounts ofprobe 1 andprobe 2 on the chip. This is because the stabilities of the perfectly matched and single-mismatched oligonucleotide duplexes are close in magnitude, and therefore, nanoparticle probes (1 and 2) bound to the spots on the chips in nearly equally amounts at all of the target ratios. Under these conditions the two targets cannot be differentiated. - In each of these tests, a slide was treated with a 0.3 M NaCl PBS buffer solution containing T1 and T2 in different ratios (total concentration=1 nM) in a humidity chamber. After 2 h, the chip was washed with a 0.3 M NaCl PBS buffer to remove nonspecifically bound target. Then, the chip was treated with nanoparticle probes (P1 and P2 at 1:1 ratio, 2nM total concentration) for 1.5 h to effect hybridization with the overhanging region of the target sequences (FIG. 6). The chip was washed with 0.3 M NaNO3 PBS buffer solution to remove chloride ions and nonspecifically bound nanoparticle probes. Note that there are four possible hybridization modes, namely, T1:P1, T2:P2, T1:P2, and T2:P1 (FIG. 6). If the chip was developed by silver enhancing without prior stringency wash, the Raman measurements on the grey spots which correspond to different solution target ratios yield nearly identical spectra in all seven experiments; these spectra also are almost identical to those obtained for a sample containing a 1:1 ratio of
probe 1 and probe 2 (see Supporting Information). These data show thatprobe 1 andprobe 2 are bound to the spots on the chip in equal amounts, regardless of the target composition on the spot. - Therefore, in order to identify the target composition on the spots, a salt or temperature-based stringency wash must be applied. Accordingly, a salt stringency wash (8 mM NaCl PBS buffer) was employed to selectively denature the imperfect duplexes (T1:P2 and/or T2:P1, FIG. 6C and 6D) but not the duplexes formed from the perfectly complementary oligonucleotides (T1:P1 and/or T2:P2, FIG. 6A and 6B).9 After stringency wash and subsequent silver staining, the Raman measurements on the grey spots can be used to readily identify the target composition on the spots by the obtained spectra. In tests where only
pure RNA target probe probe 1 and I2: 1588 cm−1 from probe 2) are detected, and the intensity ratios are proportional to the ratios of the two targets in each experiment (inset of FIG. 9B). - Screening of Protein: Small Molecule Interaction
- This Raman detection format also can be used in protein microarray applications for screening protein-small molecule and protein-protein interactions. For the detection of protein-small molecule interactions, we selected three unrelated small molecules for which the specific protein receptors are commercially available: biotin and its mouse monoclonal antibody; DIG (steroid digoxigenin) and its mouse monclonal antibody; DNP (dinitrophenyl) and its mouse monoclonal antibody. The three small molecules were labeled with Raman dye-functionalized gold particles: the gold particles (13 nm in diameter) were modified with a small-molecule capped, Raman dye and alkylthiol-functionalized poly-adenine(A20) (FIG. 11A). In a typical detection experiment, the proteins from all three pairs were immobilized in triplet onto aldehyde-functionalized glass slides by spotting the protein solution (200 μg/ml, 5% glycerol) with a commercial arrayer (FIG. 11A).33,34 After 4-hour incubation in a humidity chamber, the protein chip was washed with PBS buffer (0.173 M NaCl, 0.027 M KCl, 4.3 mM Na2BPO4, 1.4 mM KH2PO4, pH=7.4) containing 0.5% bovine serum albumin (BSA), and immersed into such solution for 4 hour to passivate the unreacted aldehydes on the protein chip. After being washed with a PBS solution (0.173 M NaCl, 0.027 M KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH=7.4), the protein chip was treated with Raman labeled small molecule probes (for 2 hours at 4° C. After washed with a buffer solution (0.2 M NaNO3, 5 mM phosphate, pH=7.4), the gold particle functionallized protein chip was treated with the silver enhancement solution for 8 minutes and washed with Nanopure water. Before Raman measurements, the silver stained chip was immersed in a 2×PBS solution for 10 minutes.
- In the first test, the protein chip was exposed to all a solution containing all three Raman-labeled small molecule probes. After silver enhancement, the triplet dot array is clearly visible, even to the naked eye (FIG. 11B-1). When measuring the Raman spectra of the dots, we obtained the correct probe spectra with no evidence of cross reactivity (i.e. less than 1%, Cy3 for biotin, Cy3.5 for DIG, and Cy5 for DNP). Next we studied the same type chip but in the presence of the DIG and DNP probes, and gain obtained the expected results, FIG. 11B-2 and C2). We did this for all other possible two probe combinations and again obtained the expected results, demonstrating the high selectivity of the system (FIG. 11B-3, C-3 and 11B-4, C-4). In the two probe experiments, one probe for the array is absent, serving as a control for screening the other interaction pairs.
- Screening Protein-Protein Interactions
- For screening protein-protein interactions, we chose three pairs of proteins to study: mouse immunoglobulin G (IgG) and its antibody; ubiquitin and its antibody; human protein C and its antibody. Mouse IgG, ubiqutin, and human protein C were spotted in quadruplicate on aldehyde slides, respectively. Gold nanoparticles were first functionalized with antibodies and then with Raman-dye labeled oligonucleotides. The labeling procedure is shown in FIG. 12: an antibody (10 μg, pH=9.2) was put into a solution of gold particles (13 nm, 10 nM, 1 mL, pH=9.2) for 20 minutes, and then the Raman dye capped-alkylthiol-functionalized poly-adenine (A10, 0.2 OD at 260 mn) was added to the solution. After 12 hours, 10% BSA solution (0.3 mL) was added to the solution to further passivate the surface of the gold particles. The solution was allowed to stand for 10 minutes. The Raman-dye capped gold particle-antibody conjugates were purified by centrifugation (14,000 rpm), which precipittaes the particles. The supernatant containing excess oligonucleotide, BSA, and antibodies can be decanted from the particles. The particle probes are then be redispersed in PBS buffer. The probes (2 nM for gold nanoparticles, about 2 μg/ml for the antibodies) were then used to develop the protein chips. The protocol for screening the protein-protein interactions is similar to that for protein-small molecule interactions (described above).
- The chip in FIG. 13 A-4 was probed with all the three Raman labeled antibodies simultaneously. After silver enhancement, all three two-by-two dots array are clearly visible after silver develioping. Raman analysis shows no detectable cross reactivity and all of the correct dyes are in the correct spots (FIG. 13).
- Just like fluorophore-based methods, this new scanometric detection format provides a general approach for genomic and porteomic detection but with a higher sensitivity and a higher multiple labeling capability. The number of available Raman dyes is much larger than the number of available and discernable fluorescent dyes.20,21 A Raman dye can be a fluorescent dye and also a non-fluorescent dye. A small modification of a dye can lead to a new dye with different Raman spectra and even the dyes which show undistinguishable fluorescent spectra can be distinguished by Raman spectroscopy.16 In the conventional multicolor fluorescent dyes labeling format, the data readout requires multi-lasers and multiple scans.1 By contrast, only a single laser and individual scan are required in this Raman scanometric detection format, suggesting a potential for a high throughput reading process. Although quantum-dot-labeled fluorescence detection requires only single laser, multicolors are usually generated from different size and shape quantum-dot nanoparticles.6,7 Different sized and shaped nanoparticles associated biological labels will have different thermodynamic and kinetic properties, which are problematic for parallel microarray biological detection. In the Raman scanometric detection format, in contrast, only one-sized gold nanoparticle (13 nm, here) carriers are required, and labeling information from different Raman dyes. Therefore, most of the labels described here have similar thermodynamic and kinetic target binding properties, which are essential for faster, more-accurate, high-throughput microarray based mapping and screening of biomolecules.1
- Multiple Raman-Dye Labeled Nanoparticle Probes
- All the Raman labels described above are single-dye systems: one carrier and Raman dye. One can load two or multiple Raman dyes onto a nano-sized nanoparticle carrier. Massively encoded Raman labels can be generated by tailoring the ratio between the components (FIG. 14 and15). In a two-dye system, two alkylthiol capped-oligonucleotide strands with same base sequences but different the Raman labels (Cy3 and TMR) were used to modify 13-nm gold nanoparticles simultaneously, and therefore a composite Raman label was generated. This two-dye labeled nanopaticle probe has similar thermodynamic and kinetic properties as the single-dye labeled nanoparticle probe (i.e. same hybridization kinetics and melting temperatures with identical strands). In a typical DNA detection experiment (target concentration is 100 pM, FIG. 1), a Raman spectrum from a silver-stained spot clearly shows characteristic Raman lines from both of Cy3 and TMR (FIG. 16, left). By varying the ratio of Cy3 and TMR, different composite Raman spectra are obtained (FIG. 16, right). These Raman spectra are distinguishable from each other by differences in relative intensities for the main bands in the region interrogated. The multiple reference windows increase the accuracy for identifying different Raman labels, making this two dye Raman labeling methodology practically usable. Beyond two-dye systems, two examples of three-dye labels, which have different amount ratio between Cy3, TMR and Cy3.5, are shown in FIG. 17.
- One can use one-dye, two-dye, three-dye and even larger combination-labeled systems. A significant question is: how many labels can be achievable in this Raman labeling system? In a two-dye system, assuming five intensity levels (0, 1, 2, 3, 4), there are 13 labels that can be generated. Five million labels and three billion labels can be generated with 10-dye and 14-dye systems, respectively (FIG. 15).
- Microbead-Based Biological Detection
- Large numbers of parallel labeling techniques are of particular importance in microbead-based biological detection strategies. Microbead technology is emerging as an important biological analysis format for gene expression monitoring, SNP genotyping, proteomic screening, and drug discovering.1,13 Compared with the microarray technique, microbead detection shows more flexibility in hybridization-based procedures, faster analyte diffusion kinetics, and they are easier and cheaper to produce. The microbead detection without the positional encoding in the microarrys, however, must rely on some sort of barcoding strategy for the particle probes. A major problem in the current fluorescent-dye-based encoding approach is that the number of distinguishable labels are limited due to the broad emission spectra and energy transfer between organic dyes.11 Raman labeling, in contrast, can overcome these difficulties.
- For a typical DNA target detection system, a three-component sandwich assay format can be used. In our experiments, glass microbeads (210-250 mm in diameter) were functionalized with oligonucleotide capture strands (FIG. 18). Gold nanoparticles (13 nm in diameter) modified with pure or mixed Raman dye-labeled and alkylthiol-capped oligonucleotides probe strands were synthesized. Then the Raman dye and gold particle associated probes (2 nM for gold particles) co-hybridized with the target strands onto the surface of the capture strand oligonucleotide functionalized glass microbeads in a 4×PBS buffer solution (0.6 M of NaCl, 10 mM of phosphate buffer( pH=7) ) for 2 hours and washed with a second buffer solution (0.6 M NaNO3, 10 mM phosphate) to remove chloride ions, and non-specifically bound nanoparticle labels, and immediately treated with a silver enhancement solution (from BBInternational) for 8 minutes. Before Raman measurements, the microbeads were immersed in a 2×PBS buffer for 10 min to further enhance Raman scattering signal.
- To demonstrate the multiplexing capabilities of the novel scanning Raman technique in microbead detection format, we chose an eight-target analyte detection experiment. The sequences of target, capture and probe oligonucleotide strands are shown in FIG. 19a and b. The corresponding Raman spectra (marked by colored circle and rectangular boxes) are listed in FIG. 20. In a typical experiment, eight capture strands were loaded onto microbeads, respectively. Mixing all the microbeads together, a flexible “random microarray” was built. Then the eight targets (100 pM) and Raman-labeled nanoparticle probes (2 nM) are introduced to the random microarray solution under hybridization conditions as described above. After washing and silver staining, the microbeads are show up as dark-grey spheres and exhibit the expected Raman signatures (FIG. 21). To achieve an easy readout process, we can align these microbeads mechanically (FIG. 22 top) and read them in serial fashion via scanning Raman spectroscopy. (FIG. 22, bottom). Moreover, the Raman fingerprints of the micorbeads can also be read out by fiber optics (FIG. 23).
- Beside this new Raman labeling technique, two recent strategies show the practical potential for massively parallel labeling abilities: quantum-dot-tagged microbeads and submicrometer metallic barcodes.11,35 However, both of these strategies achieve multiple labeling based on micron-size structures. In contrast, Raman labeling here is a nano-size labeling methodology, and has much more flexibility than those micro-size ones. In particular, the footprints of the probes are smaller and the specificity and sensitivity of systems based on the probes can be dramatically improved over the systems based upon larger structures. This new nanoparticle-based methodology is important for a variety of reasons. First, in contrast with conventional fluorescence-based chip detection, the ratio of Raman intensities can be extracted from a single Raman spectrum using single laser excitation. Second, the number of available Raman dyes is much larger than the number of available and discernable fluorescent dyes.20,21,26 Indeed, a Raman dye can be either fluorescent or non-fluorescent, but a minor chemical modification of a dye molecule can lead to a new dye with a different Raman spectrum even though the two dyes exhibit virtually indistinguishable fluorescence spectra.26 Therefore, this fingerprinting method offers potentially greater flexibility, a larger pool of available and non-overlapping probes, and higher multiplexing capabilities than conventional fluorescence-based detection approaches. Finally, the method incorporates all of the previous advantages of gold-nanoparticle based detection, including several orders of magnitude higher sensitivity and many orders of magnitude higher selectivity than the analogous molecular fluorescence based approach.8,9
- References
- 1. G. Gibson, S. V. Muse,A Primer of Genome Science (Sinauer Associates, Inc. Sunderland, Mass., 2002).
- 2. M. Schena, D. Shalon, R. W. Davis, P. O. Brown,Science 1995, 270,467.
- 3. M. Chee, R. Yang, E. Hubbell, A. Berno, X. C. Huang, D. Stem, J. Winkler, D. J. Lockhart, M. S. Morris, S. P. A. Fodor,Science 1996, 274, 610.
- 4. M. Snyder, et al.,Science 2001, 293, 2101.
- 5. R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger, C. A. Mirkin,Science 1997, 277, 1078.
- 6. M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos,Science 1998, 281, 2013.
- 7. W. C. W. Chan, S. Nie,Science 1998, 281, 2016.
- 8. T. A. Taton, C. A. Mirkin, R. L. Letsinger,
Science 2000, 289, 1757. - 9. S. J. Park, T. A. Taton, C. A. Mirkin,Science, 2002,295, 1503.
- 10. L. He, M. D. Musick, S. R. Nicewarner, F. G. Salinas, S. J. Benkovic, M. J. Natan, C. D. Keating,J. Am. Chem. Soc. 2000,122, 9071.
- 11. M. Han, X. Gao, J. Z. Su, S. Nie,
Nature Biotechnology 2001, 19, 631. - 12. I. Willner, F. Patolsky, J. Wasserman,Angew. Chem. Int. Ed. 2001, 40, 2261.
- 13. H. Mattoussi, J. M. Mauro, E. R. Goldman, G. P. Anderson, V. C. Sundar, F. V. Mikulec, M. G. Bawendi,J. Am. Chem. Soc. 2000, 122, 12142.
- 14. S. Pathak, S. K. Choi, N. Amnheim, M. E. Thompson,J. Am. Chem. Soc. 2001, 123, 4103.
- 15 S. Schultz, D. R. Smith, J. J. Mock, D. A. Schultz,Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 996.
- 16. C. M. Niemeyer,Angew. Chem. Int. Ed. 2001, 40, 4128.
- 17. See website: www.nanosphere-inc.com.
- 18. C. A. Mirkin, AACC Meeting, San Diego, Calif., 2001.
- 19. D. R. Walt,
Science 2000, 287, 451. - 20. D. Graham, B. J. Mallinder, W. E. Smith,Angew. Chem. Int. Ed. 2000, 39, 1061.
- 21. D. Graham, W. E. Smith, A. M. T. Linacre, C. H. Munro, N. D. Watson, P. C. White,Anal. Chem. 1997, 69, 4703.
- 22. L. M. Demers, C. A. Mirkin, R. C. Mucic, R. A. Reynolds III, R. L. Letsinger, R. Elghanian, G. Viswanadham,Anal. Chem. 2000, 72, 5535.
- 23. R. L. McCreery,Raman spectroscopy for chemical analysis (John Wiley & Sons, New York, 2000).
- 24. G. C. Schatz, R. P. Van Duyne, inHandbook of Vibrational Spectroscopy, J. M. Chalmers and P. R. Griffiths, Ed., Wiley, New York, 2002, page:759-774.
- 25. A. Carapion, P. Kambhampati.Chem. Soc. Rev. 1998, 27, 241.
- 26. K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari, M. S. Feld,Chem. Rev. 1999, 99, 2957.
- 27. S. R. Emory, S. Nie,J. Phys. Chem. B 1998, 102, 493.
- 28. R. C. Freeman, K. G. Grabar, K. A. Allison, R. M. Bright, J. A. Davis, A. P. Guthrie, M. B. Hommer, M. A. Jackson, P. C. Smith, D. G. Walter, M. J. Natan,Science 1995, 267, 1629.
- 29. M. D. Musick, C. D. Keating, M. H. Keefe, M. J. Natan,Chem. Mater. 1997, 9, 1499.
- 30. A. M. Michaels, M. Nirmal, L. E. Brus,J. Am. Chem. Soc. 1999, 121, 9932.
- 31. A. M. Michaels, J. Jiang, L. Brus,J. Phys.
Chem. B 2000,104, 11965. - 32. From the web site of National Center for Biotechnology Information (NCBI): http://www2.ncbi.nhn.nih.gov/Genbank/index.html.
- 33. G. MacBeath, S. L. Schreiber,Science 2001, 289, 1760.
- 34. B. B. Haab, M. J. Dunham, and P. O. Brown,Genome Biology 2001, 2(2): research0004.1-0004.13.
- 35. S. R. Nicewarner-Peñña, R. G. Freeman, B. D. Reiss, L. He, D. J. Peñña, I. D. Walton, R. Cromer, C. D. Keating, M. J. Natan.Science 2001, 294, 137.
-
-
1 27 1 15 DNA Hepatitis A virus 1 tccatgcaac tctaa 15 2 15 DNA Hepatitis B virus 2 ataactgaaa gccaa 15 3 15 DNA Ebola virus 3 tccaacattt actcc 15 4 15 DNA Bacillus anthracis 4 taacaataat ccctc 15 5 15 DNA Variola virus 5 tcttccgtta caact 15 6 18 DNA Human immunodeficiency virus type 1 6 ttattccaaa tatcttct 18 7 12 DNA Francisella tularensis 7 agccacctaa cc 12 8 15 DNA Hog cholera virus 8 acatgtccaa tttcc 15 9 15 DNA Hepatitis A virus 9 agaaagagga gttaa 15 10 15 DNA Hepatitis B virus 10 taccacatca tccat 15 11 15 DNA Ebola virus 11 ttgttgatac tgttc 15 12 15 DNA Bacillus anthracis 12 atcctttaca atatt 15 13 15 DNA Variola virus 13 ctgattacta ttgca 15 14 15 DNA Human immunodeficiency virus type 1 14 tgcatccagg tcatg 15 15 15 DNA Francisella tularensis 15 cttttgcatc atcag 15 16 15 DNA Hog cholera virus 16 tggttcacct ttgac 15 17 30 DNA Hepatitis A virus 17 ttagagttgc atggattaac tcctctttct 30 18 30 DNA Hepatitis B virus 18 ttggctttca gttatatgga tgatgtggta 30 19 30 DNA Ebola virus 19 ggagtaaatg ttggagaaca gtatcaacaa 30 20 30 DNA Bacillus anthracis 20 gagggattat tgttaaatat tgtaaaggat 30 21 30 DNA Variola virus 21 agttgtaacg gaagatgcaa tagtaatcag 30 22 33 DNA Human immunodeficiency virus type 1 22 agaagatatt tggaataaca tgacctggat gca 33 23 27 DNA Francisella tularensis 23 ggttaggtgg ctctgatgat gcaaaag 27 24 30 DNA Hog cholera virus 24 ggcaattgga catgtgtgaa aggtgaacca 30 25 15 DNA Artificial Sequence misc_feature (1)..(15) Synthetic probe strand 25 taacaataat ccctc 15 26 15 DNA Artificial Sequence misc_feature (1)..(15) Synthetic capture strand 26 atccttatca atatt 15 27 30 DNA Artificial Sequence misc_feature (1)..(30) Synthetic target sequence 27 gagggattat tgttaaatat tgataaggat 30
Claims (23)
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/172,428 US20030211488A1 (en) | 2002-05-07 | 2002-06-14 | Nanoparticle probs with Raman spectrocopic fingerprints for analyte detection |
PCT/US2003/014100 WO2003095973A2 (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
EP03736550A EP1499889B1 (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
JP2004503919A JP2005524849A (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes for analyte detection with fingerprints for Raman spectroscopy |
AT03736550T ATE515698T1 (en) | 2002-05-07 | 2003-05-07 | NANOPARTICLE PROBE WITH RAMAN SPECTROSCOPIC FINGERPRINTS FOR ANALYTE DETECTION |
CA2483697A CA2483697C (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
US10/431,341 US7985539B2 (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
AU2003237180A AU2003237180B2 (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US37853802P | 2002-05-07 | 2002-05-07 | |
US38363002P | 2002-05-28 | 2002-05-28 | |
US10/172,428 US20030211488A1 (en) | 2002-05-07 | 2002-06-14 | Nanoparticle probs with Raman spectrocopic fingerprints for analyte detection |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/431,341 Continuation-In-Part US7985539B2 (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
Publications (1)
Publication Number | Publication Date |
---|---|
US20030211488A1 true US20030211488A1 (en) | 2003-11-13 |
Family
ID=29407691
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/172,428 Abandoned US20030211488A1 (en) | 2002-05-07 | 2002-06-14 | Nanoparticle probs with Raman spectrocopic fingerprints for analyte detection |
US10/431,341 Expired - Fee Related US7985539B2 (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/431,341 Expired - Fee Related US7985539B2 (en) | 2002-05-07 | 2003-05-07 | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
Country Status (7)
Country | Link |
---|---|
US (2) | US20030211488A1 (en) |
EP (1) | EP1499889B1 (en) |
JP (1) | JP2005524849A (en) |
AT (1) | ATE515698T1 (en) |
AU (1) | AU2003237180B2 (en) |
CA (1) | CA2483697C (en) |
WO (1) | WO2003095973A2 (en) |
Cited By (83)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020137071A1 (en) * | 1996-07-29 | 2002-09-26 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20020192687A1 (en) * | 2000-03-28 | 2002-12-19 | Mirkin Chad A. | Bio-barcodes based on oligonucleotide-modified nanoparticles |
US20030054358A1 (en) * | 1996-07-29 | 2003-03-20 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030129608A1 (en) * | 2001-05-25 | 2003-07-10 | Mirkin Chad A | Non-alloying core shell nanoparticles |
US20030207296A1 (en) * | 1996-07-29 | 2003-11-06 | So-Jung Park | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20040023287A1 (en) * | 2002-08-02 | 2004-02-05 | Oliver Harnack | Method of attaching hydrophilic species to hydrophilic macromolecules and immobilizing the hydrophilic macromolecules on a hydrophobic surface |
US20040038255A1 (en) * | 2001-05-25 | 2004-02-26 | Northwestern University | Non-alloying core shell nanoparticles |
US20040053222A1 (en) * | 2002-07-02 | 2004-03-18 | Nanosphere, Inc. | Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes |
US20040072231A1 (en) * | 1996-07-29 | 2004-04-15 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20040101889A1 (en) * | 2000-07-11 | 2004-05-27 | Northwestern University | Method of detection by enhancement of silver staining |
US20040110220A1 (en) * | 1996-07-29 | 2004-06-10 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20040179195A1 (en) * | 2003-03-12 | 2004-09-16 | Xing Su | Chemical enhancement in surface enhanced raman scattering using lithium salts |
US20050037397A1 (en) * | 2001-03-28 | 2005-02-17 | Nanosphere, Inc. | Bio-barcode based detection of target analytes |
US20050064435A1 (en) * | 2003-09-24 | 2005-03-24 | Xing Su | Programmable molecular barcodes |
WO2005031301A2 (en) * | 2003-07-10 | 2005-04-07 | Parallax Biosystems, Inc. | Raman signature probes and their use in the detection and imaging of molecular processes and structures |
US20050130174A1 (en) * | 2003-02-27 | 2005-06-16 | Nanosphere, Inc. | Label-free gene expression profiling with universal nanoparticle probes in microarray assay format |
US20050142567A1 (en) * | 2003-12-29 | 2005-06-30 | Intel Corporation | Composite organic-inorganic nanoparticles and methods for use thereof |
US20050147976A1 (en) * | 2003-12-29 | 2005-07-07 | Xing Su | Methods for determining nucleotide sequence information |
US20050148100A1 (en) * | 2003-12-30 | 2005-07-07 | Intel Corporation | Methods and devices for using Raman-active probe constructs to assay biological samples |
US20050191665A1 (en) * | 2003-12-29 | 2005-09-01 | Xing Su | Composite organic-inorganic nanoclusters |
US20050221506A1 (en) * | 2004-03-30 | 2005-10-06 | Intel Corporation | Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS) |
US20050221360A1 (en) * | 2004-03-31 | 2005-10-06 | Takara Bio Inc. | Method for purifying microbeads |
US20050233474A1 (en) * | 2004-04-14 | 2005-10-20 | Roitman Daniel B | Surface-enhanced Raman spectroscopy for biosensor systems and methods for determining the presence of biomolecules |
DE102004015272A1 (en) * | 2004-03-29 | 2005-11-03 | Infineon Technologies Ag | Biosensor to determine the presence of DNA comprises trap molecules that hybridize with the target molecules over photo diode detector |
US20050250159A1 (en) * | 2003-12-30 | 2005-11-10 | Intel Corporation | Methods for using Raman spectroscopy to obtain a protein profile of a biological sample |
US20050250094A1 (en) * | 2003-05-30 | 2005-11-10 | Nanosphere, Inc. | Method for detecting analytes based on evanescent illumination and scatter-based detection of nanoparticle probe complexes |
US20060040286A1 (en) * | 2001-03-28 | 2006-02-23 | Nanosphere, Inc. | Bio-barcode based detection of target analytes |
US20060046313A1 (en) * | 2004-08-26 | 2006-03-02 | Intel Corporation | Cellular analysis using Raman surface scanning |
US20060046311A1 (en) * | 2004-08-26 | 2006-03-02 | Intel Corporation | Biomolecule analysis using Raman surface scanning |
WO2006028601A2 (en) * | 2004-07-26 | 2006-03-16 | Nanosphere, Inc. | Method for distinguishing methicillin resistant s. aureus from methicillin sensitive s. aureus in a mixed culture |
US20060068378A1 (en) * | 1996-07-29 | 2006-03-30 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20060073336A1 (en) * | 2003-12-29 | 2006-04-06 | Jingwu Zhang | External modification of composite organic inorganic nanoclusters |
WO2006037036A2 (en) * | 2004-09-27 | 2006-04-06 | Purdue Research Foundation | Quantitative proteomics with isotopic substituted raman active labeling |
US20060084181A1 (en) * | 2004-10-18 | 2006-04-20 | Stuart Farquharson | Method and apparatus for rapid extraction and analysis, by SERS, of drugs in saliva |
WO2006066180A1 (en) * | 2004-12-17 | 2006-06-22 | Intel Corporation | Detection of enhanced multiplex signals by surface enhanced raman spectroscopy (sers) |
US20060147941A1 (en) * | 2004-12-30 | 2006-07-06 | Intel Corporation | Methods and apparatus for SERS assay of biological analytes |
US7098320B1 (en) | 1996-07-29 | 2006-08-29 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20060234248A1 (en) * | 2003-12-29 | 2006-10-19 | Lei Sun | Composite organic inorganic nanoclusters |
KR100650522B1 (en) | 2005-09-15 | 2006-11-27 | 재단법인서울대학교산학협력재단 | A new label-free high throughput screening method by using sers spectroscopic encoded bead and dielectrophoresis |
US20070048797A1 (en) * | 2004-08-11 | 2007-03-01 | Xing Su | Composite organic inorganic nanoclusters as carriers and identifiers of tester molecules |
US20070072309A1 (en) * | 2005-09-29 | 2007-03-29 | General Electric Company | Analytical compositions including nanometer-sized transducers, methods to make thereof, and devices therefrom |
US20070077429A1 (en) * | 2004-02-20 | 2007-04-05 | Mirkin Chad A | Multicomponent nanorods |
US20070111204A1 (en) * | 2003-06-27 | 2007-05-17 | Kathleen Delgrosso | Methods for detecting nucleic acid variations |
US20070134815A1 (en) * | 2005-07-11 | 2007-06-14 | Danielle Chamberlin | Sensitivity enhancement of POCT devices using gold and silver nanoparticles on substrates containing nanostructures or nanoparticles that interact with labeling particles |
US20070154903A1 (en) * | 2005-06-23 | 2007-07-05 | Nanosphere, Inc. | Selective isolation and concentration of nucleic acids from complex samples |
US20070155022A1 (en) * | 2005-12-30 | 2007-07-05 | Mineo Yamakawa | Degenerate binding detection and protein identification using Raman spectroscopy nanoparticle labels |
DE102006000775A1 (en) * | 2006-01-04 | 2007-07-12 | Julius-Maximilians-Universität Würzburg | Diagnostic imaging techniques, useful for representing distribution of substances in immobilized cell, comprises displacing immobilized cell with a substance and bonding a marker for surface enhanced vibrational spectroscopy on substance |
US20070172834A1 (en) * | 2004-10-21 | 2007-07-26 | Patrick Englebienne | Stable metal/conductive polymer composite colloids and methods for making and using the same |
US20070298433A1 (en) * | 2003-12-31 | 2007-12-27 | President And Fellows Of Harvard College | Assay Device and Method |
US7393692B2 (en) * | 2004-10-18 | 2008-07-01 | Real-Time Analyzers, Inc. | SERS method for rapid pharmacokinetic analysis of drugs in saliva |
WO2008097328A2 (en) * | 2006-06-23 | 2008-08-14 | Northwestern University | Asymmetric functionalized nanoparticles and methods of use |
US20080266555A1 (en) * | 2004-12-13 | 2008-10-30 | University Of South Carolina | Surface Enhanced Raman Spectroscopy Using Shaped Gold Nanoparticles |
WO2009040114A2 (en) | 2007-09-24 | 2009-04-02 | Julius-Maximilians-Universität Würzburg | Compounds and markers for surface-enhanced raman scattering |
US20090111094A1 (en) * | 2005-08-19 | 2009-04-30 | Nanosphere, Inc. | Methods for preparing hybrid substrates comprising DNA and antibodies and uses thereof |
WO2009086509A2 (en) * | 2007-12-27 | 2009-07-09 | Purdue Research Foundation | Reagents for biomolecular labeling, detection and quantification employing raman spectroscopy |
US7738096B2 (en) | 2004-10-21 | 2010-06-15 | University Of Georgia Research Foundation, Inc. | Surface enhanced Raman spectroscopy (SERS) systems, substrates, fabrication thereof, and methods of use thereof |
EP2227681A2 (en) * | 2007-12-31 | 2010-09-15 | The Regents of the University of California | Sers-based, single step, real-time detection of protein kinase and/or phosphatase activity |
EP2245186A2 (en) * | 2008-01-14 | 2010-11-03 | Ultrapid Nanodiagnostics, Inc. | Rapid test including genetic sequence probe |
US20100291599A1 (en) * | 2009-05-18 | 2010-11-18 | Bruker Optics, Inc. | Large area scanning apparatus for analyte quantification by surface enhanced raman spectroscopy and method of use |
US7879625B1 (en) | 2008-12-03 | 2011-02-01 | The United States Of America As Represented By The Secretary Of The Navy | Preparation of SERS substrates on silica-coated magnetic microspheres |
WO2011047662A2 (en) | 2009-10-20 | 2011-04-28 | Friedrich-Schiller-Universität Jena | Sers substrate |
US20110124008A1 (en) * | 2008-05-07 | 2011-05-26 | Seoul National University Industry Foundation | NOVEL Au/Ag CORE-SHELL COMPOSITE USEFUL FOR BIOSENSOR |
WO2011078794A1 (en) | 2009-12-22 | 2011-06-30 | Agency For Science, Technology And Research | Sers-based analyte detection |
US20110171749A1 (en) * | 2009-03-02 | 2011-07-14 | Board Of Trustees Of Michigan State University | Nanoparticle tracer-based electrochemical dna sensor for detection of pathogens-amplification by a universal nano-tracer (aunt) |
US7985539B2 (en) | 2002-05-07 | 2011-07-26 | Northwestern University | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
US20110224417A1 (en) * | 2003-12-19 | 2011-09-15 | Kankyo Engineering Co., Ltd. | Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefore |
US20110294691A1 (en) * | 2008-10-15 | 2011-12-01 | Cornell University | Enhanced on-chip sers based biomolecular detection using electrokinetically active microwells |
US20120156804A1 (en) * | 2009-06-12 | 2012-06-21 | Agency For Science, Technology And Research | Method for determining protein-nucleic acid interaction |
US20120164717A1 (en) * | 2007-07-18 | 2012-06-28 | Joseph Irudayaraj | Identity profiling of cell surface markers |
US20120276523A1 (en) * | 2009-10-20 | 2012-11-01 | Vanderbilt University | Liquid drop diagnostic assays |
WO2015024863A1 (en) * | 2013-08-21 | 2015-02-26 | Mycartis Nv | Heterogenous surface functionalization |
US20160266104A1 (en) * | 2008-05-07 | 2016-09-15 | Korea Research Institute Of Chemical Technology | Heterodimeric core-shell nanoparticle in which raman-active molecules are located at a binding portion of a nanoparticle heterodimer, use thereof, and method for preparing same |
WO2016205678A1 (en) * | 2015-06-19 | 2016-12-22 | Emory University | Methods of measuring cell purity for making quality control determinations and related compositions |
US9823253B2 (en) | 2007-03-20 | 2017-11-21 | Becton, Dickinson And Company | Assays using surface-enhanced raman spectroscopy (SERS)-active particles |
US20180149646A1 (en) * | 2015-05-29 | 2018-05-31 | Kyocera Corporation | Detection method and detection device |
WO2018162742A1 (en) | 2017-03-10 | 2018-09-13 | Universität Duisburg-Essen | Efficient ligand exchange of a detergent bilayer on the surface of metal nanoparticles for molecular functionalization and assembly, corresponding functionalized nanoparticles and nanoparticle assemblies, and their use in plasmonic applications including surface-enhanced raman spectroscopy |
US10436707B2 (en) * | 2014-09-25 | 2019-10-08 | Centre National de la Recherche Scientifique—CNRS | Detection of analytes using nanoparticles as light scattering enhancers |
US20190323069A1 (en) * | 2015-11-18 | 2019-10-24 | University Of Florida Research Foundation, Inc. | Devices for detecting target biological molecules from cells and viruses |
CN110749585A (en) * | 2019-09-11 | 2020-02-04 | 亳州市新健康科技有限公司 | Biosensor based on surface enhanced Raman technology and preparation method thereof |
CN113039255A (en) * | 2018-09-26 | 2021-06-25 | 中国农业大学 | Detection kit and detection method for simultaneously detecting drift or deposition of multiple spraying droplets |
CN113433100A (en) * | 2021-05-25 | 2021-09-24 | 上海市公共卫生临床中心 | Plasma tryptophan and albumin joint detection method based on photochemical reaction of DNA synthesized silver nanoclusters and tryptophan |
WO2022090747A1 (en) * | 2020-11-02 | 2022-05-05 | The University Of Birmingham | Single point variant detection |
CN115855928A (en) * | 2023-02-27 | 2023-03-28 | 合肥工业大学 | Mercury ion detection method and kit based on nucleic acid macroarray and bifunctional molecules |
Families Citing this family (79)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030113740A1 (en) * | 2001-04-26 | 2003-06-19 | Mirkin Chad A. | Oligonucleotide-modified ROMP polymers and co-polymers |
US7160577B2 (en) | 2002-05-02 | 2007-01-09 | Micron Technology, Inc. | Methods for atomic-layer deposition of aluminum oxides in integrated circuits |
US8020433B2 (en) * | 2003-03-25 | 2011-09-20 | Tearlab Research, Inc. | Systems and methods for a sample fluid collection device |
US7810380B2 (en) | 2003-03-25 | 2010-10-12 | Tearlab Research, Inc. | Systems and methods for collecting tear film and measuring tear film osmolarity |
CA2508359A1 (en) * | 2002-12-12 | 2004-06-24 | Nanosphere, Inc. | Direct snp detection with unamplified dna |
GB0319949D0 (en) * | 2003-08-26 | 2003-09-24 | Univ Strathclyde | Nucleic acid sequence identification |
EP1678495A4 (en) * | 2003-10-20 | 2007-06-27 | Univ California | Nanoscale transduction systems for detecting molecular interactions |
WO2005069006A1 (en) * | 2004-01-06 | 2005-07-28 | Bwt Property Inc. | Novel antibody mediated surface enhanced raman scattering (sers) immunoassay and multiplexing schemes |
US20050225758A1 (en) * | 2004-03-23 | 2005-10-13 | Knopp Kevin J | Raman optical identification tag |
US20050221507A1 (en) * | 2004-03-30 | 2005-10-06 | Intel Corporation | Method to detect molecular binding by surface-enhanced Raman spectroscopy |
ES2752457T3 (en) * | 2004-04-23 | 2020-04-06 | Becton Dickinson Co | Surface-powered spectroscopically active compound nanoparticles |
US7588988B2 (en) | 2004-08-31 | 2009-09-15 | Micron Technology, Inc. | Method of forming apparatus having oxide films formed using atomic layer deposition |
WO2006030993A1 (en) * | 2004-09-14 | 2006-03-23 | Jin-Ho Choy | Information code system using dna sequences |
US7285427B2 (en) * | 2004-10-06 | 2007-10-23 | General Electric Company | Raman-active particles and methods of making and using them |
US7583379B2 (en) | 2005-07-28 | 2009-09-01 | University Of Georgia Research Foundation | Surface enhanced raman spectroscopy (SERS) systems and methods of use thereof |
WO2006060171A2 (en) * | 2004-11-16 | 2006-06-08 | Board Of Regents, The University Of Texas System | Methods and compositions related to phage-nanoparticle assemblies |
US20060134806A1 (en) * | 2004-12-20 | 2006-06-22 | General Electric Company | Method of separating unattached Raman-active tag from bioassay or other reaction mixture |
US7687409B2 (en) | 2005-03-29 | 2010-03-30 | Micron Technology, Inc. | Atomic layer deposited titanium silicon oxide films |
US7662729B2 (en) | 2005-04-28 | 2010-02-16 | Micron Technology, Inc. | Atomic layer deposition of a ruthenium layer to a lanthanide oxide dielectric layer |
US7572695B2 (en) | 2005-05-27 | 2009-08-11 | Micron Technology, Inc. | Hafnium titanium oxide films |
JP2006349462A (en) * | 2005-06-15 | 2006-12-28 | Canon Inc | Surface reinforcing raman spectroscopic analyzing jig and its manufacturing method |
US7927948B2 (en) | 2005-07-20 | 2011-04-19 | Micron Technology, Inc. | Devices with nanocrystals and methods of formation |
US7575978B2 (en) * | 2005-08-04 | 2009-08-18 | Micron Technology, Inc. | Method for making conductive nanoparticle charge storage element |
US7410763B2 (en) * | 2005-09-01 | 2008-08-12 | Intel Corporation | Multiplex data collection and analysis in bioanalyte detection |
JPWO2007049508A1 (en) * | 2005-10-24 | 2009-04-30 | 株式会社アイ・ピー・ビー | Long-chain DNA base sequence analysis apparatus and method |
US20090298197A1 (en) * | 2005-11-15 | 2009-12-03 | Oxonica Materials Inc. | Sers-based methods for detection of bioagents |
EP1977242B1 (en) * | 2006-01-27 | 2016-08-03 | Becton Dickinson and Company | Lateral flow immunoassay with encapsulated detection modality |
WO2007092941A2 (en) * | 2006-02-08 | 2007-08-16 | Oxonica, Inc. | Sers nanotag assays |
JP5277165B2 (en) * | 2006-07-24 | 2013-08-28 | ベクトン・ディキンソン・アンド・カンパニー | Analytical particle agglomeration and imaging apparatus and method |
WO2008105814A2 (en) * | 2006-08-22 | 2008-09-04 | Los Alamos National Security, Llc | Miniturized lateral flow device for rapid and sensitive detection of proteins or nucleic acids |
US7502106B2 (en) * | 2006-09-21 | 2009-03-10 | Honeywell International Inc. | SERS analyzer |
US7727776B2 (en) * | 2006-10-24 | 2010-06-01 | Honeywell International Inc. | Core-shell nanoparticles for detection based on SERS |
EP2104742B1 (en) * | 2006-11-21 | 2014-05-07 | Jawaharlal Nehru Centre for Advanced Scientific Research | A high sensitivity assay for molecular typing of biological sample, probes and a kit thereof |
US7763511B2 (en) * | 2006-12-29 | 2010-07-27 | Intel Corporation | Dielectric barrier for nanocrystals |
US7869030B2 (en) * | 2007-01-03 | 2011-01-11 | Research Foundation Of State University Of New York | Aggregates of plural transition metal nanoparticles and plural cyanine dye molecules |
US8343627B2 (en) | 2007-02-20 | 2013-01-01 | Research Foundation Of State University Of New York | Core-shell nanoparticles with multiple cores and a method for fabricating them |
US20090017562A1 (en) * | 2007-03-13 | 2009-01-15 | Iowa State University Research Foundation, Inc. | Raman-active reagents |
EP1992938A1 (en) * | 2007-05-14 | 2008-11-19 | Koninklijke Philips Electronics N.V. | Improved methods of SE(R)RS detection using multiple labels |
US8367506B2 (en) * | 2007-06-04 | 2013-02-05 | Micron Technology, Inc. | High-k dielectrics with gold nano-particles |
AU2008289383A1 (en) * | 2007-07-24 | 2009-02-26 | Northwestern University | Coated colloidal materials |
ITPO20070023A1 (en) | 2007-09-26 | 2009-03-27 | Hospitex Diagnostics S R L | METHOD AND DEVICE FOR THE HIGH SENSITIVITY MULTIPLE ANALYSIS OF BIOLOGICAL SAMPLES |
US7829735B2 (en) * | 2007-10-26 | 2010-11-09 | Northwestern University | Universal phosphoramidite for preparation of modified biomolecules and surfaces |
US20100105024A1 (en) * | 2008-01-14 | 2010-04-29 | Transgenex Nanobiotech, Inc. | Rapid test including genetic sequence probe |
US20100279272A1 (en) * | 2008-02-13 | 2010-11-04 | Michael Craig Burrell | Multiplexed analysis methods using sers-active nanoparticles |
US20090253121A1 (en) * | 2008-04-04 | 2009-10-08 | Micah Halpern | Method for amt-rflp dna fingerprinting |
EP3067694A1 (en) | 2008-05-05 | 2016-09-14 | Los Alamos National Security, LLC | Lateral flow-based nucleic acid sample preparation device, integrated with passive fluid flow control |
JP5391744B2 (en) * | 2009-03-09 | 2014-01-15 | 住友ベークライト株式会社 | Array substrate |
CA2783788C (en) * | 2009-12-11 | 2015-02-10 | Korea Research Institute Of Chemical Technology | Dimeric core-shell nanostructure labeled with raman active molecule localized at interparticle junction, use thereof, and method for preparing the same |
US8416609B2 (en) | 2010-02-15 | 2013-04-09 | Micron Technology, Inc. | Cross-point memory cells, non-volatile memory arrays, methods of reading a memory cell, methods of programming a memory cell, methods of writing to and reading from a memory cell, and computer systems |
FR2963106A1 (en) * | 2010-07-21 | 2012-01-27 | Ecole Polytechnique Dgar | METHOD AND DEVICE FOR ANALYZING MOLECULAR INTERACTIONS, AND USES THEREOF |
KR101229991B1 (en) | 2010-10-06 | 2013-02-05 | 단국대학교 산학협력단 | Simultaneous measuring sensor system of LSPR and SERS signal based on optical fiber |
DK2699700T3 (en) | 2011-04-20 | 2016-08-01 | Mesa Biotech Inc | Integrated device for nukleinsyreregistrering and identification |
GB201207821D0 (en) * | 2012-04-05 | 2012-06-13 | Renishaw Diagnostics Ltd | Dye sets for surface enhanced resonant raman spectroscopy |
KR101460439B1 (en) * | 2012-05-14 | 2014-11-12 | 서울대학교산학협력단 | Nanoprobe and method for detecting target substance using the same |
CN102692405B (en) * | 2012-06-07 | 2015-05-27 | 中国科学院合肥物质科学研究院 | Rice-grain-shaped fluoride/silver composite nanometer material and preparation method and application thereof |
JP6429318B2 (en) * | 2012-08-17 | 2018-11-28 | 国立研究開発法人科学技術振興機構 | Biomolecule analysis method and apparatus using Raman spectroscopy |
US9512468B2 (en) | 2012-11-06 | 2016-12-06 | Industrial Technology Research Institute | Detection method uses magnetic and detectable nanoparticles with oligonucleotides attached thereto |
WO2014089698A1 (en) * | 2012-12-11 | 2014-06-19 | Zamecnik Colin R | Encapsulated dye coated noble metal nanoparticles with increased surface enhanced raman scattering properties as contrast agents |
US10961564B2 (en) | 2014-05-28 | 2021-03-30 | University Of Houston System | Nanoporous gold and silver nanoparticles and substrates for molecular and biomolecular sensing |
CN104142375B (en) * | 2014-08-20 | 2016-01-20 | 中华人民共和国潍坊出入境检验检疫局 | A kind ofly utilize mould metabolic product to set up finger-print and utilize the method for fingerprint identification mold species |
EP3198257B1 (en) | 2014-09-23 | 2020-08-12 | Tearlab Research, Inc. | System for integration of microfluidic tear collection and lateral flow analysis of analytes of interest |
US10105080B1 (en) * | 2014-10-24 | 2018-10-23 | Verily Life Sciences Llc | Interstitial fluid sampling above microneedle array |
JP2016156807A (en) * | 2015-02-23 | 2016-09-01 | 株式会社堀場製作所 | Analysis method and analysis device |
CN106596927B (en) * | 2016-12-26 | 2018-10-19 | 中国科学院长春光学精密机械与物理研究所 | A kind of optical fiber bio probe and preparation method thereof and whole blood test method |
US10156658B1 (en) * | 2017-06-06 | 2018-12-18 | Saudi Arabian Oil Company | Detecting a tracer in a hydrocarbon reservoir |
CN111033260A (en) * | 2017-06-30 | 2020-04-17 | 韩国化学研究院 | Diagnostic kit for sepsis and diagnostic method using the same |
CN110554018A (en) * | 2018-05-31 | 2019-12-10 | 上海市刑事科学技术研究院 | Surface enhanced Raman material for detecting 4-bromomethcathinone in aqueous solution and preparation method thereof |
US20220187211A1 (en) * | 2019-04-03 | 2022-06-16 | Atonarp Inc. | Apparatus including analyzer unit |
GB201907007D0 (en) * | 2019-05-17 | 2019-07-03 | Nano Biosols Ltd | A simple method and kit for quantifying gold nanoparticles in a liquid sample |
CN110455754B (en) * | 2019-07-15 | 2022-03-18 | 深圳大学 | DNA probe and manufacturing method thereof |
CN110646399B (en) * | 2019-09-29 | 2021-08-13 | 吉林大学 | SERS-based high-throughput protein detection chip |
CN110702665B (en) * | 2019-11-14 | 2022-03-22 | 济南大学 | Preparation of paper-based coupling enhanced Raman sensor and application of paper-based coupling enhanced Raman sensor in okadaic acid detection |
WO2021193589A1 (en) * | 2020-03-23 | 2021-09-30 | 積水化学工業株式会社 | Testing method, testing kit, and testing system |
CN112698020B (en) * | 2020-11-12 | 2022-08-26 | 中山大学 | Multimodal coupling analysis method of cross response system based on DNA-AuNP coding |
CN113189078B (en) * | 2021-03-04 | 2024-04-16 | 吉林大学 | High-throughput screening method of targeted drugs |
US11796517B2 (en) | 2021-11-09 | 2023-10-24 | Saudi Arabian Oil Company | Multifunctional magnetic tags for mud logging |
US11725139B2 (en) | 2021-12-13 | 2023-08-15 | Saudi Arabian Oil Company | Manipulating hydrophilicity of conventional dye molecules for water tracer applications |
WO2023205784A2 (en) * | 2022-04-21 | 2023-10-26 | Emory University | Heteromultivalent nucleic acid functionalized materials to detect mutations and use in diagnostic applications |
KR20230165011A (en) * | 2022-05-26 | 2023-12-05 | 단국대학교 산학협력단 | Digital localized surface plasmon resonance sensor using optical fiber bundle and manufacturing method |
Citations (68)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4193983A (en) * | 1978-05-16 | 1980-03-18 | Syva Company | Labeled liposome particle compositions and immunoassays therewith |
US4256834A (en) * | 1979-04-09 | 1981-03-17 | Syva Company | Fluorescent scavenger particle immunoassay |
US4261968A (en) * | 1979-05-10 | 1981-04-14 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
US4313734A (en) * | 1978-07-13 | 1982-02-02 | Akzona Incorporated | Metal sol particle immunoassay |
US4318707A (en) * | 1978-11-24 | 1982-03-09 | Syva Company | Macromolecular fluorescent quencher particle in specific receptor assays |
US4650770A (en) * | 1981-04-27 | 1987-03-17 | Syntex (U.S.A.) Inc. | Energy absorbing particle quenching in light emitting competitive protein binding assays |
US4713348A (en) * | 1983-04-05 | 1987-12-15 | Syntex (U.S.A.) Inc. | Fluorescent multiparameter particle analysis |
US4853335A (en) * | 1987-09-28 | 1989-08-01 | Olsen Duane A | Colloidal gold particle concentration immunoassay |
US4868104A (en) * | 1985-09-06 | 1989-09-19 | Syntex (U.S.A.) Inc. | Homogeneous assay for specific polynucleotides |
US4996143A (en) * | 1985-12-23 | 1991-02-26 | Syngene, Inc. | Fluorescent stokes shift probes for polynucleotide hybridization |
US5203989A (en) * | 1991-01-30 | 1993-04-20 | Reidy James J | Portable air-water generator |
US5225064A (en) * | 1992-01-15 | 1993-07-06 | Enzyme Technology Research Group, Inc. | Peroxidase colloidal gold oxidase biosensors for mediatorless glucose determination |
US5266498A (en) * | 1989-10-27 | 1993-11-30 | Abbott Laboratories | Ligand binding assay for an analyte using surface-enhanced scattering (SERS) signal |
US5284748A (en) * | 1986-03-25 | 1994-02-08 | Immunotronics, Inc. | Method for electrical detection of a binding reaction |
US5288609A (en) * | 1984-04-27 | 1994-02-22 | Enzo Diagnostics, Inc. | Capture sandwich hybridization method and composition |
US5294369A (en) * | 1990-12-05 | 1994-03-15 | Akzo N.V. | Ligand gold bonding |
US5306403A (en) * | 1992-08-24 | 1994-04-26 | Martin Marietta Energy Systems, Inc. | Raman-based system for DNA sequencing-mapping and other separations |
US5360895A (en) * | 1987-04-22 | 1994-11-01 | Associated Universities, Inc. | Derivatized gold clusters and antibody-gold cluster conjugates |
US5376556A (en) * | 1989-10-27 | 1994-12-27 | Abbott Laboratories | Surface-enhanced Raman spectroscopy immunoassay |
US5384265A (en) * | 1993-03-26 | 1995-01-24 | Geo-Centers, Inc. | Biomolecules bound to catalytic inorganic particles, immunoassays using the same |
US5460831A (en) * | 1990-06-22 | 1995-10-24 | The Regents Of The University Of California | Targeted transfection nanoparticles |
US5472881A (en) * | 1992-11-12 | 1995-12-05 | University Of Utah Research Foundation | Thiol labeling of DNA for attachment to gold surfaces |
US5508164A (en) * | 1990-10-29 | 1996-04-16 | Dekalb Genetics Corporation | Isolation of biological materials using magnetic particles |
US5514602A (en) * | 1986-06-09 | 1996-05-07 | Ortho Diagnostic Systems, Inc. | Method of producing a metal sol reagent containing colloidal metal particles |
US5521289A (en) * | 1994-07-29 | 1996-05-28 | Nanoprobes, Inc. | Small organometallic probes |
US5543158A (en) * | 1993-07-23 | 1996-08-06 | Massachusetts Institute Of Technology | Biodegradable injectable nanoparticles |
US5599668A (en) * | 1994-09-22 | 1997-02-04 | Abbott Laboratories | Light scattering optical waveguide method for detecting specific binding events |
US5609907A (en) * | 1995-02-09 | 1997-03-11 | The Penn State Research Foundation | Self-assembled metal colloid monolayers |
US5637508A (en) * | 1993-03-26 | 1997-06-10 | Geo-Centers, Inc. | Biomolecules bound to polymer or copolymer coated catalytic inorganic particles, immunoassays using the same and kits containing the same |
US5665582A (en) * | 1990-10-29 | 1997-09-09 | Dekalb Genetics Corp. | Isolation of biological materials |
US5681943A (en) * | 1993-04-12 | 1997-10-28 | Northwestern University | Method for covalently linking adjacent oligonucleotides |
US5751018A (en) * | 1991-11-22 | 1998-05-12 | The Regents Of The University Of California | Semiconductor nanocrystals covalently bound to solid inorganic surfaces using self-assembled monolayers |
US5814516A (en) * | 1995-10-13 | 1998-09-29 | Lockheed Martin Energy Systems, Inc. | Surface enhanced Raman gene probe and methods thereof |
US5830986A (en) * | 1996-10-28 | 1998-11-03 | Massachusetts Institute Of Technology | Methods for the synthesis of functionalizable poly(ethylene oxide) star macromolecules |
US5900481A (en) * | 1996-11-06 | 1999-05-04 | Sequenom, Inc. | Bead linkers for immobilizing nucleic acids to solid supports |
US5922537A (en) * | 1996-11-08 | 1999-07-13 | N.o slashed.AB Immunoassay, Inc. | Nanoparticles biosensor |
US5939021A (en) * | 1997-01-23 | 1999-08-17 | Hansen; W. Peter | Homogeneous binding assay |
US5972615A (en) * | 1998-01-21 | 1999-10-26 | Urocor, Inc. | Biomarkers and targets for diagnosis, prognosis and management of prostate disease |
US5990479A (en) * | 1997-11-25 | 1999-11-23 | Regents Of The University Of California | Organo Luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes |
US6025202A (en) * | 1995-02-09 | 2000-02-15 | The Penn State Research Foundation | Self-assembled metal colloid monolayers and detection methods therewith |
US6127120A (en) * | 1995-07-25 | 2000-10-03 | University Of Strathclyde | Detection of nucleic acids and nucleic acid units |
US6149868A (en) * | 1997-10-28 | 2000-11-21 | The Penn State Research Foundation | Surface enhanced raman scattering from metal nanoparticle-analyte-noble metal substrate sandwiches |
US6214560B1 (en) * | 1996-04-25 | 2001-04-10 | Genicon Sciences Corporation | Analyte assay using particulate labels |
US6242265B1 (en) * | 1989-01-19 | 2001-06-05 | Dade Behring Marburg Gmbh | Use of doubly or triply charged cations in immunochemical assays |
US6251303B1 (en) * | 1998-09-18 | 2001-06-26 | Massachusetts Institute Of Technology | Water-soluble fluorescent nanocrystals |
US6264825B1 (en) * | 1998-06-23 | 2001-07-24 | Clinical Micro Sensors, Inc. | Binding acceleration techniques for the detection of analytes |
US6277489B1 (en) * | 1998-12-04 | 2001-08-21 | The Regents Of The University Of California | Support for high performance affinity chromatography and other uses |
US6306610B1 (en) * | 1998-09-18 | 2001-10-23 | Massachusetts Institute Of Technology | Biological applications of quantum dots |
US6361944B1 (en) * | 1996-07-29 | 2002-03-26 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6365418B1 (en) * | 1998-07-14 | 2002-04-02 | Zyomyx, Incorporated | Arrays of protein-capture agents and methods of use thereof |
US20020137070A1 (en) * | 1996-07-29 | 2002-09-26 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20020177143A1 (en) * | 2001-05-25 | 2002-11-28 | Mirkin Chad A. | Non-alloying core shell nanoparticles |
US20020192687A1 (en) * | 2000-03-28 | 2002-12-19 | Mirkin Chad A. | Bio-barcodes based on oligonucleotide-modified nanoparticles |
US6506564B1 (en) * | 1996-07-29 | 2003-01-14 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030068638A1 (en) * | 2001-08-03 | 2003-04-10 | William Cork | Nanoparticle imaging system and method |
US20030087242A1 (en) * | 1996-07-29 | 2003-05-08 | Mirkin Chad A. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030113740A1 (en) * | 2001-04-26 | 2003-06-19 | Mirkin Chad A. | Oligonucleotide-modified ROMP polymers and co-polymers |
US20030129608A1 (en) * | 2001-05-25 | 2003-07-10 | Mirkin Chad A | Non-alloying core shell nanoparticles |
US20030143598A1 (en) * | 2001-11-09 | 2003-07-31 | Viswanadham Garimella | Bioconjugate-nanoparticle probes |
US6602669B2 (en) * | 2000-07-11 | 2003-08-05 | Northwestern University | Method of detection by enhancement of silver staining |
US20030207296A1 (en) * | 1996-07-29 | 2003-11-06 | So-Jung Park | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20040053222A1 (en) * | 2002-07-02 | 2004-03-18 | Nanosphere, Inc. | Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes |
US20040072231A1 (en) * | 1996-07-29 | 2004-04-15 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6726847B2 (en) * | 2000-12-06 | 2004-04-27 | Northwestern University | Silver stain removal by chemical etching and sonication |
US20040086897A1 (en) * | 2002-05-07 | 2004-05-06 | Mirkin Chad A. | Nanoparticle probes with Raman Spectroscopic fingerprints for analyte detection |
US6767702B2 (en) * | 1996-07-29 | 2004-07-27 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6773884B2 (en) * | 1996-07-29 | 2004-08-10 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20060073336A1 (en) * | 2003-12-29 | 2006-04-06 | Jingwu Zhang | External modification of composite organic inorganic nanoclusters |
Family Cites Families (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8800702D0 (en) | 1988-01-13 | 1988-02-10 | Nycomed As | Test method & reagent kit therefor |
WO1990002205A1 (en) | 1988-08-25 | 1990-03-08 | Angenics, Inc. | Detection of nucleic acid sequences using particle agglutination |
EP0630974A3 (en) | 1993-06-25 | 1995-11-15 | Clinical Diagnostic Syst | Method and test kit for the detection of inorganic orthophosphate by-product from amplification of target nucleic acid. |
JPH07227299A (en) | 1994-02-14 | 1995-08-29 | Kyoto Daiichi Kagaku:Kk | Method for detecting specific base sequence of dna and device therefor |
US6242264B1 (en) | 1996-09-04 | 2001-06-05 | The Penn State Research Foundation | Self-assembled metal colloid monolayers having size and density gradients |
US6203989B1 (en) | 1998-09-30 | 2001-03-20 | Affymetrix, Inc. | Methods and compositions for amplifying detectable signals in specific binding assays |
DE69936534T2 (en) | 1998-11-30 | 2008-03-13 | Nanosphere, Inc., Northbrook | NANOPARTICLES WITH POLYMERSHOLES |
JP2003503699A (en) * | 1999-06-25 | 2003-01-28 | ナノスフェアー インコーポレイテッド | Oligonucleotide-Attached Nanoparticles and Methods of Use |
ATE487136T1 (en) | 2000-03-28 | 2010-11-15 | Nanosphere Inc | NANOPARTICLES WITH BONDED OLIGONUCLEOTIDES AND USES THEREOF |
EP1362121A2 (en) | 2000-08-11 | 2003-11-19 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US7829348B2 (en) * | 2000-09-22 | 2010-11-09 | Iowa State University Research Foundation, Inc. | Raman-active reagents and the use thereof |
WO2002046475A2 (en) | 2000-12-08 | 2002-06-13 | Curagen Corporation | Method of detecting and treating tuberous sclerosis complex associated disorders |
AU2002230593A1 (en) | 2000-12-08 | 2002-06-18 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
JP4146239B2 (en) | 2001-03-28 | 2008-09-10 | ナノスフェアー インコーポレイテッド | Bio barcode based on oligonucleotide modified particles |
AU2002367709A1 (en) | 2001-04-26 | 2003-10-27 | Nanosphere, Inc. | Oligonucleotide-modified romp polymers and co-polymers |
JP4347049B2 (en) | 2001-10-09 | 2009-10-21 | ナノスフェアー インコーポレイテッド | Nanoparticles with oligonucleotides attached and methods of use |
ATE479775T1 (en) | 2002-11-26 | 2010-09-15 | Univ Maryland Biotechnology | HIGHLY SENSITIVE ASSAY FOR PATHOGENE DETECTION USING METAL ENHANCED FLUORESCENCE |
CA2508359A1 (en) | 2002-12-12 | 2004-06-24 | Nanosphere, Inc. | Direct snp detection with unamplified dna |
-
2002
- 2002-06-14 US US10/172,428 patent/US20030211488A1/en not_active Abandoned
-
2003
- 2003-05-07 JP JP2004503919A patent/JP2005524849A/en active Pending
- 2003-05-07 AT AT03736550T patent/ATE515698T1/en not_active IP Right Cessation
- 2003-05-07 US US10/431,341 patent/US7985539B2/en not_active Expired - Fee Related
- 2003-05-07 AU AU2003237180A patent/AU2003237180B2/en not_active Ceased
- 2003-05-07 CA CA2483697A patent/CA2483697C/en not_active Expired - Fee Related
- 2003-05-07 EP EP03736550A patent/EP1499889B1/en not_active Expired - Lifetime
- 2003-05-07 WO PCT/US2003/014100 patent/WO2003095973A2/en active IP Right Grant
Patent Citations (96)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4193983A (en) * | 1978-05-16 | 1980-03-18 | Syva Company | Labeled liposome particle compositions and immunoassays therewith |
US4313734A (en) * | 1978-07-13 | 1982-02-02 | Akzona Incorporated | Metal sol particle immunoassay |
US4318707A (en) * | 1978-11-24 | 1982-03-09 | Syva Company | Macromolecular fluorescent quencher particle in specific receptor assays |
US4256834A (en) * | 1979-04-09 | 1981-03-17 | Syva Company | Fluorescent scavenger particle immunoassay |
US4261968A (en) * | 1979-05-10 | 1981-04-14 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
US4650770A (en) * | 1981-04-27 | 1987-03-17 | Syntex (U.S.A.) Inc. | Energy absorbing particle quenching in light emitting competitive protein binding assays |
US4713348A (en) * | 1983-04-05 | 1987-12-15 | Syntex (U.S.A.) Inc. | Fluorescent multiparameter particle analysis |
US5288609A (en) * | 1984-04-27 | 1994-02-22 | Enzo Diagnostics, Inc. | Capture sandwich hybridization method and composition |
US4868104A (en) * | 1985-09-06 | 1989-09-19 | Syntex (U.S.A.) Inc. | Homogeneous assay for specific polynucleotides |
US4996143A (en) * | 1985-12-23 | 1991-02-26 | Syngene, Inc. | Fluorescent stokes shift probes for polynucleotide hybridization |
US5284748A (en) * | 1986-03-25 | 1994-02-08 | Immunotronics, Inc. | Method for electrical detection of a binding reaction |
US5514602A (en) * | 1986-06-09 | 1996-05-07 | Ortho Diagnostic Systems, Inc. | Method of producing a metal sol reagent containing colloidal metal particles |
US5571726A (en) * | 1986-06-09 | 1996-11-05 | Ortho Diagnostic Systems, Inc. | Kit containing glutaraldehyde coated colloidal metal particles of a preselected size |
US5360895A (en) * | 1987-04-22 | 1994-11-01 | Associated Universities, Inc. | Derivatized gold clusters and antibody-gold cluster conjugates |
US4853335A (en) * | 1987-09-28 | 1989-08-01 | Olsen Duane A | Colloidal gold particle concentration immunoassay |
US6242265B1 (en) * | 1989-01-19 | 2001-06-05 | Dade Behring Marburg Gmbh | Use of doubly or triply charged cations in immunochemical assays |
US5266498A (en) * | 1989-10-27 | 1993-11-30 | Abbott Laboratories | Ligand binding assay for an analyte using surface-enhanced scattering (SERS) signal |
US5376556A (en) * | 1989-10-27 | 1994-12-27 | Abbott Laboratories | Surface-enhanced Raman spectroscopy immunoassay |
US5445972A (en) * | 1989-10-27 | 1995-08-29 | Abbott Laboratories | Raman label and its conjugate in a ligand-binding assay for a test sample analyte |
US5460831A (en) * | 1990-06-22 | 1995-10-24 | The Regents Of The University Of California | Targeted transfection nanoparticles |
US5665582A (en) * | 1990-10-29 | 1997-09-09 | Dekalb Genetics Corp. | Isolation of biological materials |
US5508164A (en) * | 1990-10-29 | 1996-04-16 | Dekalb Genetics Corporation | Isolation of biological materials using magnetic particles |
US5294369A (en) * | 1990-12-05 | 1994-03-15 | Akzo N.V. | Ligand gold bonding |
US5384073A (en) * | 1990-12-05 | 1995-01-24 | Akzo N.V. | Ligand gold bonding |
US5203989A (en) * | 1991-01-30 | 1993-04-20 | Reidy James J | Portable air-water generator |
US5751018A (en) * | 1991-11-22 | 1998-05-12 | The Regents Of The University Of California | Semiconductor nanocrystals covalently bound to solid inorganic surfaces using self-assembled monolayers |
US5225064A (en) * | 1992-01-15 | 1993-07-06 | Enzyme Technology Research Group, Inc. | Peroxidase colloidal gold oxidase biosensors for mediatorless glucose determination |
US5306403A (en) * | 1992-08-24 | 1994-04-26 | Martin Marietta Energy Systems, Inc. | Raman-based system for DNA sequencing-mapping and other separations |
US5472881A (en) * | 1992-11-12 | 1995-12-05 | University Of Utah Research Foundation | Thiol labeling of DNA for attachment to gold surfaces |
US5384265A (en) * | 1993-03-26 | 1995-01-24 | Geo-Centers, Inc. | Biomolecules bound to catalytic inorganic particles, immunoassays using the same |
US5637508A (en) * | 1993-03-26 | 1997-06-10 | Geo-Centers, Inc. | Biomolecules bound to polymer or copolymer coated catalytic inorganic particles, immunoassays using the same and kits containing the same |
US5681943A (en) * | 1993-04-12 | 1997-10-28 | Northwestern University | Method for covalently linking adjacent oligonucleotides |
US5543158A (en) * | 1993-07-23 | 1996-08-06 | Massachusetts Institute Of Technology | Biodegradable injectable nanoparticles |
US5521289A (en) * | 1994-07-29 | 1996-05-28 | Nanoprobes, Inc. | Small organometallic probes |
US5599668A (en) * | 1994-09-22 | 1997-02-04 | Abbott Laboratories | Light scattering optical waveguide method for detecting specific binding events |
US5609907A (en) * | 1995-02-09 | 1997-03-11 | The Penn State Research Foundation | Self-assembled metal colloid monolayers |
US6025202A (en) * | 1995-02-09 | 2000-02-15 | The Penn State Research Foundation | Self-assembled metal colloid monolayers and detection methods therewith |
US6127120A (en) * | 1995-07-25 | 2000-10-03 | University Of Strathclyde | Detection of nucleic acids and nucleic acid units |
US5814516A (en) * | 1995-10-13 | 1998-09-29 | Lockheed Martin Energy Systems, Inc. | Surface enhanced Raman gene probe and methods thereof |
US6214560B1 (en) * | 1996-04-25 | 2001-04-10 | Genicon Sciences Corporation | Analyte assay using particulate labels |
US6610491B2 (en) * | 1996-07-29 | 2003-08-26 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030124528A1 (en) * | 1996-07-29 | 2003-07-03 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6777186B2 (en) * | 1996-07-29 | 2004-08-17 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6773884B2 (en) * | 1996-07-29 | 2004-08-10 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6767702B2 (en) * | 1996-07-29 | 2004-07-27 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6759199B2 (en) * | 1996-07-29 | 2004-07-06 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6750016B2 (en) * | 1996-07-29 | 2004-06-15 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6740491B2 (en) * | 1996-07-29 | 2004-05-25 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6730269B2 (en) * | 1996-07-29 | 2004-05-04 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20040072231A1 (en) * | 1996-07-29 | 2004-04-15 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6720411B2 (en) * | 1996-07-29 | 2004-04-13 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6361944B1 (en) * | 1996-07-29 | 2002-03-26 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6720147B2 (en) * | 1996-07-29 | 2004-04-13 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6417340B1 (en) * | 1996-07-29 | 2002-07-09 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20020137070A1 (en) * | 1996-07-29 | 2002-09-26 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20020155461A1 (en) * | 1996-07-29 | 2002-10-24 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20020160381A1 (en) * | 1996-07-29 | 2002-10-31 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6709825B2 (en) * | 1996-07-29 | 2004-03-23 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6495324B1 (en) * | 1996-07-29 | 2002-12-17 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6682895B2 (en) * | 1996-07-29 | 2004-01-27 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6506564B1 (en) * | 1996-07-29 | 2003-01-14 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030054358A1 (en) * | 1996-07-29 | 2003-03-20 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030068622A1 (en) * | 1996-07-29 | 2003-04-10 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6677122B2 (en) * | 1996-07-29 | 2004-01-13 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030087242A1 (en) * | 1996-07-29 | 2003-05-08 | Mirkin Chad A. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6673548B2 (en) * | 1996-07-29 | 2004-01-06 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6582921B2 (en) * | 1996-07-29 | 2003-06-24 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses thereof |
US6645721B2 (en) * | 1996-07-29 | 2003-11-11 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030207296A1 (en) * | 1996-07-29 | 2003-11-06 | So-Jung Park | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030148282A1 (en) * | 1996-07-29 | 2003-08-07 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030143538A1 (en) * | 1996-07-29 | 2003-07-31 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US5830986A (en) * | 1996-10-28 | 1998-11-03 | Massachusetts Institute Of Technology | Methods for the synthesis of functionalizable poly(ethylene oxide) star macromolecules |
US5900481A (en) * | 1996-11-06 | 1999-05-04 | Sequenom, Inc. | Bead linkers for immobilizing nucleic acids to solid supports |
US5922537A (en) * | 1996-11-08 | 1999-07-13 | N.o slashed.AB Immunoassay, Inc. | Nanoparticles biosensor |
US5939021A (en) * | 1997-01-23 | 1999-08-17 | Hansen; W. Peter | Homogeneous binding assay |
US6149868A (en) * | 1997-10-28 | 2000-11-21 | The Penn State Research Foundation | Surface enhanced raman scattering from metal nanoparticle-analyte-noble metal substrate sandwiches |
US5990479A (en) * | 1997-11-25 | 1999-11-23 | Regents Of The University Of California | Organo Luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes |
US5972615A (en) * | 1998-01-21 | 1999-10-26 | Urocor, Inc. | Biomarkers and targets for diagnosis, prognosis and management of prostate disease |
US6264825B1 (en) * | 1998-06-23 | 2001-07-24 | Clinical Micro Sensors, Inc. | Binding acceleration techniques for the detection of analytes |
US6365418B1 (en) * | 1998-07-14 | 2002-04-02 | Zyomyx, Incorporated | Arrays of protein-capture agents and methods of use thereof |
US6251303B1 (en) * | 1998-09-18 | 2001-06-26 | Massachusetts Institute Of Technology | Water-soluble fluorescent nanocrystals |
US6306610B1 (en) * | 1998-09-18 | 2001-10-23 | Massachusetts Institute Of Technology | Biological applications of quantum dots |
US6277489B1 (en) * | 1998-12-04 | 2001-08-21 | The Regents Of The University Of California | Support for high performance affinity chromatography and other uses |
US20020192687A1 (en) * | 2000-03-28 | 2002-12-19 | Mirkin Chad A. | Bio-barcodes based on oligonucleotide-modified nanoparticles |
US20040101889A1 (en) * | 2000-07-11 | 2004-05-27 | Northwestern University | Method of detection by enhancement of silver staining |
US6602669B2 (en) * | 2000-07-11 | 2003-08-05 | Northwestern University | Method of detection by enhancement of silver staining |
US6726847B2 (en) * | 2000-12-06 | 2004-04-27 | Northwestern University | Silver stain removal by chemical etching and sonication |
US20030113740A1 (en) * | 2001-04-26 | 2003-06-19 | Mirkin Chad A. | Oligonucleotide-modified ROMP polymers and co-polymers |
US20020177143A1 (en) * | 2001-05-25 | 2002-11-28 | Mirkin Chad A. | Non-alloying core shell nanoparticles |
US20040038255A1 (en) * | 2001-05-25 | 2004-02-26 | Northwestern University | Non-alloying core shell nanoparticles |
US20030129608A1 (en) * | 2001-05-25 | 2003-07-10 | Mirkin Chad A | Non-alloying core shell nanoparticles |
US20030068638A1 (en) * | 2001-08-03 | 2003-04-10 | William Cork | Nanoparticle imaging system and method |
US20030143598A1 (en) * | 2001-11-09 | 2003-07-31 | Viswanadham Garimella | Bioconjugate-nanoparticle probes |
US20040086897A1 (en) * | 2002-05-07 | 2004-05-06 | Mirkin Chad A. | Nanoparticle probes with Raman Spectroscopic fingerprints for analyte detection |
US20040053222A1 (en) * | 2002-07-02 | 2004-03-18 | Nanosphere, Inc. | Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes |
US20060073336A1 (en) * | 2003-12-29 | 2006-04-06 | Jingwu Zhang | External modification of composite organic inorganic nanoclusters |
Cited By (170)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7250499B2 (en) | 1996-07-29 | 2007-07-31 | Nanosphere Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20060068378A1 (en) * | 1996-07-29 | 2006-03-30 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030054358A1 (en) * | 1996-07-29 | 2003-03-20 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030068622A1 (en) * | 1996-07-29 | 2003-04-10 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US7169556B2 (en) | 1996-07-29 | 2007-01-30 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20030207296A1 (en) * | 1996-07-29 | 2003-11-06 | So-Jung Park | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20020137071A1 (en) * | 1996-07-29 | 2002-09-26 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6986989B2 (en) | 1996-07-29 | 2006-01-17 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US7098320B1 (en) | 1996-07-29 | 2006-08-29 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20040072231A1 (en) * | 1996-07-29 | 2004-04-15 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6969761B2 (en) | 1996-07-29 | 2005-11-29 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US20040110220A1 (en) * | 1996-07-29 | 2004-06-10 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6962786B2 (en) | 1996-07-29 | 2005-11-08 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US7208587B2 (en) | 1996-07-29 | 2007-04-24 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US8323888B2 (en) | 1996-07-29 | 2012-12-04 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US7259252B2 (en) | 1996-07-29 | 2007-08-21 | Nanosphere, Inc. | Nanoparticles having oligonucleotides attached thereto and uses therefor |
US6974669B2 (en) | 2000-03-28 | 2005-12-13 | Nanosphere, Inc. | Bio-barcodes based on oligonucleotide-modified nanoparticles |
US20020192687A1 (en) * | 2000-03-28 | 2002-12-19 | Mirkin Chad A. | Bio-barcodes based on oligonucleotide-modified nanoparticles |
US20040101889A1 (en) * | 2000-07-11 | 2004-05-27 | Northwestern University | Method of detection by enhancement of silver staining |
US20060040286A1 (en) * | 2001-03-28 | 2006-02-23 | Nanosphere, Inc. | Bio-barcode based detection of target analytes |
US20050037397A1 (en) * | 2001-03-28 | 2005-02-17 | Nanosphere, Inc. | Bio-barcode based detection of target analytes |
US20030129608A1 (en) * | 2001-05-25 | 2003-07-10 | Mirkin Chad A | Non-alloying core shell nanoparticles |
US7135055B2 (en) | 2001-05-25 | 2006-11-14 | Nanosphere, Inc. | Non-alloying core shell nanoparticles |
US7147687B2 (en) | 2001-05-25 | 2006-12-12 | Nanosphere, Inc. | Non-alloying core shell nanoparticles |
US20070190551A1 (en) * | 2001-05-25 | 2007-08-16 | Nanosphere, Inc. | Non-alloying core shell nanoparticles |
US7238472B2 (en) | 2001-05-25 | 2007-07-03 | Nanosphere, Inc. | Non-alloying core shell nanoparticles |
US20040038255A1 (en) * | 2001-05-25 | 2004-02-26 | Northwestern University | Non-alloying core shell nanoparticles |
US7985539B2 (en) | 2002-05-07 | 2011-07-26 | Northwestern University | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection |
US20040053222A1 (en) * | 2002-07-02 | 2004-03-18 | Nanosphere, Inc. | Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes |
US7253277B2 (en) | 2002-07-02 | 2007-08-07 | Nanosphere, Inc. | Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes |
US7785901B2 (en) * | 2002-08-02 | 2010-08-31 | Sony Deutschland Gmbh | Method of attaching hydrophilic species to hydrophilic macromolecules and immobilizing the hydrophilic macromolecules on a hydrophobic surface |
US20040023287A1 (en) * | 2002-08-02 | 2004-02-05 | Oliver Harnack | Method of attaching hydrophilic species to hydrophilic macromolecules and immobilizing the hydrophilic macromolecules on a hydrophobic surface |
US20050130174A1 (en) * | 2003-02-27 | 2005-06-16 | Nanosphere, Inc. | Label-free gene expression profiling with universal nanoparticle probes in microarray assay format |
US20040179195A1 (en) * | 2003-03-12 | 2004-09-16 | Xing Su | Chemical enhancement in surface enhanced raman scattering using lithium salts |
US7019828B2 (en) | 2003-03-12 | 2006-03-28 | Intel Corporation | Chemical enhancement in surface enhanced raman scattering using lithium salts |
US20050250094A1 (en) * | 2003-05-30 | 2005-11-10 | Nanosphere, Inc. | Method for detecting analytes based on evanescent illumination and scatter-based detection of nanoparticle probe complexes |
US20070111204A1 (en) * | 2003-06-27 | 2007-05-17 | Kathleen Delgrosso | Methods for detecting nucleic acid variations |
WO2005031301A2 (en) * | 2003-07-10 | 2005-04-07 | Parallax Biosystems, Inc. | Raman signature probes and their use in the detection and imaging of molecular processes and structures |
US20070134733A1 (en) * | 2003-07-10 | 2007-06-14 | Parallax Biosystems, Inc. | Raman signature probes and their use in the detection and imaging of molecular processes and structures |
WO2005031301A3 (en) * | 2003-07-10 | 2007-06-14 | Parallax Biosystems Inc | Raman signature probes and their use in the detection and imaging of molecular processes and structures |
US20070054288A1 (en) * | 2003-09-24 | 2007-03-08 | Intel Corporation | Programmable molecular barcodes |
US20050064435A1 (en) * | 2003-09-24 | 2005-03-24 | Xing Su | Programmable molecular barcodes |
US20060199216A1 (en) * | 2003-09-24 | 2006-09-07 | Intel Corporation | Programmable molecular barcodes |
US20110224417A1 (en) * | 2003-12-19 | 2011-09-15 | Kankyo Engineering Co., Ltd. | Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefore |
US20050147963A1 (en) * | 2003-12-29 | 2005-07-07 | Intel Corporation | Composite organic-inorganic nanoparticles and methods for use thereof |
US20070031861A1 (en) * | 2003-12-29 | 2007-02-08 | Intel Corporation | Methods for determining nucleotide sequence information |
US20050142567A1 (en) * | 2003-12-29 | 2005-06-30 | Intel Corporation | Composite organic-inorganic nanoparticles and methods for use thereof |
US20090069481A1 (en) * | 2003-12-29 | 2009-03-12 | Jingwu Zhang | External modification of composite organic inorganic nanoclusters |
GB2427469B (en) * | 2003-12-29 | 2008-10-29 | Intel Corp | Composite organic-inorganic nanoclusters |
US20060073336A1 (en) * | 2003-12-29 | 2006-04-06 | Jingwu Zhang | External modification of composite organic inorganic nanoclusters |
US7790286B2 (en) | 2003-12-29 | 2010-09-07 | Intel Corporation | External modification of composite organic inorganic nanoclusters |
US20050147976A1 (en) * | 2003-12-29 | 2005-07-07 | Xing Su | Methods for determining nucleotide sequence information |
US20060234248A1 (en) * | 2003-12-29 | 2006-10-19 | Lei Sun | Composite organic inorganic nanoclusters |
US20100240870A1 (en) * | 2003-12-29 | 2010-09-23 | Xing Su | Composite organic-inorganic nanoclusters |
US7361410B2 (en) * | 2003-12-29 | 2008-04-22 | Intel Corporation | External modification of composite organic inorganic nanoclusters comprising raman active organic compound |
US20050191665A1 (en) * | 2003-12-29 | 2005-09-01 | Xing Su | Composite organic-inorganic nanoclusters |
GB2427469A (en) * | 2003-12-29 | 2006-12-27 | Intel Corp | Composite organic-inorganic nanoclusters |
WO2005090948A3 (en) * | 2003-12-29 | 2006-02-16 | Intel Corp | Composite organic-inorganic nanoclusters |
US20060033910A1 (en) * | 2003-12-29 | 2006-02-16 | Lei Sun | Multiplexed detection of analytes in fluid solution |
WO2005090948A2 (en) * | 2003-12-29 | 2005-09-29 | Intel Corporation | Composite organic-inorganic nanoclusters |
US20080076119A9 (en) * | 2003-12-29 | 2008-03-27 | Lei Sun | Composite organic inorganic nanoclusters |
US20070128615A1 (en) * | 2003-12-29 | 2007-06-07 | Intel Corporation | Methods for determining nucleotide sequence information |
US20070279626A9 (en) * | 2003-12-29 | 2007-12-06 | Lei Sun | Multiplexed detection of analytes in fluid solution |
US20050250159A1 (en) * | 2003-12-30 | 2005-11-10 | Intel Corporation | Methods for using Raman spectroscopy to obtain a protein profile of a biological sample |
US20050148100A1 (en) * | 2003-12-30 | 2005-07-07 | Intel Corporation | Methods and devices for using Raman-active probe constructs to assay biological samples |
US20070298433A1 (en) * | 2003-12-31 | 2007-12-27 | President And Fellows Of Harvard College | Assay Device and Method |
US8574924B2 (en) | 2003-12-31 | 2013-11-05 | President And Fellows Of Harvard College | Assay device and method |
US10082507B2 (en) | 2003-12-31 | 2018-09-25 | President And Fellows Of Harvard College | Assay device and method |
US7736890B2 (en) | 2003-12-31 | 2010-06-15 | President And Fellows Of Harvard College | Assay device and method |
US20100279310A1 (en) * | 2003-12-31 | 2010-11-04 | President And Fellows Of Harvard College | Assay device and method |
US20070077429A1 (en) * | 2004-02-20 | 2007-04-05 | Mirkin Chad A | Multicomponent nanorods |
US7422696B2 (en) | 2004-02-20 | 2008-09-09 | Northwestern University | Multicomponent nanorods |
US20090155587A1 (en) * | 2004-02-20 | 2009-06-18 | Northwestern University | Multicomponent Nanorods |
DE102004015272A1 (en) * | 2004-03-29 | 2005-11-03 | Infineon Technologies Ag | Biosensor to determine the presence of DNA comprises trap molecules that hybridize with the target molecules over photo diode detector |
US7351591B2 (en) | 2004-03-30 | 2008-04-01 | Intel Corporation | Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS) |
US20050221506A1 (en) * | 2004-03-30 | 2005-10-06 | Intel Corporation | Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS) |
US7432113B2 (en) * | 2004-03-30 | 2008-10-07 | Intel Corporation | Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS) |
US7432112B2 (en) | 2004-03-30 | 2008-10-07 | Intel Corporation | Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS) |
US20050221510A1 (en) * | 2004-03-30 | 2005-10-06 | Intel Corporation | Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS) |
US7427513B2 (en) | 2004-03-30 | 2008-09-23 | Intel Corporation | Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS) |
US20050221508A1 (en) * | 2004-03-30 | 2005-10-06 | Intel Corporation | Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS) |
US20050221360A1 (en) * | 2004-03-31 | 2005-10-06 | Takara Bio Inc. | Method for purifying microbeads |
US7226794B2 (en) * | 2004-04-14 | 2007-06-05 | Agilent Technologies, Inc. | Surface-enhanced Raman spectroscopy for biosensor systems and methods for determining the presence of biomolecules |
US20050233474A1 (en) * | 2004-04-14 | 2005-10-20 | Roitman Daniel B | Surface-enhanced Raman spectroscopy for biosensor systems and methods for determining the presence of biomolecules |
WO2006028601A3 (en) * | 2004-07-26 | 2007-02-01 | Nanosphere Inc | Method for distinguishing methicillin resistant s. aureus from methicillin sensitive s. aureus in a mixed culture |
WO2006028601A2 (en) * | 2004-07-26 | 2006-03-16 | Nanosphere, Inc. | Method for distinguishing methicillin resistant s. aureus from methicillin sensitive s. aureus in a mixed culture |
US20060057613A1 (en) * | 2004-07-26 | 2006-03-16 | Nanosphere, Inc. | Method for distinguishing methicillin resistant S. aureus from methicillin sensitive S. aureus in a mixed culture |
US20070048797A1 (en) * | 2004-08-11 | 2007-03-01 | Xing Su | Composite organic inorganic nanoclusters as carriers and identifiers of tester molecules |
US7776547B2 (en) | 2004-08-26 | 2010-08-17 | Intel Corporation | Cellular analysis using Raman surface scanning |
US20060046311A1 (en) * | 2004-08-26 | 2006-03-02 | Intel Corporation | Biomolecule analysis using Raman surface scanning |
US20060046313A1 (en) * | 2004-08-26 | 2006-03-02 | Intel Corporation | Cellular analysis using Raman surface scanning |
WO2006037036A2 (en) * | 2004-09-27 | 2006-04-06 | Purdue Research Foundation | Quantitative proteomics with isotopic substituted raman active labeling |
WO2006037036A3 (en) * | 2004-09-27 | 2006-09-14 | Purdue Research Foundation | Quantitative proteomics with isotopic substituted raman active labeling |
US20090053818A1 (en) * | 2004-09-27 | 2009-02-26 | Dongmao Zhang | Quantitative proteomics with isotopic substituted raman active labeling |
US7393691B2 (en) * | 2004-10-18 | 2008-07-01 | Real-Time Analyzers, Inc. | SERS method and apparatus for rapid extraction and analysis of drugs in saliva |
US7393692B2 (en) * | 2004-10-18 | 2008-07-01 | Real-Time Analyzers, Inc. | SERS method for rapid pharmacokinetic analysis of drugs in saliva |
US20060084181A1 (en) * | 2004-10-18 | 2006-04-20 | Stuart Farquharson | Method and apparatus for rapid extraction and analysis, by SERS, of drugs in saliva |
US7618560B2 (en) | 2004-10-21 | 2009-11-17 | Pharma Diagnostics N.V. | Stable metal/conductive polymer composite colloids and methods for making and using the same |
US7738096B2 (en) | 2004-10-21 | 2010-06-15 | University Of Georgia Research Foundation, Inc. | Surface enhanced Raman spectroscopy (SERS) systems, substrates, fabrication thereof, and methods of use thereof |
US7686983B2 (en) | 2004-10-21 | 2010-03-30 | Pharma Diagnostics N.V. | Stable metal/conductive polymer composite colloids and methods for making and using the same |
US20070172834A1 (en) * | 2004-10-21 | 2007-07-26 | Patrick Englebienne | Stable metal/conductive polymer composite colloids and methods for making and using the same |
US8241922B2 (en) * | 2004-12-13 | 2012-08-14 | University Of South Carolina | Surface enhanced Raman spectroscopy using shaped gold nanoparticles |
US20110184202A1 (en) * | 2004-12-13 | 2011-07-28 | University Of South Carolina | Surface enhanced raman spectroscopy using shaped gold nanoparticles |
US20080266555A1 (en) * | 2004-12-13 | 2008-10-30 | University Of South Carolina | Surface Enhanced Raman Spectroscopy Using Shaped Gold Nanoparticles |
US20110137062A1 (en) * | 2004-12-13 | 2011-06-09 | University Of South Carolina | Surface enhanced raman spectroscopy using shaped gold nanoparticles |
US8129199B2 (en) * | 2004-12-13 | 2012-03-06 | University of South Caroliina | Surface enhanced Raman spectroscopy using shaped gold nanoparticles |
GB2439656B (en) * | 2004-12-17 | 2010-09-08 | Intel Corp | Detection of enhanced multiplex signals by surface enhanced raman spectroscopy (SERS) |
WO2006066180A1 (en) * | 2004-12-17 | 2006-06-22 | Intel Corporation | Detection of enhanced multiplex signals by surface enhanced raman spectroscopy (sers) |
US20090303461A1 (en) * | 2004-12-17 | 2009-12-10 | Lei Sun | Detection of enhanced multiplex signals by surface enhanced raman spectroscopy (sers) |
GB2439656A (en) * | 2004-12-17 | 2008-01-02 | Intel Corp | Detection of enhanced multiplex signals by surface enhanced raman spectroscopy (sers) |
US20090023135A1 (en) * | 2004-12-17 | 2009-01-22 | Lei Sun | Detection of enhanced multiplex signals by surface enhanced raman spectroscopy |
US7485471B1 (en) | 2004-12-17 | 2009-02-03 | Intel Corporation | Detection of enhanced multiplex signals by surface enhanced Raman spectroscopy |
US20060147941A1 (en) * | 2004-12-30 | 2006-07-06 | Intel Corporation | Methods and apparatus for SERS assay of biological analytes |
US20070154903A1 (en) * | 2005-06-23 | 2007-07-05 | Nanosphere, Inc. | Selective isolation and concentration of nucleic acids from complex samples |
US20070134815A1 (en) * | 2005-07-11 | 2007-06-14 | Danielle Chamberlin | Sensitivity enhancement of POCT devices using gold and silver nanoparticles on substrates containing nanostructures or nanoparticles that interact with labeling particles |
US20090111094A1 (en) * | 2005-08-19 | 2009-04-30 | Nanosphere, Inc. | Methods for preparing hybrid substrates comprising DNA and antibodies and uses thereof |
WO2007032653A1 (en) * | 2005-09-15 | 2007-03-22 | Seoul National University Industry Foundation | A new label-free high throughput screening method by using sers spectroscopic encoded bead and dielectrophoresis |
US20100072067A1 (en) * | 2005-09-15 | 2010-03-25 | Seoul National Univeristy Industry Foundation | Label-Free High Throughput Screening Method by Using Sers Spectroscopic Encoded Bead and Dielectrophoresis |
KR100650522B1 (en) | 2005-09-15 | 2006-11-27 | 재단법인서울대학교산학협력재단 | A new label-free high throughput screening method by using sers spectroscopic encoded bead and dielectrophoresis |
US20070072309A1 (en) * | 2005-09-29 | 2007-03-29 | General Electric Company | Analytical compositions including nanometer-sized transducers, methods to make thereof, and devices therefrom |
US20070155022A1 (en) * | 2005-12-30 | 2007-07-05 | Mineo Yamakawa | Degenerate binding detection and protein identification using Raman spectroscopy nanoparticle labels |
DE102006000775A1 (en) * | 2006-01-04 | 2007-07-12 | Julius-Maximilians-Universität Würzburg | Diagnostic imaging techniques, useful for representing distribution of substances in immobilized cell, comprises displacing immobilized cell with a substance and bonding a marker for surface enhanced vibrational spectroscopy on substance |
WO2008097328A3 (en) * | 2006-06-23 | 2008-10-16 | Univ Northwestern | Asymmetric functionalized nanoparticles and methods of use |
WO2008097328A2 (en) * | 2006-06-23 | 2008-08-14 | Northwestern University | Asymmetric functionalized nanoparticles and methods of use |
US9823253B2 (en) | 2007-03-20 | 2017-11-21 | Becton, Dickinson And Company | Assays using surface-enhanced raman spectroscopy (SERS)-active particles |
US11016095B2 (en) | 2007-03-20 | 2021-05-25 | Becton Dickinson And Company | Assays using surface-enhanced raman spectroscopy (SERS)-active particles |
US20120164717A1 (en) * | 2007-07-18 | 2012-06-28 | Joseph Irudayaraj | Identity profiling of cell surface markers |
WO2009040114A2 (en) | 2007-09-24 | 2009-04-02 | Julius-Maximilians-Universität Würzburg | Compounds and markers for surface-enhanced raman scattering |
US8765950B2 (en) | 2007-12-27 | 2014-07-01 | Purdue Research Foundation | Reagents for biomolecular labeling, detection and quantification employing raman spectroscopy |
WO2009086509A3 (en) * | 2007-12-27 | 2009-09-24 | Purdue Research Foundation | Reagents for biomolecular labeling, detection and quantification employing raman spectroscopy |
US8153827B2 (en) | 2007-12-27 | 2012-04-10 | Purdue Research Foundation | Reagents for biomolecular labeling, detection and quantification employing Raman spectroscopy |
WO2009086509A2 (en) * | 2007-12-27 | 2009-07-09 | Purdue Research Foundation | Reagents for biomolecular labeling, detection and quantification employing raman spectroscopy |
US20090219526A1 (en) * | 2007-12-27 | 2009-09-03 | Vincent Jo Davisson | Reagents for biomolecular labeling, detection and quantification employing raman spectroscopy |
EP2227681A4 (en) * | 2007-12-31 | 2011-01-12 | Univ California | Sers-based, single step, real-time detection of protein kinase and/or phosphatase activity |
US20110046018A1 (en) * | 2007-12-31 | 2011-02-24 | Fanqing Frank Chen | Sers-based, single step, real-time detection of protein kinase and/or phosphatase activity |
EP2227681A2 (en) * | 2007-12-31 | 2010-09-15 | The Regents of the University of California | Sers-based, single step, real-time detection of protein kinase and/or phosphatase activity |
CN101978072A (en) * | 2008-01-14 | 2011-02-16 | 超快纳米诊断公司 | Rapid test including genetic sequence probe |
EP2245186A2 (en) * | 2008-01-14 | 2010-11-03 | Ultrapid Nanodiagnostics, Inc. | Rapid test including genetic sequence probe |
EP2245186A4 (en) * | 2008-01-14 | 2011-08-24 | Ultrapid Nanodiagnostics Inc | Rapid test including genetic sequence probe |
US20110124008A1 (en) * | 2008-05-07 | 2011-05-26 | Seoul National University Industry Foundation | NOVEL Au/Ag CORE-SHELL COMPOSITE USEFUL FOR BIOSENSOR |
US20160266104A1 (en) * | 2008-05-07 | 2016-09-15 | Korea Research Institute Of Chemical Technology | Heterodimeric core-shell nanoparticle in which raman-active molecules are located at a binding portion of a nanoparticle heterodimer, use thereof, and method for preparing same |
US20110294691A1 (en) * | 2008-10-15 | 2011-12-01 | Cornell University | Enhanced on-chip sers based biomolecular detection using electrokinetically active microwells |
US9057099B2 (en) * | 2008-10-15 | 2015-06-16 | Cornell University | Enhanced on-chip SERS based biomolecular detection using electrokinetically active microwells |
US7879625B1 (en) | 2008-12-03 | 2011-02-01 | The United States Of America As Represented By The Secretary Of The Navy | Preparation of SERS substrates on silica-coated magnetic microspheres |
US20110171749A1 (en) * | 2009-03-02 | 2011-07-14 | Board Of Trustees Of Michigan State University | Nanoparticle tracer-based electrochemical dna sensor for detection of pathogens-amplification by a universal nano-tracer (aunt) |
US20100291599A1 (en) * | 2009-05-18 | 2010-11-18 | Bruker Optics, Inc. | Large area scanning apparatus for analyte quantification by surface enhanced raman spectroscopy and method of use |
US20120156804A1 (en) * | 2009-06-12 | 2012-06-21 | Agency For Science, Technology And Research | Method for determining protein-nucleic acid interaction |
US9046526B2 (en) * | 2009-06-12 | 2015-06-02 | Agency For Science, Technology And Research | Method for determining protein-nucleic acid interaction |
DE102009050287A1 (en) | 2009-10-20 | 2011-05-26 | Friedrich-Schiller-Universität Jena | SERS substrate |
US9575061B2 (en) * | 2009-10-20 | 2017-02-21 | Vanderbilt University | Liquid drop diagnostic assays |
US20120276523A1 (en) * | 2009-10-20 | 2012-11-01 | Vanderbilt University | Liquid drop diagnostic assays |
WO2011047662A2 (en) | 2009-10-20 | 2011-04-28 | Friedrich-Schiller-Universität Jena | Sers substrate |
WO2011078794A1 (en) | 2009-12-22 | 2011-06-30 | Agency For Science, Technology And Research | Sers-based analyte detection |
EP2516995A4 (en) * | 2009-12-22 | 2013-06-05 | Agency Science Tech & Res | Sers-based analyte detection |
US9689801B2 (en) | 2009-12-22 | 2017-06-27 | Agency For Science, Technology And Research | SERS-based analyte detection |
EP2516995A1 (en) * | 2009-12-22 | 2012-10-31 | Agency For Science, Technology And Research | Sers-based analyte detection |
US10001478B2 (en) | 2013-08-21 | 2018-06-19 | Mycartis Nv | Heterogenous surface functionalization |
WO2015024863A1 (en) * | 2013-08-21 | 2015-02-26 | Mycartis Nv | Heterogenous surface functionalization |
US10436707B2 (en) * | 2014-09-25 | 2019-10-08 | Centre National de la Recherche Scientifique—CNRS | Detection of analytes using nanoparticles as light scattering enhancers |
US20180149646A1 (en) * | 2015-05-29 | 2018-05-31 | Kyocera Corporation | Detection method and detection device |
US10392598B2 (en) | 2015-06-19 | 2019-08-27 | Emory University | Methods of measuring cell purity for making quality control determinations and related compositions |
WO2016205678A1 (en) * | 2015-06-19 | 2016-12-22 | Emory University | Methods of measuring cell purity for making quality control determinations and related compositions |
US20190323069A1 (en) * | 2015-11-18 | 2019-10-24 | University Of Florida Research Foundation, Inc. | Devices for detecting target biological molecules from cells and viruses |
US10968474B2 (en) * | 2015-11-18 | 2021-04-06 | University Of Florida Research Foundation Inc. | Devices for detecting target biological molecules from cells and viruses |
WO2018162742A1 (en) | 2017-03-10 | 2018-09-13 | Universität Duisburg-Essen | Efficient ligand exchange of a detergent bilayer on the surface of metal nanoparticles for molecular functionalization and assembly, corresponding functionalized nanoparticles and nanoparticle assemblies, and their use in plasmonic applications including surface-enhanced raman spectroscopy |
CN113039255A (en) * | 2018-09-26 | 2021-06-25 | 中国农业大学 | Detection kit and detection method for simultaneously detecting drift or deposition of multiple spraying droplets |
CN110749585A (en) * | 2019-09-11 | 2020-02-04 | 亳州市新健康科技有限公司 | Biosensor based on surface enhanced Raman technology and preparation method thereof |
WO2022090747A1 (en) * | 2020-11-02 | 2022-05-05 | The University Of Birmingham | Single point variant detection |
CN113433100A (en) * | 2021-05-25 | 2021-09-24 | 上海市公共卫生临床中心 | Plasma tryptophan and albumin joint detection method based on photochemical reaction of DNA synthesized silver nanoclusters and tryptophan |
CN115855928A (en) * | 2023-02-27 | 2023-03-28 | 合肥工业大学 | Mercury ion detection method and kit based on nucleic acid macroarray and bifunctional molecules |
Also Published As
Publication number | Publication date |
---|---|
WO2003095973A3 (en) | 2004-07-22 |
JP2005524849A (en) | 2005-08-18 |
ATE515698T1 (en) | 2011-07-15 |
WO2003095973A2 (en) | 2003-11-20 |
AU2003237180B2 (en) | 2007-05-24 |
US7985539B2 (en) | 2011-07-26 |
EP1499889A2 (en) | 2005-01-26 |
AU2003237180A1 (en) | 2003-11-11 |
CA2483697C (en) | 2010-03-30 |
US20040086897A1 (en) | 2004-05-06 |
EP1499889A4 (en) | 2006-11-08 |
CA2483697A1 (en) | 2003-11-20 |
EP1499889B1 (en) | 2011-07-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7985539B2 (en) | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection | |
EP1379693B1 (en) | Bio-barcodes based on oligonucleotide-modified particles | |
US7323309B2 (en) | Bio-barcodes based on oligonucleotide-modified particles | |
EP1747295B1 (en) | Bio-barcode based detection of target analytes | |
Fritzsche et al. | Metal nanoparticles as labels for heterogeneous, chip-based DNA detection | |
EP2217928B1 (en) | Alternate labeling strategies for single molecule sequencing | |
US20060040286A1 (en) | Bio-barcode based detection of target analytes | |
EP1649055B1 (en) | Bio-barcode based detection of target analytes | |
US20050037397A1 (en) | Bio-barcode based detection of target analytes | |
US20040053222A1 (en) | Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes | |
US20100081134A1 (en) | Bio-barcode based detection of target analytes | |
AU2009200803A1 (en) | Aptamer-nanoparticle conjugates and method of use for target analyte detection | |
CN101241078A (en) | SERRS reactive particles | |
CN101198707A (en) | Bio-barcode based detection of target analytes | |
CN101001960A (en) | Bio-barcode based detection of target analytes | |
AU2007211939A1 (en) | Nanoparticle probes with raman spectroscopic fingerprints for analyte detection | |
AU2002256145B2 (en) | Bio-barcodes based on oligonucleotide-modified particles | |
Schlücker et al. | Selective detection of proteins and nucleic acids with biofunctionalized SERS labels | |
AU2002256145A1 (en) | Bio-barcodes based on oligonucleotide-modified particles | |
JP2003284593A (en) | Method for detecting biochemical reactant |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: NORTHWESTERN UNIVERSITY, ILLINOIS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MIRKIN CHAD A.;CAO, YUNWEI;JIN, RONGCHAO;REEL/FRAME:013252/0474;SIGNING DATES FROM 20020813 TO 20020814 |
|
AS | Assignment |
Owner name: AIR FORCE, UNITED STATES, VIRGINIA Free format text: CONFIRMATORY LICENSE;ASSIGNOR:NORTHWESTERN UNIVERSITY;REEL/FRAME:014243/0258 Effective date: 20030515 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: NATIONAL SCIENCE FOUNDATION, VIRGINIA Free format text: CONFIRMATORY LICENSE;ASSIGNOR:NORTHWESTERN UNIVERSITY;REEL/FRAME:026772/0052 Effective date: 20030515 |
|
AS | Assignment |
Owner name: NATIONAL SCIENCE FOUNDATION, VIRGINIA Free format text: CONFIRMATORY LICENSE;ASSIGNOR:NORTHWESTERN UNIVERSITY;REEL/FRAME:026776/0051 Effective date: 20030515 |