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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54346—Nanoparticles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• • 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
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
  • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

• Bioassays are used to probe the quantity of a target analyte present in a biological sample.
• Surface-based assays in which the amount of target is quantified by capturing it on a solid support and then labeling it with a detectable label, are especially important since they allow for the facile separation of bound and unbound labels.
• Recently a number of surface-based assays have been developed that utilize different types of arrays.
• Array-based assays are of importance because they permit a very large number of interrogations to be performed simultaneously by placing different "assays" on spatially distinct locations of an array.
• Addressable arrays can be fabricated to study many different analytes including proteins, DNA and RNA.
• the sample to be tested is spread over the entire array so that target biomolecules in the sample can form complexes with their binding partner on the array.
• the target is typically labeled with some type of detectable tag (e.g., a fluorescent or radioactive label) so that the amount of each target analyte in the sample can be quantified by detecting the labeled complexes on the array.
• a surface based assay is a DNA microarray.
• DNA microarrays have become widely adopted in the study of gene expression and genotyping due to the ability to monitor large numbers of genes simultaneously (see, e.g., Schena et al. (1995) Science 270:467-470; and Pollack et al. (1999) Nat. Genet. 23:41- 46). More than 100,000 different probe sequences can be bound to distinct spatial locations across the microarray surface, each spot corresponding to a single gene (Schena et al. (1998) Tibtech 16:301-306). When a fluorescently labeled DNA target sample is placed over the surface of the array, individual DNA strands hybridize to complementary strands within each array spot.
• the level of fluorescence detected quantifies the number of copies bound to the array surface and therefore the relative presence of each gene, while the location of each spot determines the gene identity.
• arrays it is theoretically possible to simultaneously monitor the expression of all genes in an organism's genome.
• the use of DNA microarrays is an extremely powerful technique, with applications spanning all areas of genetics (see, e.g., the Chipping Forecast supplement to Nature Genetics 21 (1999)).
• Arrays can also be fabricated using other binding moieties such as antibodies, proteins, haptens or aptamers, in order to facilitate a wide variety of bioassays in array format.
• microtiter plate-based ELISAs enzyme-linked immunosorbent assays
• a protein sample is then added to each well along with a fluorescently labeled secondary antibody for each protein.
• Target proteins are captured on the surface of each well and secondarily labeled with a fluorophore. Fluorescence at the bottom of each well quantifies the amount of each target molecule in the sample.
• antibodies or DNA can be bound to a microsphere such as a polymer bead and assayed as described above.
• Dynamic range refers to the ability to simultaneously measure analyte over a wide range of concentrations. Using current detection technology, it is usually necessary to sacrifice linearity in the high concentration regime for detection sensitivity in the low concentration regime. This limits the dynamic range of a single experiment.
• the performance of an assay is typically measured by its ability to specifically and quantitatively measure vanishingly small quantities of the target species under investigation. This is especially true for genetic analysis such as gene expression or genotyping, where the available quantity of genetic material is limited.
• genetic analysis such as gene expression or genotyping, where the available quantity of genetic material is limited.
• gene expression analysis on DNA microarrays requires between 50 and 200 ⁇ g of total RNA for single array hybridization. This requires as many as 10 5 cells (Duggan et al. (1999) Nature Genetics 21(nl s): 10-14).
• samples extracted through microdissection Sgroi et al. (1999) Cancer Res. 59:5656-5661
• these large quantities of material are simply not available. This greatly complicates the detection of such samples labeled with standard organic fluorophores.
• the semiconductor nanocrystals enhance signal detection relative to conventional organic dyes.
• the semiconductor nanocrystals emit an intense signal that aids detection.
• signals are sufficiently intense that a single semiconductor nanocrystal can be detected.
• the semiconductor nanocrystals By controlling the size and composition of the semiconductor nanocrystals, one can obtain semiconductor nanocrystals that emit at particular wavelengths.
• the semiconductor nanocrystals have large absorption cross sections, they have narrow, symmetric emission spectra. This means that a number of different semiconductor nanocrystals can be excited at a single wavelength but emit at a variety of distinct wavelengths.
• the semiconductor nanocrystals can be readily attached to a variety of different biomolecules, the semiconductor nanocrystals can be utilized in a variety of different microarray analyses.
• the semiconductor nanocrystals can be utilized to label target molecules that are probed using nucleic acid arrays, protein arrays, tissue arrays or other arrays that utilize labeled targets and optical detection.
• certain methods for detecting a ligand of interest in a sample involve initially providing a first plurality of antiligands immobilized on a solid support at positionally distinct locations thereon to provide a first array, wherein the plurality of antiligands comprises a first antiligand capable of binding specifically to a first ligand of interest.
• This array is then contacted with a sample containing or suspected of containing the first ligand, wherein the first ligand is linked through a linker to a first semiconductor nanocrystal before, during or after the contacting, under conditions in which the first ligand binds specifically to the first antiligand to form a first complex. Unbound ligand is optionally removed from the a ⁇ ay. The location of the first complex is then identified by detecting, and optionally quantifying, the presence in the first complex of the first semiconductor nanocrystal.
• Certain methods are used in analyzing variations in nucleic acids such as single nucleotide polymorphisms. Some of these methods involve providing a first plurality of nucleic acid primers having a 3' end and a 5' end and which primers are immobilized on a solid support at positionally distinct locations thereon to provide a first array, wherein the plurality of primers comprise a first primer complementary to a first target nucleic acid having an allelic site.
• the first a ⁇ ay is then contacted with a sample containing or suspected of containing the first target nucleic acid, in the presence of a first terminating nucleotide linked to a first semiconductor nanocrystal through a linker, under conditions such that the first target nucleic acid hybridizes to the first primer to form a first target-primer complex and such that if the first terminating nucleotide is complementary to the nucleotide at the allelic site the first primer is extended to incorporate the first terminating nucleotide to provide an extended primer.
• the location or locations that includes extended primer is identified by detecting the presence therein of the first semiconductor nanocrystal.
• Other methods are secondary inte ⁇ ogation or sandwich type assays. These methods typically involve providing a first plurality of antiligands immobilized on a solid support at positionally distinct locations thereon to provide a first array, wherein the first plurality of antiligands comprises a first antiligand that is a binding partner of a first ligand. The a ⁇ ay is then contacted with a sample containing or suspected of containing the first ligand, whereby the first antiligand and the first ligand interact to form a first binary complex.
• the binary complex in turn is contacted with a second antiligand wherein the second antiligand is (i) a binding partner of the first ligand and (ii) linked to a first semiconductor nanocrystal through a linker, whereby the second antiligand binds to the first ligand in the first binary complex to form a first ternary complex.
• the location of the a ⁇ ay that includes the first ternary complex is identified by detecting the presence therein of the first semiconductor nanocrystal.
• Still other methods involve labeling a ligand after it has become bound to an a ⁇ ay. Certain of these methods involve providing a first plurality of antiligands immobilized on a solid support at positionally distinct locations thereon to provide a first a ⁇ ay, wherein the plurality comprises a first antiligand that is a binding partner of a first ligand. The first a ⁇ ay is then contacted with a sample containing or suspected of the first ligand, whereby the first ligand and the first antiligand interact to form a first complex. The first ligand in the first complex is subsequently labeled with a first semiconductor nanocrystal. The location of the a ⁇ ay that includes the first complex is identified by detecting the presence therein of the first semiconductor nanocrystal.
• FIG. 1 A is a graphical representation that depicts the results of a particular immunological assay involving a secondary inte ⁇ ogation of a complex between a capture antibody and a protein labeled with a semiconductor nanocrystal and a secondary antibody labeled with another semiconductor nanocrystal.
• FIG. IB is a graphical representation of a tertiary complex (capture antibody, protein labeled with semiconductor nanocrystal, secondary antibody labeled with another semiconductor nanocrystal) fonned in a secondary inte ⁇ ogation according to one method of the invention.
• FIGS. 2A-2B illustrate the optical properties associated with semiconductor nanocrystals as a consequence of the phenomenon of quantum confinement.
• FIGS. 2 A and 2B show the absorption and emission spectra from different semiconductor nanocrystal samples, illustrating how the emission wavelength varies as a function of size. Absorption spectra have been normalized to the height of the first absorption peak and have been vertically offset for clarity. Inset numbers co ⁇ espond to the average diameter of the quantum dots within each ensemble sample.
• FIG. 2C illustrates how the material from which a semiconductor nanocrystal is constructed affects the wavelength at which it emits. Emission spectrum from semiconductor nanocrystals of three different materials are shown: CdSe (visible), InP (visible-near infrared) and hiAs (infrared).
• FIG. 3 provides a graph that illustrates photodegradation of semiconductor nanocrystals vs fluorescein under identical excitation conditions. Sample concentrations were matched (-10-5 mol/1) and each was excited with ⁇ 1 W/cm 2 of 488 nm light from an Ar+ laser. Note that while fluorescein photobleaches within the first few seconds, quantum dots actually increase slightly in intensity over the first minute.
• FIGS. 4A and 4B illustrate single semiconductor nanocrystal detection.
• FIG. 4A is a photograph of single semiconductor nanocrystals using a laser epifluorescence microscope. Each individual spot co ⁇ esponds to the fluorescence from a single semiconductor nanocrystal.
• FIG. 4B depicts spectra from single semiconductor nanocrystals.
• Wavelength is dispersed on the x-axis and position on the y-axis. Each horizontal line co ⁇ esponds to the fluorescence spectrum from a single semiconductor nanocrystal. Note that different size semiconductor nanocrystals are easily identified by small changes in emission wavelength.
• FIGS. 5 A and 5B show a comparison between the absorption and emission spectra of fluorescein (FIG. 5 A) and a comparable color semiconductor nanocrystal (FIG. 5B). Note that while the emission spectrum of the semiconductor nanocrystal is significantly narrower than that for fluorescein, the absorption spectrum extends far to the blue, allowing efficient excitation with all wavelengths shorter than the emission wavelength.
• FIGS. 6A-6C illustrate the extension of dynamic range that can be achieved through single hybridization counting.
• FIG. 6A is a graphic representation of the transition from the ensemble concentration regime to the single copy hybridization regime.
• FIG. 6B is a graph showing simulated data demonstrating the improved sensitivity achieved through single hybridization detection.
• 6C is a plot of the theoretical number of discrete points detected within a 100 ⁇ m diameter array spot as the total number of bound labels increases. The calculation assumes that individual labels cannot be distinguished if they reside within the same 0.5 ⁇ m diameter region and a random distribution of label locations with an average density that is uniform across the a ⁇ ay spot. Saturation becomes significant above ⁇ 6000 as the probability of finding 2 or more labels within the same diffraction limited spot increases.
• FIG. 7 presents a schematic drawing of single quantum dot microscope.
• FIGS. 8A-8E illustrate and summarize the steps in certain automated a ⁇ ay scanning methods of the invention. Initially, sequential images are taken at periodic positions across the a ⁇ ay (FIG. 8A). The a ⁇ ay is then reconstructed (FIG. 8B). Pattern recognition is utilized to identify the location of the a ⁇ ay spots relative to alignment spots (FIG. 8C). Within each spot the average intensity is measured as well as the total number of discrete points (FIG. 8D). Values for the average intensity and the total number of discrete points are exported (FIG. 8E). DETAILED DESCRIPTION I. Definitions
• semiconductor nanocrystal semiconductor nanocrystal
• quantum dot QdotTM nanocrystal
• nanocrystal refers to an inorganic crystallite between about 1 nm and about 1000 nm in diameter or any integer or fraction of an integer therebetween, generally between about 2 nm and about 50 nm or any integer or fraction of an integer therebetween, more typically about 2 nm to about 20 nm (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm).
• a semiconductor nanocrystal is capable of emitting electromagnetic radiation upon excitation (i.e., the semiconductor nanocrystal is luminescent) and includes a "core" of one or more first semiconductor materials, and may be surrounded by a “shell” of a second semiconductor material.
• a semiconductor nanocrystal core surrounded by a semiconductor shell is refe ⁇ ed to as a "core/shell” semiconductor nanocrystal.
• the surrounding "shell” material typically has a bandgap energy that is larger than the bandgap energy of the core material and can be chosen to have an atomic spacing close to that of the "core” substrate.
• the core and/or the shell can be a semiconductor material including, but not limited to, those of the group II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, rnAs, InSb, and the like) and IN (Ge, Si, and the like) materials, and an alloy or a mixture thereof.
• group II-VI ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaS
• a semiconductor nanocrystal is, optionally, surrounded by a "coat" of an organic capping agent.
• the organic capping agent can be any number of materials, but has an affinity for the semiconductor nanocrystal surface.
• the capping agent can be an isolated organic molecule, a polymer (or a monomer for a polymerization reaction), an inorganic complex, and an extended crystalline structure.
• the coat is used to confer solubility, e.g., the ability to disperse a coated semiconductor nanocrystal homogeneously into a chosen solvent, functionality, binding properties, or the like.
• the coat can be used to tailor the optical properties of the semiconductor nanocrystal. Methods for producing capped semiconductor nanocrystals are discussed further below.
• semiconductor nanocrystal As used herein denote a coated semiconductor nanocrystal core, as well as a core/shell semiconductor nanocrystal.
• Luminescence is meant the process of emitting electromagnetic radiation (light) from an object. Luminescence results from a system which is "relaxing" from an excited state to a lower state with a co ⁇ esponding release of energy in the form of a photon. These states can be electronic, vibronic, rotational, or any combination of the tliree.
• the transition responsible for luminescence can be stimulated through the release of energy stored in the system chemically or added to the system from an external source.
• the external source of energy can be of a variety of types including chemical, thermal, electrical, magnetic, electromagnetic, physical or any other type capable of causing a system to be excited into a state higher than the ground state.
• a system can be excited by absorbing a photon of light, by being placed in an electrical field, or through a chemical oxidation-reduction reaction.
• the energy of the photons emitted during luminescence can be in a range from low-energy microwave radiation to high-energy x-ray radiation.
• luminescence refers to photons in the range from UN to IR radiation.
• "Monodisperse particles” include a population of particles wherein at least about 60% of the particles in the population, more preferably 75% to 90% of the particles in the population, or any integer in between this range, fall within a specified particle size range.
• a population of monodispersed particles deviate less than 10% rms (root-mean- square) in diameter and typically less than 5% rms.
• the phrase "one or more sizes of semiconductor nanocrystals" is used synonymously with the phrase “one or more particle size distributions of semiconductor nanocrystals.”
• One of ordinary skill in the art will realize that particular sizes of semiconductor nanocrystals are actually obtained as particle size distributions.
• FWHM half maximum peak height
• a broad wavelength band with regard to the excitation of the semiconductor nanocrystal is meant absorption of radiation having a wavelength equal to, or shorter than, the wavelength of the onset radiation (the onset radiation is understood to be the longest wavelength (lowest energy) radiation capable of being absorbed by the semiconductor nanocrystal).
• the onset radiation is understood to be the longest wavelength (lowest energy) radiation capable of being absorbed by the semiconductor nanocrystal).
• This onset occurs near to, but at slightly higher energy than the “na ⁇ ow wavelength band” of the emission.
• This is in contrast to the "na ⁇ ow absorption band” of dye molecules which occurs near the emission peak on the high energy side, but drops off rapidly away from that wavelength and is often negligible at wavelengths further than 100 nm from the emission.
• polynucleotide oligonucleotide
• nucleic acid nucleic acid molecule
• polynucleotide oligonucleotide
• nucleic acid molecule a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single- stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide.
• polynucleotide examples include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Nirals, Inc., Corvallis, Oregon, as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
• PNAs peptide nucleic acids
• these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels that are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoal
• polynucleotide analyte and “nucleic acid analyte” are used interchangeably and include a single- or double-stranded nucleic acid molecule that contains a target nucleotide sequence.
• the analyte nucleic acids may be from a variety of sources, e.g., biological fluids or solids, chromosomes, food stuffs, environmental materials, etc., and may be prepared for the hybridization analysis by a variety of means, e.g., proteinase K/SDS, chaotropic salts, or the like.
• target nucleic acid region or “target nucleotide sequence” includes a probe-hybridizing region contained within the target molecule.
• target nucleic acid sequence includes a sequence with which a probe will form a stable hybrid under desired conditions.
• nucleic acid probe or simply “probe” includes reference to a structure comprised of a polynucleotide, as defined above, that contains a nucleic acid sequence complementary to a nucleic acid sequence present in the target nucleic acid analyte.
• the polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.
• hybridizing sequences need not have perfect complementarity to provide stable hybrids. In many situations, stable hybrids will form where fewer than about 10% of the bases are mismatches, ignoring loops of four or more nucleotides. Accordingly, as used herein the term “complementary” refers to an oligonucleotide that forms a stable duplex with its “complement” under assay conditions, generally where there is about 90% or greater homology.
• An “array” broadly refers to an a ⁇ angement of antiligands in positionally distinct locations on a substrate.
• the location of the antiligands on the a ⁇ ay are spatially encoded so that the identity of an antiligand of an a ⁇ ay can be deduced from its location on the a ⁇ ay.
• a “microarray” generally refers to an a ⁇ ay in which detection requires the use of microscopic detection to detect complexes formed between antiligands and ligands.
• a “location” on an a ⁇ ay refers to a localized area on the a ⁇ ay surface that includes antiligands, each defined so that it can be distinguished from adjacent locations (e.g., being positioned on the overall a ⁇ ay or having some detectable characteristic that allows the location to be distinguished from other locations).
• each location includes a single type of antiligand.
• the location can have any convenient shape (e.g., circular, rectangular, elliptical or wedge-shaped).
• the size of an area can vary significantly. In some instances, the area of a location is greater than 1 cm 2 , such as 2-20 cm 2 , including any area within this range. More typically, the area of the location is less than 1 cm , in other instances less than 1 mm , in still other instances less than 0.5 mm , in yet still other instances less than 10,000 ⁇ m 2 , or less than 100 ⁇ m 2 .
• a “solid support” includes planar or nonplanar substrates such as glass, nitrocellulose (e.g., in membrane or microtiter well form); polyvmylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
• the terni "aptamer” is used herein to refer to a single- or double-stranded DNA or a single-stranded RNA molecule that recognizes and binds to a desired target molecule by virtue of its shape.
• aptazyme includes allosteric ribozymes that are activated in the presence of an effector molecule (either chemical or biological). Aptazymes are capable of transducing a noncovalent molecular recognition event into a catalytic event, for example, the production of a new covalent bond via ligation.
• Polypeptide and “protein” are used interchangeably herein and include a molecular chain of amino acids linked through peptide bonds. The terms do not refer to a specific length of the product.
• polypeptides include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
• protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.
• a “ligand” generally refers to any molecule that binds to an antiligand to form a ligand/antiligand pair.
• a ligand is any molecule for which there exists another molecule (i.e., the antiligand) that specifically binds to the ligand, owing to recognition of some portion or feature of the ligand.
• an “antiligand” is a molecule that specifically or nonspecifically interacts with another molecule (i.e., the ligand).
• target molecule or “analyte” refers to the species whose presence, absence and/or concentration is being detected or assayed. In the a ⁇ ay-based assays, described herein, the target molecule or analyte is also refe ⁇ ed to as the ligand.
• binding pair refers to first and second molecules that specifically bind to each other such as a ligand and an antiligand.
• binding pair or binding partners can refer to the antiligand and ligand that form a complex on an a ⁇ ay.
• the terms can also refer to a first molecule attached to a ligand and a second molecule attached to a semiconductor nanocrystal that interact such that the ligand becomes attached to the semiconductor nanocrystal via the interacting binding pair members.
• Binding partners need not necessarily be limited to pairs of single molecules.
• a single ligand can be bound by the coordinated action of two or more antiligands.
• the result of binding between bind pairs or binding partners is a binding complex, sometimes refe ⁇ ed to as a ligand/antiligand complex or simply as ligand/antiligand.
• binding pairs include: (a) any haptenic or antigenic compound in combination with a co ⁇ esponding antibody or binding portion or fragment thereof (e.g., digoxigenin and anti-digoxigenin; fluorescein and anti-fluorescein; dinitrophenol and anti-dinitrophenol; bromodeoxyuridine and anti-bromodeoxyuridine; mouse immunoglobulin and goat anti-mouse immunoglobulin), (b) nonimmunological binding pairs (e.g., biotin-avidin, biotin-streptavidin, biotin-Neutravidin); (c) hormone [e.g., thyroxine and cortisol] -hormone binding protein; (d) receptor-receptor agonist or antagonist (e.g., acetylcholine receptor-acetylcholine or an analog thereof); (e) IgG- protein A (f) lectin-carbohydrate; (g) enzyme-enzyme cofactor; (h) enzyme-en
• affinity molecule refers to a molecule that will selectively bind, through chemical or physical means to a detectable substance present in a sample.
• selective bind is meant that the molecule binds preferentially to the target of interest or binds with greater affinity to the target than to other molecules.
• an antibody will selectively bind to the antigen against which it was raised;
• a DNA molecule will bind to a substantially complementary sequence and not to unrelated sequences.
• the affinity molecule can comprise any molecule, or portion of any molecule, that is capable of being linked to a semiconductor nanocrystal and that, when so linked, is capable of recognizing specifically a detectable substance.
• affinity molecules include, by way of example, such classes of substances as antibodies, as defined below, monomeric or polymeric nucleic acids, aptamers, proteins, polysaccharides, sugars, and the like. See,, e.g.,
• a “semiconductor nanocrystal conjugate” is a semiconductor nanocrystal that is linked to or associated with a specific-binding molecule, as defined above.
• a “semiconductor nanocrystal conjugate” includes, for example, a semiconductor nanocrystal linked or otherwise associated, through the coat, to a member of a "binding pair" or a "specific-binding molecule” that will selectively bind to a detectable substance present in a sample, e.g., a biological sample as defined herein.
• the first member of the binding pair linked to the semiconductor nanocrystal can comprise any molecule, or portion of any molecule, that is capable of being linked to a semiconductor nanocrystal and that, when so linked, is capable of recognizing specifically the second member of the binding pair.
• antibody as used herein includes antibodies obtained from both polyclonal and monoclonal preparations, as well as, the following: (i) hybrid (cbimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Patent No. 4,816,567); (ii) F(ab')2 and F(ab) fragments; (iii) Fv molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659- 2662; and Ehrlich et al.
• Functional antibody fragments can be produced by cleaving a constant region, not responsible for antigen binding, from the antibody molecule, using e.g., pepsin, to produce F(ab')2 fragments. These fragments contain two antigen binding sites, but lack a portion of the constant region from each of the heavy chains. Similarly, Fab fragments, comprising a single antigen binding site, can be produced, e.g., by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments, including only the variable regions of the heavy and light chains, can also be produced, using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv.
• a single-chain Fv (“sFv” or "scFv”) polypeptide is a covalently linked NH-NL heterodimer which is expressed from a gene fusion including NH- and NL- encoding genes linked by a peptide-encoding linker.
• a number of methods have been described to discern and develop chemical structures (linkers) for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody N region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Patent Nos.
• the sFv molecules may be produced using methods described in the art. See, e.g., Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883; U.S. Patent Nos. 5,091,513, 5,132,405 and 4,946,778.
• Design criteria include detennining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Such methods have been described in the art. See, e.g., U.S. Patent Nos.
• Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility.
• minibodies are sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region.
• the oligomerization domain comprises self- associating ⁇ -helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds.
• the oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein.
• minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126.
• the term "monoclonal antibody” refers to an antibody composition having a homogeneous antibody population.
• the term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made.
• the term encompasses antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas. See, e.g., Cote, et al. Monclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p. 77.
• a semiconductor nanocrystal is "linked” or “conjugated” to, or “associated” with, a specific-binding molecule or member of a binding pair when the semiconductor nanocrystal is chemically coupled to, or associated with the specific- binding molecule.
• these terms intend that the semiconductor nanocrystal can either be directly linked to the specific-binding molecule or can be linked via a linker moiety, such as via a chemical linker described below.
• the terms indicate species that are physically linked by, for example, covalent chemical bonds, physical forces such van der Waals or hydrophobic interactions, encapsulation, embedding, or the like.
• semiconductor nanocrystals can be conjugated to molecules that can interact physically with biological compounds such as cells, proteins, nucleic acids, subcellular organelles and other subcellular components.
• semiconductor nanocrystals can be associated with biotin which can bind to the proteins, avidin and streptavidin.
• semiconductor nanocrystals can be associated with molecules that bind nonspecifically or sequence-specif ⁇ cally to nucleic acids (DNA, RNA).
• such molecules include small molecules that bind to the minor groove of DNA (for reviews, see Geierstanger and Wemmer (1995) Ann. Rev. Biophys. Biomol. Struct.
• radiomimetic DNA damaging agents such as bleomycin, neocarzinostatin and other enediynes (for a review, see Povirk (1996) Mutat. Res. 355:71- 89), and metal complexes that bind and/or damage nucleic acids through oxidation (e.g. Cu-phenanthroline, see Perrin et al. (1996) Prog. Nucleic Acid Res. Mol. Biol. 52:123- 151; Ru(II) and Os(II) complexes, see Moucheron et al. (1997) J. Photochem. Photobiol B 40:91-106; chemical and photochemical probes of DNA, see Nielsen (1990) J. Mol. Recognit. 3:1-25.
• a "biological sample” refers to a sample of isolated cells, tissue or fluid, including but not limited to, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including, but not limited to, conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).
• a "small molecule” is defined as including an organic or inorganic compound either synthesized in the laboratory or found in nature. Typically, a small molecule is characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 1500 grams/Mol.
• a "biomolecule” is a synthetic or naturally occurring molecule, such as a protein, amino acid, nucleic acid, nucleotide, carbohydrate, sugar, lipid and the like.
• each detectable label is linked to one of a plurality of first members of binding pairs each of which first members is capable of binding to a distinct co ⁇ esponding second member of the binding pair.
• a multiplexed method using semiconductor nanocrystals having distinct emission spectra can be used to detect simultaneously in the range of 2 to 1,000,000, preferably in the range of 2 to 10,000, more preferably in the range of 2 to 100, or any integer between these ranges, and even more preferably in the range of up to 10 to 20, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, of analytes, biological compounds or biological states.
• Multiplexing also includes assays or methods in which the combination of more than one semiconductor nanocrystal having distinct emission spectra can be used to detect a single analyte.
• a “site of variation,” “variant site” or “allelic site” when used with reference to a nucleic acid broadly refers to a site wherein the identity of nucleotide at the
• Polymorphic marker or “site” refers to a genetic locus at which divergence occurs. Prefe ⁇ ed markers have at least two polymorphic forms, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population.
• a genetic locus can be as small as one base pair, if the polymorphism is a nucleotide substitution or deletion, or many base pairs if the polymorphism is, e.g., deletion, inversion or duplication of part of a chromosome.
• Polymorphic markers include, e.g., restriction fragment length polymorphisms, variable number of tandem repeats (NNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu.
• One identified allelic form is arbitrarily designated as a the reference allele and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes refe ⁇ ed to as the wild-type form. Diploid organisms may be homozygous or heterozygous for allelic forms.
• a di-allelic polymorphism has two forms.
• a tri-allelic polymorphism has three forms.
• a single nucleotide polymorphism occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
• a single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymo ⁇ hic site.
• a transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine.
• a transversion is the replacement of a purine by a pyrimidine or vice versa.
• Single nucleotide polymorphisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
• a “primer” is a single-stranded polynucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
• the appropriate length of a primer depends on the intended use of the primer but typically is at least 7 nucleotides long and, more typically range from 10 to 30 nucleotides in length. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
• a primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.
• primer site or “primer binding site” refers to the segment of the target DNA to which a primer hybridizes.
• primer pair means a set of primers including a 5' "upstream primer” that hybridizes with the complement of the 5' end of the DNA sequence to be amplified and a 3' “downstream primer” that hybridizes with the 3' end of the sequence to be amplified.
• a primer that is "perfectly complementary” has a sequence fully complementary across the entire length of the primer and has no mismatches.
• the primer is typically perfectly complementary to a portion (subsequence) of a target sequence.
• a “mismatch” refers to a site at which the nucleotide in the primer and the nucleotide in the target nucleic acid with which it is aligned are not complementary.
• substantially complementary when used in reference to a primer means that a primer is not perfectly complementary to its target sequence; instead, the primer is only sufficiently complementary to hybridize selectively to its respective strand at the desired primer- binding site.
• a “specimen” is a small part, or sample, of any substance or material obtained for analysis.
• a "tissue” is an aggregation of similar cells united in the performance of a particular function.
• the four basic tissues are epithelium, connective tissues (including blood, bone and cartilage), muscle tissue and nerve tissue.
• a "cellular specimen” is one that contains whole cells, and includes tissues. Examples include, but are not limited to, cells from the skin, breast, prostrate, blood, testis, ovary and endometrium.
• a "cellular suspension” is a liquid in which cells are dispersed, and can include a uniform or non-uniform suspension.
• Examples of cellular suspensions are those obtained by fine-needle aspiration from tumor sites, cytology specimens , washes, urine that contains cells, ascitic fluid, or other bodily fluids.
• a "cytological preparation” is a pathological specimen in which a cellular suspension can be converted into a smear or other form for pathological examination or analysis.”
• a "tumor” is a neoplasm that may be either malignant or non-malignant.
• Tumors of the same tissue type refers to primary tumors originating in a particular organ (such as breast, prostrate, bladder or lung).
• the present invention provides a variety of methods for conducting assays with different types of addressable a ⁇ ays using semiconductor nanocrystals (also refe ⁇ ed to herein simply as a quantum dot or a QdotTM) as a label to enhance detection of various complexes formed on the a ⁇ ay.
• Semiconductor nanocrystals can be used to label various ligands or target molecules for use in nucleic acid a ⁇ ays, protein a ⁇ ays, tissue a ⁇ ays or essentially any other type of a ⁇ ay that utilizes optical detection methods.
• the semiconductor nanocrystal labels can be directly incorporated into, or directly attached to, the ligands of interest through covalent or non-covalent attachment,or indirectly attached via a linker.
• the methods can be used in multiplex formats to simultaneously evaluate a plurality of samples with a single a ⁇ ay. Some methods also utilize ligands that each bear a single semiconductor nanocrystal.
• semiconductor nanocrystals utilized to label ligands or antiligands for use in a ⁇ ay-based assays can be tailored to have a number of desired properties.
• semiconductor nanocrystals can be produced that have characteristic spectral emissions. These spectral emissions can be tuned to a desired wavelength by varying the particle size, size distribution and/or composition of the particle. This means that multiple emission colors can be achieved, a feature that can be utilized in separately detecting ligands from different samples.
• the emission spectra of a population of semiconductor nanocrystals can be manipulated to have linewidths as na ⁇ ow as 25-30 nm, depending on the size distribution heterogeneity of the sample population, and lineshapes that are symmetric, gaussian or nearly gaussian with an absence of a tailing region.
• the combination of tunability, na ⁇ ow linewidths, and symmetric emission spectra enables high resolution of multiply sized semiconductor nanocrystals (e.g., populations of monodisperse semiconductor nanocrystals having multiple distinct size distributions within a system) and simultaneous detection of a variety of species.
• the range of excitation wavelengths of such nanocrystals is broad and can be higher in energy than the emission wavelengths of all available semiconductor nanocrystals.
• This feature allows the use of a single energy source, such as light, usually in the ultraviolet or blue region of the spectrum, to effect simultaneous excitation of all populations of semiconductor nanocrystals in a system having distinct emission spectra.
• Semiconductor nanocrystals can also be more robust than conventional organic fluorescent dyes by having a high quantum yield, and typically are more resistant to photobleaching than the organic dyes conventionally utilized in a ⁇ ay-based assays. The robustness of the nanocrystal also alleviates the problem of contamination of degradation products of the organic dyes in the system being examined.
• semiconductor nanocrystals have a relatively large Stokes shift, thereby significantly reducing problems with autofluorescence and scattered excitation light. Therefore, a ⁇ ay- based technology used in combination with semiconductor nanocrystals can be used as a sensitive way to conduct a variety of assays and in certain instances the methods can be designed to allow for quantification of complexes formed on an a ⁇ ay.
• semiconductor nanocrystals also permit flexibility in methods for detecting and quantifying ligands as assayed using a ⁇ ays.
• the ability to detect single nanocrystals means that in some instances single ligands bound to the a ⁇ ay can be individually counted. This capability means that one can quantitate the amount of ligand bound to the a ⁇ ay, as well as quantifying the amount of ligand in the original sample containing the ligand by calibration against samples of known concentration.
• detection can involve counting of single ligands (lower densities) or determining the total emission intensity from each location of the a ⁇ ay (higher ligand density).
• the ability to chose between these detection regimes results in significant expansion of the dynamic range of detection, thus allowing a greater range in the concentration of ligands that can accurately be quantified either as attached to the a ⁇ ay or in the original sample.
• the present invention in general provides a variety of methods for assaying for ligands or target molecules using various a ⁇ ay formats.
• Semiconductor nanocrystals are used as a labeling agent to enhance detection in several respects.
• the methods utilize a ⁇ ays that include a substrate or support upon which a plurality of antiligands are placed or attached. If attached, the antiligands can be directly attached to the support, or attached via a linker.
• the a ⁇ ay includes a variety of distinct locations to which the antiligands are placed or attached, hence the identity of the antiligands on the a ⁇ ay is spatially encoded. Each location has at least one antiligand, but often there are a plurality of antiligands at each location. The antiligands at the various locations can be the same or different.
• the a ⁇ ay is contacted with a sample that contains, or potentially contains, one or more ligands.
• ligands in the sample are brought into contact with the antiligands of the a ⁇ ay, ligands and antiligands that are members of a binding pair interact to form complexes.
• the ligands can be labeled with semiconductor nanocrystals either before or after the sample containing the ligands is contacted with the a ⁇ ay.
• the a ⁇ ay is then typically rinsed to remove uncomplexed ligand and other assay components.
• Complexes formed on the a ⁇ ay are identified by detecting a signal mediated by the semiconductor nanocrystals contained within the complexes.
• the identity of antiligands that have bound to a ligand can be determined based upon the location of the antiligand on the a ⁇ ay.
• a sample containing one or more unlabeled ligands can be contacted with an a ⁇ ay including multiple antiligands.
• ligands and antiligands that are binding partners form binary complexes. Since the ligands are unlabeled, complexes can be detected by contacting the binary complexes with a sample that contains secondary antiligands labeled with semiconductor nanocrystals. The secondary antiligands can bind to ligands in the binary complexes that are binding partners to form a tertiary complex.
• Those locations of the a ⁇ ay in which an antiligand is complexed with a ligand can then be detected by a signal from the semiconductor nanocrystal in the tertiary complexes.
• This approach is a sandwich type assay in which the antiligand serves to capture a ligand which is its binding partner. The ligand is then bound to the labeled secondary antiligand such that the ligand is sandwiched between the two antiligands.
• the ability to tune different semiconductors to emit at a distinctive wavelength by adjusting their size enables a variety of different multiplex analyses to be conducted. For example, different ligands from different samples can be separately labeled and then mixed together.
• the mixture can be applied to the a ⁇ ay and complexes containing ligands from different samples identified on the basis of the color of the semiconductor nanocrystal within the complex.
• different ligands within a single sample can be differentially labeled by selectively attaching a first member of different binding pairs to the different ligands.
• the second member of the various binding pairs can than be selectively attached to different semiconductor nanocrystals.
• the resulting ligands and semiconductor nanocrystals can then be mixed.
• Different ligands within the sample become differentially labeled because each ligand only joins to a label that bears a complementary binding pair member.
• Semiconductor nanocrystals are typically nanometer sized semiconductor crystals that have optical properties that are strongly dependent on both the size and the material of the crystal (see, e.g., Alivisatos (1996) Science 271:933-937).
• One feature of semiconductor nanocrystals is that the absorption and emission spectra from semiconductor nanocrystals can be tuned across a broad range of the electromagnetic spectrum by changing their size.
• semiconductor nanocrystals manufactured from CdSe can emit light in a na ⁇ ow wavelength band at any chosen wavelength between 490 nm and 640 nm.
• FIG. 2A In the size range of semiconductor nanocrystals, the confinement energy can be extremely large, and becomes one of the dominant factors affecting the absorption and emission energies of the material. Therefore, by changing the size of the quantum dots, the absorption and emission can be modified due to changes in the confinement energy.
• Figures 2A - 2B demonstrate this effect by showing a series of absorption and emission spectra from different size semiconductor nanocrystals of the same material (CdSe). Changing the material of the semiconductor nanocrystal can also affect the emission energy. By using a few different materials, it is possible to generate semiconductor nanocrystals with emission spectra that are tunable from the ultraviolet into the infrared (see FIG. 2C).
• Semiconductor nanocrystals demonstrate quantum confinement effects in their luminescent properties.
• a secondary emission of energy occurs at a frequency that co ⁇ esponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal.
• the bandgap energy is a function of the size and/or composition of the nanocrystal.
• a mixed population of semiconductor nanocrystals of various sizes and/or compositions can be excited simultaneously using a single wavelength of light and the detectable luminescence can be engineered to occur at a plurality of wavelengths.
• the luminescent emission is related to the size and/or the composition of the constituent semiconductor nanocrystals of the population.
• each semiconductor nanocrystal distribution is capable of emitting energy in na ⁇ ow spectral linewidths, as na ⁇ ow as 12 nm to 60 nm full width of emissions at half peak height (FWHM), and with a symmetric, nearly Gaussian line shape, thus providing an easy way to identify a particular semiconductor nanocrystal.
• the linewidths are dependent on, among other things, the size heterogeneity, i.e., monodispersity, of the semiconductor nanocrystals in each preparation.
• Certain single semiconductor nanocrystal complexes have been observed to have FWHM as na ⁇ ow as 12 nm to 15 nm.
• Semiconductor nanocrystal distributions with larger linewidths in the range of 35 nm to 60 nm can be readily made and have the same physical characteristics as semiconductor nanocrystals with na ⁇ ower linewidths.
• emission characteristics of semiconductor nanocrystals are dependent upon size and composition one can detect and/or distinguish between different semiconductor nanocrystals in a number of ways, including for example, emission intensity, emission wavelength, full width at half maximum peak height, absorption, scattering, fluorescence lifetime, or any combination of the foregoing.
• a core/shell semiconductor nanocrystal is one made from one material such as CdSe that has been coated with a shell of a second, higher bandgap material such as ZnS (see, e.g., Hines et al (1996) J Phys. Chem. 100:468-471; Peng, et al. (1997) J. Am. Chem. Soc. 119:7019-7029; and Dabbousi, et al. (1991) J. Phys. Chem. B 101:9463- 9475).
• the higher bandgap shell material protects the fluorescent electron-hole pair from interacting with the surface and su ⁇ ounding environment (such interactions can produce fluorescence quenching in semiconductor nanocrystals). This results in significantly enhanced fluorescence quantum yields, typically from 50% to 80% .
• These core/shell structures have a surface that is intrinsically functionalized with organic ligands.
• Nanocrystal Colloids Manganese Doped Cadmium Selenide, (Core)Shell Composites for Biological Labeling, and Highly Fluorescent Cadmium Telluride” (1999) Doctoral dissertation, Massachusetts mstitute of Technology.
• optical properties make semiconductor nanocrystals useful for detecting complexes in a ⁇ ay-based methods. These properties include:
• FIG. 5 A shows a comparison between the absorption and emission spectra of fluorescein (FIG. 5 A) and a comparable color semiconductor nanocrystal analogue (FIG. 5B).
• Na ⁇ ow, symmetric emission spectra significantly reduce the overlap of adjacent colors in multiplexed assays, thereby increasing detection sensitivity.
• the semiconductor nanocrystal and the biomolecule have appropriate functional groups that allow the two molecules to be coupled.
• Certain biomolecules can be labeled by labeling a component of the biomolecule (e.g., a monomer of a polymer) which becomes incorporated into the final biomolecule during synthesis (e.g., incorporation of a nucleotide labeled with a semiconductor nanocrystal into a nucleic acid).
• the semiconductor nanocrystal and biomolecule can also be linked via a linker.
• the linkers typically are bifunctional, having a functional group at each end. One end of the linker becomes attached to the semiconductor nanocrystal and the other end to the biomolecule.
• the semiconductor nanocrystal can bear one member of a binding pair and the biomolecule the other member of the binding pair.
• the biomolecule and nanocrystal can thus be joined via the binding pair members.
• an a ⁇ ay broadly refers to an a ⁇ angement of biomolecules in positionally distinct locations on a substrate such that the identity of the various biomolecules in the array can be determined based upon their location in the a ⁇ ay.
• the biomolecules of the a ⁇ ay are attached to a support that maintains the relative position of the biomolecules of the a ⁇ ay either directly or via a linker.
• the support can be any material wliich can support a plurality of biomolecules and maintain the biomolecules such that they remain positionally distinct.
• the support can be manufactured from a wide variety of materials.
• the support can be made of organic, inorganic, biological, or nonbiological materials or combinations of these materials.
• suitable supports include, but are not limited to, various plastics, polymers, Pyrex®, quartz, resins, silicon, silica or silica based materials, carbon, metals, inorganic glasses, inorganic crystals, cellulose, nylon and the like.
• the form of the support can also vary.
• the support can have essentially any configuration. It may include a substantially planar surface or lack a planar surface.
• the substrate can have raised or depressed regions at which a reaction can occur or at which a solution or suspension can be placed.
• a specific example of such a support is a microtiter plate as is known in the art.
• Other suitable shapes for the support include, but are not limited to, beads, particles, strands, gels, sheets, membranes, tubing, capillaries, pads, films, plates and slides, for example.
• the a ⁇ ay can also be in the form of a bundle of optical fibers, each fiber in the bundle having an end that is substantially planar or that includes a cavity etched into the end (see, e.g., U.S. Pat. No. 5,837,196 and PCT Publication WO 98/50782).
• a sample containing or potentially containing target nucleic acids from one or more sources is contacted with an a ⁇ ay of nucleic acid probes attached at different locations on the a ⁇ ay.
• the target nucleic acids are typically labeled with one or more semiconductor nanocrystals prior to contacting the a ⁇ ay with a sample, or include a modified nucleotide that permits the facile labeling of the target nucleic acids after they have become hybridized to complementary probes attached to the a ⁇ ay.
• a modified nucleotide can be a nucleotide (e.g., dATP, dTTP, dGTP and dCTP) that has been functionalized with a group that reacts with a complementary functional group borne by the semiconductor nanocrystal.
• the modified nucleotide is attached to one member of a binding pair that specifically binds to the other member of the binding pair that is attached to a semiconductor nanocrystal.
• the a ⁇ ay is optionally washed with a stringency buffer to remove unbound or non-specifically bound target nucleic acids.
• Hybridization complexes formed on the a ⁇ ay are detected by detecting a signal associated or mediated by semiconductor nanocrystals attached to target nucleic acids that are within the hybridization complexes.
• These series of steps can be automated utilizing various automated systems. These systems can include temperature controllers and mixers to regulate the reaction conditions as appropriate to the particular analysis being conducted. The systems can be programmable to program the temperature and mixing conditions, as well as to automatically dispense reagents, wash the a ⁇ ay and perform detection assays. Information regarding such systems and components is described, for example, in PCT publication WO 95/3386.
• the a ⁇ ay typically is stringency washed following application of sample to the a ⁇ ay to remove unbound target nucleic acids and to at least partially remove target nucleic acids that are not perfectly complementary to the probe nucleic acid to which they are bound.
• the stringency of selected hybridization conditions depends on various factors known in the art, including, e.g., temperature, ionic strength and pH. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Probe, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).
• “stringent conditions” are selected to be about 5-10 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
• Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
• Stringent conditions are those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 °C for short probes (e.g.,' 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides).
• Stringent conditions can also be achieved with the addition of de-stabilizing agents such as formamide.
• a positive signal is at least two times background, preferably 10 times background hybridization.
• Modely stringent hybridization conditions include hybridization in a buffer of 40% formamide, 1 M NaCI, 1% SDS at 37 °C, and a wash in IX SSC at 45 °C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
• a variety of methods can be utilized to incorporate semiconductor nanocrystals into the target nucleic acids to be analyzed by use of an a ⁇ ay.
• One approach involves enzymatically incorporating semiconductor nanocrystal-dNTPs into reverse- transcribed cDNA by reverse transcribing a template nucleic acid in the presence of dNTPs labeled with semiconductor nanocrystals.
• the desired cDNA is amplified using polymerase chain reaction (PCR) primers labeled with one or more semiconductor nanocrystals. Because the intensity of the emission of certain semiconductor nanocrystals is sufficiently high such that a single semiconductor nanocrystal can be detected, in some methods it is advantageous to incorporate just a single semiconductor into the nucleic acid.
• PCR polymerase chain reaction
• Incorporation of a single semiconductor nanocrystal into a nucleic acid can be achieved using primers that are labeled with a single semiconductor nanocrystal during amplification of target.
• labeling with a single semiconductor nanocrystal allows one to quantify the amount of ligand (e.g., cDNA) that has hybridized to the array using the total fluorescence intensity.
• ligand e.g., cDNA
• a third nucleic acid labeling approach involves synthesizing cDNA from active group-functionalized dNTP using reverse transcriptase. The resulting unlabeled form is then labeled by directly conjugating the active groups to the surface of semiconductor nanocrystals.
• an "active group” or “functional group” has means an atom or group of atoms that define the structure of a particular molecule or family of molecules and, at the same time, determines their properties. Exemplary functional groups include hydroxyl, sulfhydryl, carbonyl, carboxyl, amino and double or triple bonds.
• an amine-functionalized dNTP is covalently bound to succinamidyl ester-functionalized semiconductor nanocrystals.
• Another option is to postpone labeling until after a target has become hybridized to a complementary probe nucleic acid borne by the a ⁇ ay.
• a target has become hybridized to a complementary probe nucleic acid borne by the a ⁇ ay.
• the synthesized cDNA is then hybridized to probes on the a ⁇ ay, followed by conjugation of the semiconductor nanocrystals to the functional groups borne by the dNTPs that were inco ⁇ orated into the synthesized cDNA.
• four semiconductor nanocrystals each of a different color and each with a different surface functionalization can be washed over a hybridized a ⁇ ay to label four different sets of cDNA.
• This approach has the advantage that the semiconductor nanocrystals are not present during the hybridization step, thereby minimizing potential interference by the nanocrystals with hybridization.
• This method also minimizes the possibility of cross-linking different cDNA strands during labeling if individual semiconductor nanocrystals have more than one functional group on the surface.
• This type of labeling can also be done for single color detection by using a single active group and conjugating after hybridization.
• Another labeling approach is to fragment transcribed DNA and then end- label the fragments with a semiconductor nanocrystal-dTTP conjugate using terminal transferase.
• multiplexed assays can be performed by preparing and labeling different cDNA samples separately, and then blending the samples together prior to hybridization on the a ⁇ ay.
• linker can be any of a number of different homo- and hetero-bifunctional moieties that include a functional group at either end of a chain of molecules; the functional groups at each end can be the same or different.
• suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic linkers and peptide linkers. Suitable linkers are available from Pierce Chemical Company in Rockford, Illinois and are described in EPA 188,256; U.S. Pat. Nos.
• binding pair members in which one member of the pair is attached to the semiconductor nanocrystal and the other member is attached to a nucleic acid or nucleotide inco ⁇ orated therein.
• the binding pair members can be any set of molecules that specifically bind to one another. Suitable binding pair members include, but are not limited to, antigen/antibodies, biotin/streptavidin (or avidin or neutravidin) and oligosaccharide/lectin.
• labeling methods include the following.
• One option is to bind streptavidin-coated semiconductor nanocrystals to biotinylated cDNA.
• the reverse approach can also be taken in which biotin-coated semiconductor nanocrystals are bound to biotinylated cDNA through a streptavidin bridge.
• Another approach is to bind antibody-labeled semiconductor nanocrystals to antigen-labeled cDNA. For instance, digoxigenin-labeled cDNA can be bound to semiconductor nanocrystal-labeled anti-digoxigenin antibody.
• the member of the binding pair can be inco ⁇ orated at internal locations within a cDNA by conducting the reverse transcription of a nucleic acid template in the presence of dNTPs labeled with the binding pair member(s).
• the binding pair member can be inco ⁇ orated near the terminus of the target by using primers labeled with the binding pair member to amplify the target nucleic acid.
• Target nucleic acids bearing multiple labels can be prepared by conducting transcription in the presence of dNTPs bearing a binding pair member or by amplifying the target with primers bearing multiple binding pairs.
• Target nucleic acids bearing a single semiconductor nanocrystal can be prepared by using primers bearing a single binding pair member to conduct amplification of the target nucleic acid.
• attachment of the semiconductor nanocrystals bearing a binding pair member to target nucleic acids bearing the other binding pair member can be done either before or after hybridization of target nucleic acids to probes on the a ⁇ ays.
• target nucleic acids can be differentially labeled by adding different semiconductor nanocrystals into the different reaction vessels. The resulting labeled target nucleic acids can then be mixed together prior to applying the labeled target nucleic acids to the a ⁇ ay.
• the a ⁇ ays typically utilized when assaying target nucleic acids according to the methods of the present invention typically include some type of solid support to which nucleic acid probes are attached.
• the nucleic acid probes attached to the solid support are generally nucleic acids or uncharged nucleic acid analogs such as, for example, peptide nucleic acids that are disclosed in International Publication No. WO 92/20702; mo ⁇ holino analogs that are described in U.S. Patents Nos. 5,185,444, 5,034,506 and 5, 142,047.
• Nucleic acid a ⁇ ays can be prepared in two general ways.
• One approach involves binding DNA from genomic or cDNA libraries to some type of solid support, such as glass for example.
• solid support such as glass for example.
• the second general approach involves the synthesis of nucleic acid probes.
• One method involves synthesis of the probes according to standard automated techniques and then post-synthetic attachment of the probes to a support. See for example,
• a second category is the so-called "spatially directed" oligonucleotide synthesis approach. Methods falling within this category further include, by way of illustration and not limitation, light-directed oligonucleotide synthesis, microlithography, application by ink jet, microchannel deposition to specific locations and sequestration by physical barriers.
• a ⁇ ays can also be synthesized utilizing combinatorial chemistry by utilizing mechanically constrained flowpaths or microchannels to deliver monomers to cells of a support (see, e.g., Wihkler et al, EP 624,059; WO 93/09668; and U.S. Pat. No. 5,885,837).
• the samples contain such a low level of target nucleic acids that it is useful to conduct a pre-amplification reaction to increase the concentration of the target nucleic acids.
• amplification using primers or nucleotides labeled with semiconductor nanocrystals also provides a facile way to label the target nucleic acids of interest.
• amplification is typically conducted using the polymerase chain reaction (PCR) according to known procedures.
• PCR polymerase chain reaction
• PCR Technology Principles and Applications for DNA Amplification (H.A. Erlich, Ed.) Freeman Press, NY, NY (1992); PCR Protocols: A Guide to Methods and Applications (Innis, et al, Eds.) Academic Press, San Diego, CA (1990); Mattila et al, Nucleic Acids Res. 19: 4967 (1991); Eckert et al, PCR Methods and Applications 1: 17 (1991); PCR (McPherson et al. Ed.), TRL Press, Oxford; and U.S. Patent Nos. 4,683,202 and
• LCR ligase chain reaction
• transcription amplification see, e.g., Kwoh et al, Proc. Natl. Acad. Sci. USA 86:1173 (1989)
• self-sustained sequence replication see, e.g., Guatelli et al, Proc. Natl. Acad. Sci. USA, 87:1874 (1990)
• NABSA nucleic acid based sequence amplification
• Certain methods of the present invention involve the analysis of gene expression levels.
• expression analysis involves the detection and quantification of mRNA levels (or cDNA derived therefrom) in one or more samples.
• differently colored labels are used simultaneously to quantitate changes in gene expression levels, or to estimate changes in expression of one gene relative to another, in the cell under different conditions.
• cDNA prepared from a first specimen under one set of environmental conditions can be labeled with a first color tag and the cDNA derived from a second specimen, or the same first specimen under a different set of environmental conditions, can be labeled with a second color tag.
• Both samples are co-hybridized to the a ⁇ ay and the differentially labeled first and second cDNA molecules compete to bind at each spot.
• the ratio of the two colors at each location gives a quantitative measure of the relative change in expression for the gene.
• Expression analysis provides key insight into a variety of biological phenomenon. Cellular development and differentiation is one area in which expression analysis finds particular utility. In any given cell, only a fraction of all the encoded genes are expressed. The levels and timing of expression control cellular development, differentiation, function and physiology. Thus, monitoring gene expression can be used to analyze these processes. As an example, expression analysis can be utilized to assess differences in expression between different types of tissue. Expression studies can also provide important information into the genetic basis for aging and phenotypic differences.
• Expression analysis can also be of value in studies of various diseases. For instance, expression analysis can be used to analyze the development and progression of cancer, since these events are accompanied by complex changes in the pattern of gene expression. Such studies can involve, for example, a comparison of gene expression in diseased tissue and normal tissue or infected tissue and normal tissue. Expression analysis can also be used in other clinical applications, including evaluating the effects of various drug treatments on expression. For instance, differences in gene expression for normal tissue treated with drugs or a drug candidate and normal tissue can be compared. Similar drug studies could be performed with diseased tissue and normal tissue. By determining wliich genes are expressed in various diseases, one can identify genes or their protein products that have potential utility as drugs or as drug targets. In other clinical applications, expression analysis can be used in toxicological evaluations by comparing expression levels between tissue treated with potential poisons or toxins and normal tissue. Of course, expression analysis can be used in a variety of other comparative studies to assess the impact of variations in gene expression.
• Samples can be obtained from essentially any source from which nucleic acids can be obtained.
• Cells in the sample can be disrupted in a variety of ways to release the RNA therein (see for example, Watson, et al, Recombinant DNA, 2 n & Edition,
• nucleic acids may be released by mechanical disruption (such as repeated freeze/thaw cycles, abrasion and sonication), physical/chemical disruption, such as treatment with detergents (e.g., Triton, Tween, or sodium dodecylsulfate), osmotic shock, heat, or enzymatic lysis (e.g., lysosyme, proteinase K, and pepsin).
• mechanical disruption such as repeated freeze/thaw cycles, abrasion and sonication
• physical/chemical disruption such as treatment with detergents (e.g., Triton, Tween, or sodium dodecylsulfate), osmotic shock, heat, or enzymatic lysis (e.g., lysosyme, proteinase K, and pepsin).
• detergents e.g., Triton, Tween, or sodium dodecylsulfate
• osmotic shock e.g
• nucleic acids typically are reversed transcribed into cDNA, although mRNA can be used directly.
• cDNA generally sequences of interest are amplified according to any of the various amplification techniques that are known in the art such as those described infra.
• Labeled RNA can be prepared from a cDNA template using RNA polymerase, or methods known in the art.
• Differential gene expression analysis in which gene expression under differing sets of conditions is monitored can be accomplished in two general ways.
• One approach, and the approach traditionally utilized, is to use multiple arrays.
• a different a ⁇ ay is utilized for each of the different samples, each sample co ⁇ esponding to a different set of conditions.
• one sample might contain nucleic acids obtained from a healthy cell, while a second sample contains nucleic acids from a diseased cell.
• one a ⁇ ay would be used to determine expression in the healthy cell and the other a ⁇ ay used to determine expression in the diseased cell.
• the problem with this approach is that each measurement made for the different a ⁇ ays has some e ⁇ or associated with it.
• the e ⁇ or in each measurement becomes cumulative, thereby increasing the total e ⁇ or.
• Semiconductor nanocrystals can be used to reduce e ⁇ or generated in this approach.
• the nucleic acids from each sample can be labeled with one or more semiconductor nanocrystals according to any of the direct or indirect methods just described, either before or after hybridization of the nucleic acids to the a ⁇ ay.
• such e ⁇ or reduction can be accomplished using samples individually labeled with detectably distinct semiconductor nanocrystals and by making simultaneous measurements with the same a ⁇ ay.
• semiconductor nanocrystals can readily be tuned to emit at different wavelengths, one can simultaneously analyze numerous different samples on a single a ⁇ ay by differentially labeling the target nucleic acids from different samples using the labeling methods set forth above. This ability with the use of semiconductors afford results in significant improvements in the noise level when comparing hybridization results from different expression conditions because the cumulative e ⁇ or associated with measurements for multiple a ⁇ ays is avoided.
• SNPs have been co ⁇ elated with various human diseases (see, e.g., Publication WO 93/02216 wliich provides an extensive list of such SNPs). Because SNPs appear regularly throughout the genome, they also serve as useful genetic markers. The ability to detect specific nucleotide alterations or mutations in DNA sequences has a number of medical and non-medical utilities. For example, methods capable of identifying nucleotide alterations provide a means for screening and diagnosing many common diseases that are associated with SNPs. Such methods are also valuable in identifying individuals susceptible to disease, those who could benefit from prophylactic measures, and thus obtaining information useful in patient counseling and education.
• Methods for detecting alterations and mutations have further value in the detection of microorganisms, and making co ⁇ elations between the DNA in a particular sample and individuals having related DNA. This latter capability can be useful in resolving paternity disputes and in forensic analysis.
• the invention provides a number of different methods for detecting one or more target nucleic acids having a particular sequence.
• these methods involve providing an a ⁇ ay that bears a plurality of nucleic acid probes having different sequences. Normally, probes of different sequence are positioned at different locations so that the identity of the probe is spatially encoded.
• a sample containing target nucleic acids labeled with semiconductor nanocrystals is contacted with the probe.
• Hybridization complexes between complementary probes and target nucleic acids is detected by detecting a signal associated with the semiconductor nanocrystal.
• one can detect the presence or absence of a particular target nucleic acid of interest.
• Allele Specific Hybridization One method for detecting a target nucleic acid that has a particular polymo ⁇ hic form is to utilize allele specific probes that each specifically hybridize with a particular polymo ⁇ hic form of a target nucleic acid. By detecting which of the probes on the a ⁇ ay form hybridization complexes, one can detennine the presence or absence of particular target nucleic acids. Samples from different individuals can be probed on a single a ⁇ ay by differentially labeling nucleic acids from the different individuals with different semiconductor nanocrystals.
• the labeled probes necessary for conducting the reaction can be prepared according to the methods set forth above, by synthesizing the probes with functionalized nucleotides that permit the post-synthetic attachment of the semiconductor nanocrystals or using one or more labeled nucleotides in the synthesis of the probes by standard methods.
• the group of probes attached to the a ⁇ ay support can include all the allelic probes that specifically hybridize to each of the different polymo ⁇ hic forms of a target nucleic acid. Since most polymo ⁇ hisms are biallelic, this means that the a ⁇ ay includes two probes for each polymo ⁇ hic form.
• the target nucleic acid is triallelic, then three probes each complementary to one of the three polymo ⁇ hic forms can be utilized. Similarly, if the target nucleic acid is tetra-allelic, then the four probes complementary to the four different polymo ⁇ hic forms can be included in the array.
• the target nucleic acids By labeling the target nucleic acids with semiconductor nanocrystals, detection of the hybridization complexes is enhanced; the ability to use different colored semiconductor nanocrystals means that samples from a number of individuals can be analyzed simultaneously.
• Allele-Specific Ligation Other methods of the invention utilize semiconductor labeled probes to conduct allele-specific ligation reactions to detect the presence or absence of a particular target nucleic acid and distinguish between different polymo ⁇ hic forms.
• these methods involve contacting a target nucleic acid (which can be amplified prior to analysis) with a first probe that is complementary to a sequence adjacent the polymo ⁇ hic site under hybridization conditions. This first probe hybridizes to a sequence that is possessed by all the target nucleic acids regardless of the nucleotide at the polymo ⁇ hic site (i.e., regardless of the polymo ⁇ hic form of the target).
• the target nucleic acid is also contacted with a second probe that is an allele-specific probe, i.e., a probe that only hybridizes to a particular polymo ⁇ bic form of the target nucleic acid.
• the first and second probes are selected such that when the allele-specific probe is complementary with the nucleotide at the polymo ⁇ hic site the two probes hybridize directly adjacent one another. In particular, the 3' terminus of one probe is immediately adjacent the 5' terminus of the other probe. So long as the allele-specific probe is complementary to the nucleotide at the polymo ⁇ hic site, added ligase can join the two probes. By labeling either or both of the probes with semiconductor nanocrystals, detection of ligated product can be enhanced.
• the probes necessary for conducting these types of analyses can be prepared as described above in the section on allele-specific hybridization.
• one of the probes is attached to a solid support.
• the ligation reaction is conducted in solution and the ligated products are detected after being captured by capture reagents attached to an a ⁇ ay.
• the capture reagents specifically recognize a tag attached to one or both of the probes.
• Different targets can be distinguished by different tags and/or by labeling different target nucleic acids with different semiconductor nanocrystals.
• These analyses can be conducted in multiplex format by differentially labeling the probes with different semiconductor nanocrystals. For example, the presence or absence of specific alleles can rapidly be determined by differentially labeling the allele-specific probes used to conduct the ligation reaction.
• Certain methods of the invention involve conducting mini-sequencing reactions or primer extension reactions to identify the nucleotide present at a polymo ⁇ hic site in a target nucleic acid.
• a primer complementary to a segment of a target nucleic acid is extended if the reaction is conducted in the presence of a nucleotide that is complementary to the nucleotide at the polymo ⁇ hic site.
• the primer extension assays or mini-sequencing assays of the invention typically involve hybridizing a primer to a complementary target nucleic acid such that the 3' end of the primer is immediately adjacent the polymo ⁇ hic site or is a few bases upstream of the polymo ⁇ hic site.
• the extension reaction is conducted in the presence of one or more nucleotides labeled with a semiconductor nanocrystal and a polymerase. Often the nucleotide is a dideoxynucleotide that prevents further extension by the polymerase once it is inco ⁇ orated onto the 3' end of the primer.
• a labeled nucleotide is inco ⁇ orated onto the 3' end of the primer to generate a labeled extension product. Because the inco ⁇ orated nucleotide is complementary to the nucleotide at the polymo ⁇ hic site, extended primers provide an indication of which nucleotide is present at the polymo ⁇ hic site of target nucleic acids. Methods utilizing this general approach are discussed, for example, in U.S. Patent Nos.
• the primers are typically attached to a support. These primers can be of random sequence or selected to be complementary to the target nucleic acids of interest.
• a sample containing target nucleic acids is contacted with the a ⁇ ay of primers under conditions in which target nucleic acids become hybridized to complementary primers.
• Primers of the appropriate sequence hybridize to the target nucleic acid so that the 3' end of the primer is adjacent to the polymo ⁇ hic site of the target.
• the 3' end of the primer is immediately adjacent (but does not span) the polymo ⁇ hic site (i.e., the 3' end hybridizes to the nucleotide just upstream of the polymo ⁇ hic site).
• one or more nucleotides labeled with semiconductor nanocrystals are added. As indicated above, if the labeled nucleotides added include a nucleotide complementary to the nucleotide at the polymo ⁇ hic site, the primer is extended by inco ⁇ oration of a nucleotide bearing a semiconductor nanocrystal.
• each attached to a different nucleotide e.g., ddATP, ddTTP, ddCTP and ddGTP
• all possible alleles can be tested on a single a ⁇ ay simultaneously.
• methods can be performed with primers that simply hybridize adjacent to, but do not span, the polymo ⁇ hic site. This is possible so long as none of the nucleotides on the target nucleic acid located between the 3' end of the primer and the polymo ⁇ hic site are the same as the nucleotide at the variant site.
• the extension reaction mixture in such instances must also include nucleotides complementary to those nucleotides positioned between the 3' primer end and the polymo ⁇ hic site.
• the primers include two general regions: a 5' end region that includes a tag, and a 3' region that is complementary to a target nucleic acid of interest.
• the a ⁇ ay includes one or more capture reagents that can specifically bind with a tag borne by the primers.
• different capture reagents specific for different extension products are positioned at different locations on the a ⁇ ay so that the identity of the capture reagents is spatially encoded.
• the tag and capture reagent can be selected from any type of binding pairs in which the members of the pair specifically bind to one another.
• the capture reagent can be a nucleic acid that is complementary to a nucleic acid segment of a primer (i. e. , the primer tag).
• the extension reactions described above can be conducted in solution rather than on the a ⁇ ay.
• the reactions are conducted in the presence of one or more nucleotides (typically ddNTPs) labeled with a semiconductor nanocrystal.
• the extension products in the extension reaction are contacted with the a ⁇ ay.
• the capture reagents on the a ⁇ ay capture primers bearing tags that are specifically recognized by the capture reagent. Those primers that have been extended can be detected by the semiconductor nanocrystal inco ⁇ orated into the primer.
• the identity of the nucleotide at the polymo ⁇ hic site of the target nucleic acid can be determined from the location on the a ⁇ ay at which the extended product binds (see, e.g., U.S. Pat. No. 5,981,176).
• an a ⁇ ay of nucleic acid probes that are complementary to subsequences of a target sequence can be utilized to determine the identity of a target sequence, measure its amount, and detect differences between the target and a reference sequence using a procedure refe ⁇ ed to as "tiling.”
• tiling strategies utilize a tiled a ⁇ ay in which multiple nucleic acids that are identical except for one location are utilized.
• a ⁇ ay that typically is used for SNP analyses
• there is a set of four probes of relatively short length for example, 15-mers
• a perfectly complementary probe binds more tightly to a target nucleic acid than those probes that have a single mismatch.
• the labeled probe generating the most intense signal co ⁇ esponds to the probe having a nucleotide complementary to the nucleotide at the polymo ⁇ hic site.
• the target nucleic acids can be labeled with semiconductor nanocrystals either before or after target nucleic acids have hybridized to the nucleic acid probes of the a ⁇ ay.
• the labeled nucleotides utilized in the primer extension reactions can be . prepared by directly attaching a semiconductor nanocrystal to the nucleotides via functional groups present on the naturally occurring nucleotides, or through different functional groups introduced onto the nucleotides.
• different nucleotides can bear different binding pair members (e.g., biotin or antibodies); the other complementary binding pair members are attached to the semiconductor nanocrystals. If functionalized nucleotides or nucleotides bearing binding pair members are used to conduct the extension reaction, then extension products can be labeled either before or after extension has occu ⁇ ed.
• Target Nucleic Acid Preparation The target nucleic acids utilized in SNP analyses can be extracted and isolated according to the methods generally described supra for expression analysis. If necessary, target nucleic acids can be amplified according to the various amplification methods also described above.
• SBH uses a set of short nucleic acid probes of defined sequence to probe for complementary sequences on a longer target nucleic acid strand. The defined sequences that hybridize to the target can then be aligned using computer algorithms to construct the sequence of the target nucleic acid.
• SBH can be performed in two fo ⁇ nats.
• Hybridization methodology can be carried out by attaching target DNA to a surface. The target is then inte ⁇ ogated with a set of oligonucleotide probes, one at a time [see Strezoska et al, Proc. Natl. Acad. Sci. USA 88:10089-10093 (1991); and Drmanac et al, Science 260:1649-1652, (1993)].
• this approach can be implemented with well established methods of immobilization and hybridization detection, it involves a large number of manipulations. For example, to probe a sequence utilizing a full set of octanucleotides, tens of thousands of hybridization reactions must be performed.
• SBH is carried out by attaching probes to a surface in an a ⁇ ay format where the identity of the probes at each site is known.
• Target nucleic acid typically fragmented, is then added to the array of probes.
• the hybridization pattern determined in a single experiment can directly reveal the identity of all complementary probes.
• the methods of the invention utilize target nucleic acids labeled with semiconductor nanocrystals using either of these two formats. Most typically, however, the latter approach is utilized.
• sequencing begins with the fragmenting of the target nucleic acid into fragments using various techniques known in the art (e.g., the use of restriction enzymes, or heating in the presence of high salt concentrations).
• the resulting fragments are labeled with semiconductor nanocrystals, diluted in buffer and then applied to an a ⁇ ay bearing nucleic acid probes.
• the fragments are allowed to hybridize to the probes, typically using an automated apparatus to control temperature and sample mixing.
• the a ⁇ ay is then optionally rinsed with a stringency buffer to remove unbound fragments and hybridization complexes detected by detecting a signal from the semiconductor nanocrystal used to label the fragments.
• the target nucleic acid can be labeled with semiconductor nanocrystals using any of the various methods described supra in the section on expression analysis.
• PSBH positional SBH
• enzymatic ligation of the target to the duplex probe This approach is designed to reduce mismatches (see for example, Broude, et al, Proc. Natl. Acad. Sci. USA 91:3072- 3076 (1994); and U.S. Patent No. 5,631,134 to Cantor).
• PSBH itself has been further modified so that following the ligation reaction, DNA polymerase is added to extend the immobilized probe as a way of further reducing mismatches during capture of the target [see, e.g., Kuppuswamy, et al, Proc. Natl. Acad. Sci. USA 86:1143-1147 (1991)].
• the target nucleic acid fragments, probes or nucleotides used in such approaches can be labeled with semiconductor nanocrystals to enhance detection.
• Aptamers are single- or double-stranded DNA or single-stranded RNA molecules that recognize and bind to a desired target molecule by virtue of their shapes. See, e.g., PCT Publication Nos. WO92/14843, WO91/19813, and WO92/05285.
• the SELEX procedure described in U.S. Patent No. 5,270,163 to Gold et al, Tuerk et al. (1990) Science 249:505-510, Szosiak et al. (1990) Nature 346:818-822 and Joyce (1989) Gene 82:83-87, can be used to select for RNA or DNA aptamers that are target-specific.
• an oligonucleotide pool is constructed wherein an n- mer, typically a randomized sequence of nucleotides thereby forming a "randomer pool" of oligonucleotides, is flanked by two polymerase chain reaction (PCR) primers.
• the oligonucleotides in the pool are labeled with semiconductor nanocrystals according the methods described above.
• the a ⁇ ay utilized in the screening of aptamers typically bears the target molecules (e.g., proteins or small molecules) to be screened.
• the a ⁇ ay is then contacted with the oligonucleotide pool under conditions that favor binding of the oligonucleotides to the target molecules on the a ⁇ ay.
• Those oligonucleotides that bind the target molecule are separated from non-binding oligonucleotide using stringency washes.
• Those oligonucleotides that bind to the target molecules on the a ⁇ ay are dissociated from the a ⁇ ay using known techniques and then amplified using conventional PCR technology to form a ligand-enriched pool of oligonucleotides. Further rounds of binding, separation, dissociation and amplification are performed until an aptamer with the desired binding affinity, specificity or both is achieved.
• the final aptamer sequence identified can then be prepared chemically or by in vitro transcription.
• Protein a ⁇ ays can be used to investigate interactions between proteins and a wide variety of different types of molecules such as nucleic acids, various small molecules and other proteins, for example. Protein a ⁇ ays can be designed according to the aims of the particular investigation. For instance, an a ⁇ ay can contain all the combinatorial variants of a bioactive protein or specific variants of a single protein species (e.g., splice variants, domains, or mutants), a family of protein orthologs from different species, a protein pathway, or even the entire protein complement of an organism. The a ⁇ ays can also include antibodies, recombinant proteins, purified proteins and receptors, for example. Protein a ⁇ ays can be assigned to two general types.
• a ⁇ ay One type is refe ⁇ ed to as a nonliving or chemical a ⁇ ay. These protein arrays are composed of synthetic proteins. Arrays of this type are useful to investigate specific interactions between relatively small proteins with other proteins, particular nucleic acids or metal ions, for example.
• the second type is the biological or living protein a ⁇ ay. These a ⁇ ays include living entities that express proteins, including, but not limited to, viruses or cells. The arrays can include pools of proteins, cell fractions or intact cells. This type of a ⁇ ay can be used to investigate more complex biological activities, including, but not limited to, activities involving multicomponent complexes or multistep enzymatic or signaling pathways. Regardless of type, the proteins typically are placed at positionally distinct locations on the a ⁇ ay so that the different proteins are spatially encoded.
• semiconductor nanocrystals make them useful in protein a ⁇ ays. For example, tunability permits multicolor simultaneous detection and, hence, multiple sample detection. There is also no need for enzyme development as with certain traditional ELISA methods.
• the semiconductor nanocrystals have increased photostability relative to organic fluorophores, thereby increasing detection sensitivity by virtue of the ability to monitor the signal over a long period of time.
• Peptide a ⁇ ays can then be synthesized via the cellulose-bound alanine following deprotection (see, e.g., Gausepohl, H., et al, Pept. Res. 5:315-320 (1992); and Kramer, A. and Schneider-Mergener, J., Methods in Molecular Biology 87:25-39 (1998)).
• Other methods can be used to synthesize proteins on polymeric rods using solid state chemistry (see, e.g., Geysen, et al, Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1984)).
• Proteins can be synthesized directly on a solid support using various photolithographic methods. These techniques allow the number of proteins synthesized per unit area to be greatly increased. Examples of such methods are discussed, for example, in U.S. Pat. Nos. 5,143,854; 5,489,678; and 5,744,305; PCT Publications WO 90/15070 and WO 92/10092; and by Fodor, S.P. et al, Science 251:161-113 (1991).
• preformed proteins can be directly deposited on a support to form the a ⁇ ays.
• recombinant proteins, purified proteins and the like can be blotted onto a support.
• the proteins synthesized for use in non-living protein a ⁇ ays need not be limited to proteins composed of the L-amino acids. Other building blocks, such as D- amino acids and modified amino acids can also be used. Living a ⁇ ays can also be prepared utilizing different methods. As indicated above, the a ⁇ ays can be formed from pools of proteins, cellular extracts or intact cells. The pools, extracts or intact cells typically are placed in some type of support having a series of depressions (e.g., a microtiter plate) to contain the proteins. Methods for producing genome- wide protein a ⁇ ays have also been described.
• Certain of these methods involves transformation events in which one of the open-reading frames (ORF) from an organism (e.g., yeast) is inserted into plasmid encoding for glutathione-S- transferase (GST). This plasmid is subsequently used to transform the organism which then expresses different GST-ORF fusion proteins.
• the resulting transformed cell cultures or colonies can be used as an element of an a ⁇ ay (i.e., different colonies or groups of colonies are placed at different locations of the a ⁇ ay; see, e.g., Martzen, M.R. , et al, (1999) Science 286:1153-1155).
• Protein a ⁇ ays can be used to assay or screen for a variety of different types of activity or to conduct other types of analyses.
• protein a ⁇ ays can be used to: (1) screen for various molecules that interact with a protein of interest (e.g., agonists or antagonists).
• Molecules to be screened can include, but are not limited to, other proteins, nucleic acids, drugs, macromolecules or small molecules (see, e.g., Kramer, A., et al, (1993) Pept. Res.
• the immunoassays can be conducted in a variety of different formats.
• the ligands (potential antigens) to be screened are attached to an a ⁇ ay and then contacted with antibodies that are labeled with semiconductor nanocrystals.
• antibodies can be positioned on a support and then contacted with samples containing ligands that are labeled with semiconductor nanocrystals.
• the ligand being screened is an antibody and it is attached to a support and then screened with a known antigen of interest.
• ligands or antibodies that do not form a binding complex on the a ⁇ ay are typically washed from the a ⁇ ay.
• Complexes on the a ⁇ ay are then detected by detecting a signal from a semiconductor nanocrystal within a complex.
• the assays can also be performed in a "sandwich" type format in which antibodies positioned on an a ⁇ ay are contacted with a sample containing ligands.
• the ligands in the sample can be labeled or unlabeled.
• Ligands that specifically bind to an antibody form a binary antibody-ligand complex.
• the antibodies on the a ⁇ ay act to capture a ligand to which it specifically binds; hence, such antibodies are sometimes called "capture antibodies.”
• the binary complex formed between a capture antibody and a ligand can optionally be detected if the ligands are labeled.
• the a ⁇ ay is also contacted with a secondary antibody that is labeled with a semiconductor nanocrystal.
• a tertiary complex is formed.
• Tertiary complexes can be detected by detecting an emission mediated by the semiconductor nanocrystal in the tertiary complex.
• the wells of a microtiter plate are coated with a selected antigen.
• a biological sample containing or suspected of containing antibodies to the antigen is then added to the coated wells.
• the plate(s) can be washed to remove unbound antibodies and other sample components and a detection moiety labeled with a semiconductor nanocrystal is added. The detection moiety is allowed to react with any captured sample antibodies, the plate washed and the presence of the secondary binding molecule detected as described above.
• the presence of antibodies bound to antigens immobilized on a solid support can be readily detected using a detection moiety that comprises an antibody labeled with a semiconductor nanocrystal that specifically binds to the antigen/antibody complex.
• an immunoaffinity matrix can be provided, wherein a polyclonal population of antibodies from a biological sample suspected of containing a particular antigen is immobilized to a substrate.
• an initial affinity purification of the sample can be carried out using immobilized antigens.
• the resultant sample preparation thus only contains specific antibodies, avoiding potential nonspecific binding properties in the affinity support.
• immobilized protein A or protein G can be used to immobilize immunoglobulins.
• semiconductor nanocrystal-labeled proteins i.e., potential antigens
• the presence of bound antigen can be determined by assaying for a signal mediated by a semiconductor nanocrystal.
• antibodies raised to particular antigens can be used in the above-described assays in order to detect the presence of a protein of interest in a given sample. These assays are performed essentially as described above and are well known to those of skill in the art.
• Suitable supports for use in the methods of the invention include, but are not limited to, nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchlori.de (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads; and magnetically responsive beads.
• Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.
• BSA bovine serum albumin
• Other reagents that can be used to bind molecules to the support include polysaccharides, polylactic acids, polyglycohc acids, polymeric amino acids and amino acid copolymers. Additional details regarding molecules and coupling methods are provided by, for example, Brinkley, M.A. (1992) Bioconjugate Chem. 3:2-13; Hashida et al. (1984) J. Appl. Biochem. 6:56-63; and Anjaneyulu and Staros (1987) InternationalJ. of Peptide and Protein Res. 30:117-124.
• Antibodies that are used in the methods of the invention are produced using established techniques and disclosed in, for example, U.S. Patent Nos. 4,011,308;
• polyclonal antibodies are generated by im imizing a suitable animal, such as a mouse, rat, rabbit, sheep or goat, with an antigen of interest.
• a suitable animal such as a mouse, rat, rabbit, sheep or goat
• the antigen can be linked to a carrier prior to immunization.
• carrier are well known to those of ordinary skill in the art.
• Immunization is generally performed by mixing or emulsifying the antigen in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). The animal is generally boosted 2-6 weeks later with one or more injections of the antigen in saline, preferably using Freund's incomplete adjuvant.
• Antibodies can also be generated by in vitro immunization, using methods known in the art. Polyclonal antiserum is then obtained from the immunized animal.
• Monoclonal antibodies can be prepared using the method of Kohler and Milstein (1975) Nature 256:495-497, or a modification thereof.
• a mouse or rat is immunized as described above.
• the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells.
• the spleen cells can be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the antigen.
• B-cells, expressing membrane-bound immunoglobulin specific for the antigen will bind to the plate, and are not rinsed away with the rest of the suspension.
• Resulting B-cells, or all dissociated spleen cells are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT").
• a selective medium e.g., hypoxanthine, aminopterin, thymidine medium, "HAT”
• the resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the irnmunizing antigen (and which do not bind to unrelated antigens).
• the selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in mice).
• Human monoclonal antibodies are obtained by using human rather than murine hybridomas. See, e.g., Cote, et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
• Monoclonal antibodies or portions thereof can be identified by first screening a B-cell cDNA library for DNA molecules that encode antibodies that specifically bind to pi 85, according to the method generally set forth by Huse et al. (1989) Science 246:1275-1281. The DNA molecule can then be cloned and amplified to obtain sequences that encode the antibody (or binding domain) of the desired specificity. As indicated supra, a variety of other types of antibodies can also be utilized. For example, the antibodies can be recombinant antibodies from phage libraries. A variety of antibody fragments can also be used. Such fragments include Fab fragments, scFv fragments and mini-antibodies, for example.
• the antibodies can be labeled with semiconductor nanocrystals using the conjugation methods set forth infra. D. Exemplary Assays
• Certain techniques can be utilized to prepare large numbers of fusion proteins that subsequently can be screened for their ability to interact with macromolecules such as other proteins, nucleic acids, small molecules, oligosaccharides, drugs and other biological molecules.
• macromolecules such as other proteins, nucleic acids, small molecules, oligosaccharides, drugs and other biological molecules.
• an a ⁇ ay of cells expressing different fusion proteins comprising different ORFs from various organisms e.g., yeast
• the proteins expressed by the cells can then be screened with ligands of interest to identify those capable of interacting with the expressed proteins.
• Such methods allow one to identify unknown ligands to known proteins, as well permitting one to identify unknown proteins (i.e., ho activity has yet been assigned to the ORF) capable of binding known or unknown ligands.
• transposon constructs that include a transposon flanked by recombination sites are prepared.
• the construct also includes an epitope tag segment adjacent one of the recombination sites.
• a ⁇ ay of such cells can then be assayed to determine the subcellular localization of various proteins. Since in frame insertions of the construct generates fusion proteins that include the epitope, the fusion proteins can be localized by contacting cells with antibodies that specifically bind to the epitope. In this way, one can localize proteins to various regions of the cell, such as the nucleus, mitochondria and plasma membrane, for example. See, e.g., Ross-Macdonald, P., et al. (1999) Nature 402:413- 418. Detection of complexes formed between the antibodies and the localized proteins is enhanced by using antibodies that are labeled with semiconductor nanocrystals. 3. Epitope Determination
• Semiconductor labeled antibodies can also be utilized to determine the antigenic epitope of antigens of interest.
• An antigenic epitope is defined as a region of a protein to which an antibody can bind.
• An immunogenic epitope refers to those parts of a protein that elicit the antibody response when the whole protein is the immunogen. Certain of these methods involve the synthesis of overlapping proteins that cover the entire amino acid sequence of protein known to elicit an antigenic response. These proteins can be synthesized in an a ⁇ ay format using solid state protein synthesis methods such as described supra. The immobilized proteins are then tested for antigenicity using established ELISA techniques and various different anti-sera in which the antibodies are labeled with semiconductor nanocrystals.
• a replacement set of proteins is generated.
• the replacement set includes all the proteins co ⁇ esponding to the identified protein except that a single amino acid replacement is introduced.
• Each replacement set also can be synthesized as an a ⁇ ay.
• the replacement set is then rescreened using antibodies labeled with semiconductor nanocrystals to determine if antigenicity is retained. From the collective results, one can determine the location and identity of amino acids that play a critical role in antigenicity. See, e.g., Geysen, H. M., et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002.
• Histopathological examination in which tissue specimens are subjected to microscopic examination has enabled the biological mechanisms of many diseases to be clarified and consequently aided in the development of effective medical treatment for a variety of illnesses.
• a diagnosis is made on the basis of cell mo ⁇ hology and staining characteristics. Such analyses can sometimes be limited because of the sensitivity of standard staining procedures.
• tissue a ⁇ ays can be utilized to permit the rapid screening of a large number of tissue samples.
• Semiconductor nanocrystals can be used in all of the foregoing methods to enhance detection, sensitivity and, in some instances, to provide quantitative information.
• the semiconductor nanocrystals can be conjugated to a number of different species to stain particular cellular components.
• semiconductor nanocrystals can be conjugated to nucleic acids and antibodies and then used to probe the presence or absence of particular proteins or nucleic acids within a tissue specimen.
• Tissue a ⁇ ays can be prepared in a number of different ways.
• a clinician can obtain tissue samples from various sources using standard anatomical procedures and individually place the tissue samples at different locations on some type of a support (e.g., a glass slide or microtiter wells).
• the process can be automated in certain respects using devices designed to obtain tissue samples and then place the samples within an a ⁇ ay.
• An example of suitable devices for preparing a ⁇ ays in this manner are discussed in PCT publications WO 00/24940, 99/44062 and 99/44063. In general these devices utilize a punch apparatus to bore into a tissue sample and then dislodge the sample into a receptacle in an a ⁇ ay support.
• the a ⁇ ay can be prepared in a variety of different formats.
• the a ⁇ ay can include tissue samples from a single individual. Different labeled biomolecules bearing a semiconductor nanocrystal can be added to each location to detect the presence of a different cellular species.
• Other a ⁇ ays can include tissue samples from a number of different individuals.
• the a ⁇ ay can include tissue samples obtained from the same type of tissue from different individuals to screen for particular disease markers.
• a specific example of such an a ⁇ ay is an a ⁇ ay of breast tissue samples taken from women suspected of having breast cancer.
• Sets of tissue a ⁇ ays can also be prepared, each set of a ⁇ ays including the same tissue samples.
• each a ⁇ ay can be subjected to a different type of analysis to detect different markers. In this way, results for a number of different markers can rapidly be compiled for a population of different individuals.
• the multicolor capabilities of semiconductor nanocrystals permits a number of different types of inte ⁇ ogations to be conducted with a single tissue sample.
• utilization of the multiplexing capabilities of semiconductor nanocrystals coupled with an a ⁇ ay format in which sets of a ⁇ ays having the same tissue samples on each a ⁇ ay allows for very high throughput analysis of a number of different markers.
• tissue samples can be utilized. Suitable tissue samples include, but are not limited to, tissue sections excised using known surgical procedures, one or more whole cells, cellular suspensions, cytological preparations (e.g., smears obtained from cellular suspensions), as well as cellular extracts or homogenates.
• Cell suspensions can be utilized, for example, by pelleting the suspension and then fixing it to a support.
• the samples can also be obtained from specific tissues (e.g., skin, breast, prostrate, testes and ovaries) or from various bodily fluids (e.g., blood, plasma, urine that contains cells, semen, vaginal fluids, bronchial washings and ascitic fluids).
• the cells can be directly obtained from an organism or can be obtained from a cell culture.
• Tissue a ⁇ ays can be used to rapidly profile hundreds or thousands of tissue samples at the DNA, RNA and protein levels.
• the assays are not limited to detecting these three major classes of biomolecules.
• the presence and concentration of oligosaccharides, glycoproteins, particular fatty acids and other targets can all be detected using an appropriate molecule that specifically binds to the target of interest
• semiconductor nanocrystals can be tuned to a variety of different wavelengths, it is possible to use semiconductor nanocrystal labeled nucleic acids, proteins (e.g., antibodies) and other biomolecules to probe multiple different DNAs, RNAs, proteins and/or other biomolecules within a single tissue sample. The results from such investigations can be compiled in a database.
• semiconductor nanocrystal-conjugates either a single semiconductor nanocrystal conjugated to biomolecules or a plurality of semiconductor nanocrystals
• semiconductor nanocrystal-conjugates allow specific, sensitive, photostable detection of target molecules, factors that can be problematic using currently available stains.
• semiconductor nanocrystals i.e., single excitation source, na ⁇ ow, gaussian spectra and tunability of emission wavelength, mean that many more colors are resolved than with conventional fluorescent dyes.
• samples can be inte ⁇ ogated to detect the levels of target nucleic acids using either semiconductor labeled nucleic acids whose sequence is complementary to the target nucleic acid being inte ⁇ ogated or a labeled protein that specially binds to the target nucleic acid.
• semiconductor labeled nucleic acids whose sequence is complementary to the target nucleic acid being inte ⁇ ogated
• a labeled protein that specially binds to the target nucleic acid.
• an antibody typically an antibody that specifically binds to the target protein and bears a semiconductor nanocrystal is used to detect the presence of the target protein.
• the target protein is a DNA binding protein
• the nucleic acid that binds to the protein can be labeled with a semiconductor nanocrystal and used to probe for the target protein.
• analyses can be carried out in a two- step reaction in which a primary antibody is initially contacted with the tissue sample, followed by addition of a semiconductor nanocrystal-conjugated antibody.
• an antibody (or other biomolecule) semiconductor nanocrystal conjugate can be used to directly label proteins of interest within the sample.
• five (or more with increased spectral use or reduced spectral separation) different populations of semiconductor nanocrystals can be synthesized with emission spectra that are spaced at 40 nm intervals from, e.g., 490-650 nm.
• Each spectrally distinct population of semiconductor nanocrystals is conjugated to a different molecule that specifically recognizes a biomolecule of interest that may or may not be present in the sample to be analyzed.
• the sample is labeled with the semiconductor nanocrystals and analyzed for the location and quantity of the target molecule. This analysis may be carried out by conventional fluorescent microscopy techniques or by use of a spectral scanning device.
• semiconductor nanocrystals can be generated that are spectrally distinct, it is possible to label different biomolecules such as multiple antibodies and/or multiple nucleic acid probes that can then be used to measure the position and quantity of cellular compounds.
• biomolecules such as multiple antibodies and/or multiple nucleic acid probes that can then be used to measure the position and quantity of cellular compounds.
• semiconductor nanocrystal colors can be used by taking advantage of the known spatial separation of the targets to be analyzed. For example, no overlap would occur between nuclear localized targets and membrane localized targets.
• organelle- specific groups of semiconductor nanocrystals can be employed to increase dramatically the number of discernable targets.
• the multi-color capability of semiconductor nanocrystals can be used, for example, to conduct multi-color in-situ hybridization to measure the levels of tRNA, mRNA, DNA, protein or any other cellular compound that can be stained in a tissue a ⁇ ay.
• multi-color in-situ hybridization to measure the levels of tRNA, mRNA, DNA, protein or any other cellular compound that can be stained in a tissue a ⁇ ay.
• semiconductor nanocrystal conjugates that include a single semiconductor nanocrystal, one can make quantitative measurements of the target biomolecule(s) under investigation. Multiplexing also allows rapid analysis of many molecular markers in the same set of specimens with greater accuracy.
• tissue a ⁇ ays in various types of co ⁇ elation studies. For instance, by obtaining samples from individuals known to have a particular disease, analyses with various semiconductor nanocrystal conjugates can be used to identify potential markers associated with the disease or illness. In general, such methods involve the use of semiconductor nanocrystal conjugates to determine the levels of selected DNA, RNA and/or protein levels in tissues from diseased individuals. Detection of elevated or reduced levels of various genes or gene products can be used to make initial co ⁇ elations between such genes or proteins with the particular disease under investigation
• tissue array assays utilizing semiconductor nanocrystal conjugates can be used to establish co ⁇ elations between certain markers and patient prognosis.
• the levels of various DNA, RNA and/or protein levels can be monitored over time in tissue samples obtained from patients known to have a disease as the patients receive different treatments. By monitoring the health history of the patients and the various levels of markers over time, one can establish co ⁇ elations between certain markers and disease outcomes. Similarly, co ⁇ elations between levels of markers and the efficacy of different therapeutic treatments can established. In this instance, the level of different markers is tracked for individuals receiving different treatments.
• the photostability of semiconductor nanocrystals allows tissue a ⁇ ays t be read repeatedly and archived for the pmpose of comparison at a later date.
• semiconductor nanocrystal conjugates that specially recognize such markers can be used to screen and identify individuals that have a disease or are susceptible to acquiring the disease.
• assays utilizing semiconductor nanocrystal conjugates can be utilized to determine the marker profile for an individual and thus identify the most appropriate treatment option.
• Tissue a ⁇ ay analysis can also be used in combination with other analyses.
• the initial co ⁇ elation studies just described can be conducted using the differential gene expression methods described supra.
• nucleic acid a ⁇ ays can be utilized to identify a nucleic acid whose expression is altered in diseased individuals. Additional validation studies can then be conducted using tissue arrays. If the nucleic acid is shown to be a bonafide marker for a disease, then semiconductor nanocrystal conjugates that specifically bind to the marker can be used to screen individuals for the presence of, or susceptibility to, the disease.
• Semiconductor nanocrystals can be utilized to label various target biomolecules for use in various types of secondary inte ⁇ ogations. Such investigations generally involve conducting an additional analysis once a binding a binding complex between two or more biomolecules have already been formed.
• the a ⁇ ay in such investigations typically bears a biomolecule that captures a target molecule in preparation for a secondary inte ⁇ ogation.
• Suitable targets in this type of study include, but are not limited to, nucleic acids (e.g., DNA, RNA), proteins, or antibodies.
• a specific example of a secondary inte ⁇ ogation is the use of an a ⁇ ay of antibodies to probe for multiple epitopes of a protein. In this instance, each spot on the a ⁇ ay contains a different antibody.
• a complex protein target is labeled with a single color semiconductor nanocrystal and allowed to bind to the a ⁇ ay bearing the different antibodies.
• the three shaded regions co ⁇ espond to the positive signals from the a ⁇ ay.
• a secondary antibody labeleled with a semiconductor nanocrystal having a second color
• FIG. IB shows the specific example shown in FIG. 1 A, different colors are used to label each of the three active antibodies identified in the first round of screening.
• This procedure can be used, for example, to find complementary antibody pairs.
• the first step several antibodies are identified that bind efficiently to the target molecule. Since each of these antibodies may bind to a different epitope of the target molecule, the second step can inte ⁇ ogate the bound proteins by contacting the a ⁇ ay with the same antibodies identified in the first step. Those locations on the a ⁇ ay that emit signals from both semiconductor nanocrystals are those locations in which there are two antibodies that recognize different epitopes of the target molecule.
• This technique of capture and secondary inte ⁇ ogation can also be used to do simultaneous genotyping of multiple SNPs.
• both polymo ⁇ hic sites can be inte ⁇ ogated simultaneously by using a nucleic acid probe on an a ⁇ ay that is complimentary to the first SNP to capture the target nucleic acid labeled with a semiconductor nanocrystal.
• a nucleic acid probe on an a ⁇ ay that is complimentary to the first SNP to capture the target nucleic acid labeled with a semiconductor nanocrystal.
• the labeled target nucleic acid can be probed with a second set of labeled probes that is complimentary to the various polymo ⁇ hic forms of the second SNP.
• Probes for different polymo ⁇ hic forms can each be labeled with a different color.
• semiconductor nanocrystals can provide significant improvement in both sensitivity and dynamic range.
• the fluorescence from semiconductor nanocrystals is extremely bright and stable, and permits routine detection of single semiconductor nanocrystals (see, e.g., Empedocles et al. (1999a), "Three- dimensional orientation measurements of symmetric single chromophores using polarization microscopy," Nature 399:126-130; Empedocles et al. (1999b), "Spectroscopy of Single CdSe Nanocrystallites,” Ace. Chem. Res. 32:389-396; Empedocles et al.
• the single target counting assays and methods thereof described herein do not necessarily have to be performed using semiconductor nanocrystal labels.
• Any fluorescent label capable of being detected on the single molecule level can be utilized for the type of measurement described herein.
• suitable labels include, but is not limited to, organic dye molecules, metal colloid scattering particles, and surface-enhanced Raman spectroscopy (SERS) particles.
• SERS surface-enhanced Raman spectroscopy
• the many unique features of semiconductor nanocrystals described supra make them particularly useful as labels in single target counting assays.
• certain methods described herein do not require the ability to detect a single label, but rather a single target molecule. Therefore, the methods described herein can be used to detect single target molecules that are labeled with a single detectable label, or with multiple detectable labels.
• the high stability, detection sensitivity and ease of multiplexing make semiconductor nanocrystals useful as multi-color fluorophores for use in ultra-sensitive surface based assays.
• the ability to easily detect single semiconductor nanocrystals means that semiconductor nanocrystals are particularly useful as fluorophores in bioassays in which single target molecules bound to the assay surface are counted one at a time.
• Single target counting does not mean the counting of all of the target molecules within a sample, but rather the counting of the target molecules that are bound to the surface of the a ⁇ ay substrate. While this number is not necessarily the same as the total number of target molecules in the sample, the actual target level can be determined through calibration against a sample of known concentration. By enabling the detection and counting of single bound target molecules, one can extend the sensitivity of surface- based assays beyond what is possible using current detection techniques. For instance, current microarray technology allows the detection of target at a density of as low as 0.1 labels/ ⁇ m 2 ( -8 labels per lO ⁇ m diameter confocal spot).
• the left side co ⁇ esponds to the high concentration regime (ensemble regime), in which the entire a ⁇ ay spot is covered with target and the average emission intensity is dependent on the average density of label across the surface of the a ⁇ ay.
• sample concentration is proportional to average emission intensity (ensemble intensity).
• the right side co ⁇ esponds to the single copy counting regime, where individual bound target molecules are separated from each other by distances that are greater than the diffraction limit of light and can be detected one at a time. In this regime, sample concentration is proportional to the number of individual targets counted on the surface of the a ⁇ ay.
• Figure 6B shows data simulating the relative signal vs concentration detected using ensemble intensity and single copy counting over the entire concentration range.
• Ensemble measurements yield a linear concentration dependence at high concentrations, but saturate at low concentrations. This saturation occurs when the total signal from bound target in the detection region is lower than the noise generated from the integrated background across that entire region.
• Detecting single molecules bound to the a ⁇ ay with high-resolution microscopy can dramatically reduce the integrated background noise by comparing the signal from a single fluorophore to the background from an extremely small (diffraction limited) area of the a ⁇ ay spot.
• the background generated over a standard lO ⁇ m diameter ensemble probe spot is 400 times higher than the background generated from a high resolution image of a single fluorophore ( ⁇ 0.5 ⁇ m diameter). This results in a decrease in noise (and therefore an increase in sensitivity) of a factor of 20. This effect is further enhanced if the ensemble signal is integrated over the entire a ⁇ ay spot.
• the background signal is 40000 times higher than for a diffraction limited spot resulting in approximately 200 times higher sensitivity.
• the background over the bottom of an entire well of a 96 well plate is -10 8 times higher yielding an enhancement of 10 4 .
• the single target counting signal saturates at high concentrations. This occurs when the concentration increases to the point where individual target molecules are so close together that they can not be distinguished. This means that some individual spots actually contain more than one bound target molecule and therefore results in an undercounting of the total number of target molecules. The result is an underestimate of the total sample concentration (see FIG. 6C).
• the concentration is low enough to count individual targets, but high enough to be detectable in an ensemble measurement. This is refe ⁇ ed to as the transition regime.
• detection sensitivity can be increased, as well as the dynamic range of microa ⁇ ay assays.
• detection sensitivity at the low end is achieved at the expense of dynamic range at the high end due to detector saturation.
• single target counting with ensemble intensity measurements, one can cover the entire dynamic range in a single experiment. The reason is that in the single copy counting regime, as the concentration increases, the peak intensity does not — only the number of detected spots increases. As such the entire dynamic range of the detector can be used to cover the ensemble concentration regime, where peak intensity varies linearly with concentration.
• a detection system capable of detecting the fluorescence from single semiconductor nanocrystals over the entire area of a lOO ⁇ m microa ⁇ ay spot, with a spatial resolution of less than 0.5 ⁇ m.
• This system uses a 2- dimensional CCD camera with a dynamic range of 65,536 counts per pixel and a read noise of -2 counts/pixel. If excitation intensity and integration time are selected to yield 30 counts/pixel/semiconductor nanocrystal, then in the single copy counting regime, individual semiconductor nanocrystals are detected with a signal to noise ratio of -15.
• Single molecule fluorescence detection can be achieved using either laser scanning confocal microscopy or wide-field imaging with a 2D CCD camera.
• One distinct advantage of wide-field imaging over scanning confocal microscopy for these applications is that fluorescence can be collected from all points witbin one or more a ⁇ ay spots simultaneously. This means that the signal can be integrated for relatively long periods of time without increasing the read-time for the a ⁇ ay. This is particularly beneficial when detecting semiconductor nanocrystals, since they do not photobleach. It is therefore possible to integrate the signal from each a ⁇ ay spot for a relatively long time compared to organic dyes.
• the same image of a single spot can be obtained in a single 100 ms exposure. Once taken, the a ⁇ ay can be translated to an adjacent region and the next image acquired. By precisely controlling the scanning stage and stitching the images together, the entire a ⁇ ay image can be produced. This procedure dramatically decreases the total read-time, allowing an entire a ⁇ ay to be read in less than 20 minutes. In addition, in some instances multiple a ⁇ ay spots can be imaged simultaneously, further reducing the total collection time.
• the methods of the present invention focus on a ⁇ ay based assays in which an assay occurs on an a ⁇ ay surface, the methods also apply to other bioassays performed on a surface support such as the bottom of a microtitre plate or a polymer bead.
• the considerations just described apply generally to any of the assays set forth above.
• Assays typically are conducted with semiconductor nanocrystals, but as noted above, can also be performed with other fluorophores such as an organic dye or metal colloid.
• Semiconductor nanocrystals can be inco ⁇ orated into the ligands or antiligands of the assay via a plurality of techniques described herein. Each bound target molecule is labeled with 1 or more semiconductor nanocrystals.
• the fluorescence from the sample is detected. If the density of bound target molecules is greater than -1 target/ ⁇ m 2 , then the assay signal is measured and calibrated using the total emission intensity from the entire assay region (e.g. the total signal from a single microa ⁇ ay spot or from an entire microtitre well). If the target density is less than -1 target/ ⁇ m 2 , so that individual target molecules can be spatially resolved using standard far-field optics, then the assay signal is measured and calibrated by counting the total number of bound target molecules. The assay signal can be measured from all assays using both ensemble and single target counting methods. A calibration curve can then be used to identify which assays fall in the ensemble, single and transition regimes.
• an optical detection system capable of detecting the fluorescence from single semiconductor nanocrystals (or other labels) with a spatial resolution of l ⁇ m or less.
• this optical system is comprised of a wide-field imaging system with a 2D CCD camera and a high numerical aperture microscope objective.
• a laser based microscope system capable of detecting and spectrally resolving the fluorescence from single semiconductor nanocrystals can also be utilized (see, e.g., Empedocles et al. (1999a), Nature 399:126-130; Empedocles et al. (1999b), Ace. Chem. Res. 32:389-396; Empedocles et al (1991), Science 278:2114-2117; and Empedocles et al. (1996), Phys. Rev. Lett. 77:3873-3876).
• FIG. 7 is a schematic drawing of the significant optical components of such a laser microscope system 100.
• Excitation light 102 from a laser source (488 nm Ar + ) 104 is transmitted through a dispersing prism 106 and a 500 nm short pass dichroic minor 108 at an angle of 45°.
• the excitation light is then focused by a high numerical aperture microscope objective 110 onto the sample surface 112.
• An additional lens in the excitation path i.e., the dispersing prism 106) causes the laser 104 to illuminate a wide area of the sample surface 112.
• the fluorescent image is collected by the same objective lens 110.
• the image is reflected by the dichroic minor 108, passes through a wavelength specific filter 114 to remove any excitation light, and is focused by a final lens 116 onto the detection system 118.
• the detection system 118 consists of a 2D CCD camera 120 and a tunable bandpass filter 122. Spectral images are obtained by acquiring multiple images at a different wavelength. With this system, it is possible to simultaneously obtain spectra at every point within the image with a spectral resolution of 2 nm and a spatial resolution of less than ⁇ 0.5 ⁇ m.
• Uniform excitation intensity in this system can be generated either through the use of a lamp light source or a laser excitation source that has been transformed from a Gaussian intensity profile to a "top-hat” profile through the use of a series of 2 Powel lenses each oriented at 90 degrees relative to each other.
• the optical system can be comprised of a scanning confocal microscope system with a spatial resolution of less than ⁇ 0.5 ⁇ m.
• Another detection option utilizes an optical system that comprises a microscope with an immersion microscope objective in which the sample is viewed from the backside of the sample substrate (e.g. from the underside of a microa ⁇ ay substrate or from the bottom of a microtitre well).
• detection can be with a water- or other fluid-immersion lens, also detecting from the back-side of the sample substrate.
• Autofluorescence from the a ⁇ ay substrate and assay materials can be minimized by (a) using low fluorescence a ⁇ ay substrates such as quartz or low fluorescence glass, (b) choosing a fluorescent label that does not overlap significantly with the autofluorescence from the substrate and assay materials, and (c) choosing an excitation wavelength that does not significantly excite autofluorescence. Since semiconductor nanocrystals can be synthesized to absorb and emit at any wavelengths, they are an ideal fluorophore for minimizing interference from autofluorescence.
• kinematic alignment of the a ⁇ ay slide combined with the use of "alignment spots" is used to automatically locate the edges of the a ⁇ ay and register the first image so that the a ⁇ ay spots are each located within the center of each image.
• Alignment spots are a ⁇ ay spots that are not complementary to any sequence of interest.
• To each hybridization sample one can add a labeled target that is specific for these alignment spots at a known concentration. These spots therefore have a high signal and can be detected and used for alignment pu ⁇ oses.
• a pattern of alignment spots can be placed across each anay that unambiguously identify the absolute position of the anay.
• Software can then be used to locate and analyze each spot within the anay.
• the alignment spots can be identified and all other spot locations will be determined from the known periodicity of the a ⁇ ay.
• each spot on the a ⁇ ay can be located according to its position within a periodic lattice.
• the radius of all spots is the same and can be predetermined or extracted from the radius of the alignment spots.
• Two separate algorithms can then be used to analyze the signal from within each spot area. First, the total integrated signal from within each spot can be measured and compared to either an equivalent area outside of the a ⁇ ay spot or to a calibration spot of known intensity. Second, an algorithm can be used to count individual fluorescent point within each a ⁇ ay spot.
• the algorithm can identify and count fluorescent points that fit a set of predetermined characteristics of shape, size and threshold intensity that are specific for the fluorescence from single semiconductor nanocrystals.
• a data file can be exported containing the ensemble intensity and the "count number" (i.e. the number of discrete fluorescent points) for each spot.
• Figures 8A-8E describe the complete a ⁇ ay scanning procedure.
• a second semiconductor nanocrystal color that does not spectrally overlap with the detection label. This second color can be added to each bead, either internally, or bound to the surface at a known concentration. This color can then be used to locate individual beads. Once found, a bandpass filter can be used to block the fluorescence from the alignment color and allow single target detection of only the label semiconductor nanocrystals. This 2- color technique can also be used for microa ⁇ ays.
• each target molecule is labeled with two different semiconductor nanocrystal colors via two different binding interactions. Specifically bound labels can then be identified through the detection of both colors colocalized within the same fluorescent spot.
• the primary shortcoming of surface based assays such as nucleic acid microa ⁇ ays is the lack of appropriate sensitivity needed to detect extremely low levels of target concentration. For instance, as much as 40% of the known genes of interest studied using gene expression microarrays are expressed at a level of between 1 and 10 copies per cell, just at or below the limit of detection using cunent detection schemes. In addition to low expression levels, the costs incu ⁇ ed in extracting material for genetic testing creates pressure to minimize sample size requirements for genetic analysis. The ability to measure vanishingly small quantities of expressed DNA significantly improves one's ability to identify and treat diseases at an early stage. Ultra-sensitive detection in microanay assays can also assist in identifying new genes of interest in all areas of disease. A system for labeling and high sensitivity detection of fluorescence from DNA microa ⁇ ays can significantly reduce the costs associated with expression analysis while simultaneously increasing the available information content.
• the prefe ⁇ ed method for detection of surface based assays such as microa ⁇ ays is by labeling target molecules with organic dyes.
• the cunent state-of-the-art detection can only detect a minimum of approximately 10 molecules in a lO ⁇ m x lO ⁇ m region of a microa ⁇ ay spot (Duggan et al. (1999) Nature Genetics 21(nls): 10-14). This means that the minimum number of bound DNA molecules required in order to detect signal from a standard lOO ⁇ m diameter microa ⁇ ay spot is approximately 1000. In order to generate this signal, more than 10 million cells may be required.
• exemplary materials for use as semiconductor nanocrystals in the biological and chemical assays of the present invention include, but are not limited to those described above, including group II-NI, III-N and group IN semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, Ga ⁇ , GaP, GaAs, GaSb, InP, InAs, InSb, A1S, AIP, AlSb, PbS, PbSe, Ge and Si and ternary and quaternary mixtures thereof.
• group II-NI, III-N and group IN semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaS
• the semiconductor nanocrystals are characterized by their uniform nanometer size. As discussed above, the selection of the composition of the semiconductor nanocrystal, as well as the size of the semiconductor nanocrystal, affects the characteristic spectral emission wavelength of the semiconductor nanocrystal. Thus, as one of ordinary skill in the art will realize, a particular composition of a semiconductor nanocrystal as listed above will be selected based upon the spectral region being monitored. For example, semiconductor nanocrystals that emit energy in the visible range include, but are not limited to, CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, and GaAs.
• Semiconductor nanocrystals that emit energy in the near IR range include, but are not limited to, InP, InAs, InSb, PbS, and PbSe.
• semiconductor nanocrystals that emit energy in the blue to near-ultraviolet include, but are not limited to, ZnS and Ga ⁇ .
• the nanocrystals For any particular composition selected for the semiconductor nanocrystals to be used in the inventive methods, it is possible to tune the emission to a desired wavelength by controlling the size of the particular composition of the semiconductor nanocrystal. In some instances, 5-20 discrete emissions (five to twenty different size populations or distributions distinguishable from one another), more preferably 10-15 discrete emissions, are obtained for any particular composition, although one of ordinary skill in the art will realize that fewer than five emissions and more than twenty emissions can be used depending on the monodispersity of the semiconductor nanocrystal particles. If high information density is required, and thus a greater number of distinct emissions, the nanocrystals are preferably substantially monodisperse within the size range given above.
• monodisperse as that term is used herein, means a colloidal system in which the suspended particles have substantially identical size and shape. In certain high information density applications, monodisperse particles deviate less than 10% rms in diameter, and preferably less than 5%. Monodisperse semiconductor nanocrystals have been described in detail in Mu ⁇ ay et al. (1993) J. Am. Chem. Soc. 115:8706, and in Mu ⁇ ay, "Synthesis and Characterization of II-NI Quantum Dots and Their Assembly into 3-D Quantum Dot Superlattices,” (1995) Doctoral dissertation, Massachusetts Institute of Technology.
• the number of discrete emissions that can be distinctly observed for a given composition depends not only upon the monodispersity of the particles, but also on the deconvolution techniques employed.
• Semiconductor nanocrystals unlike dye molecules, can be easily modeled as Gaussians and therefore are more easily and more accurately deconvoluted.
• high information density is not required and it is more economically attractive to use more polydisperse particles.
• the linewidth of the emission can be in the range of 40-60 nm.
• the surface of the semiconductor nanocrystal is modified to enhance the efficiency of the emissions, by adding an overcoating layer to the semiconductor nanocrystal.
• the overcoating layer is typically utilized because at the surface of the semiconductor nanocrystal, surface defects can result in traps for electrons or holes that degrade the electrical and optical properties of the semiconductor nanocrystal.
• An insulating layer at the surface of the semiconductor nanocrystal provides an atomically abrupt jump in the chemical potential at the interface that eliminates energy states that can serve as traps for the electrons and holes. This results in higher efficiency in the luminescent process.
• Suitable materials for the overcoating layer include semiconductor materials having a higher bandgap energy than the semiconductor nanocrystal core.
• suitable materials for the overcoating layer should have good conduction and valence band offset with respect to the core semiconductor nanocrystal.
• the conduction band is desirably higher and the valence band is desirably lower than those of the core semiconductor nanocrystal.
• a material that has a bandgap energy in the ultraviolet regions can be used.
• Exemplary materials include ZnS, GaN, and magnesium chalcogenides, e.g., MgS, MgSe, and MgTe.
• materials having a bandgap energy in the visible such as CdS or CdSe, can also be used.
• semiconductor nanocrystals are prepared in coordinating solvent, such as trioctylphosphine oxide (TOPO) and trioctyl phosphine (TOP) resulting in the formation of a passivating organic layer on the nanocrystal surface comprised of the organic solvent.
• This layer is present on semiconductor nanocrystals containing an overcoating and those that do not contain an overcoating.
• organic solvents such as toluene, chloroform and hexane.
• These functional moieties can be readily displaced or modified to provide an outer coating that renders the semiconductor nanocrystals suitable for use as the detectable labels of the present invention, as described further below.
• Furthennore based upon the desired application, a portion of the semiconductor nanocrystal functionality, or the entire surface of the semiconductor nanocrystal functionality can be modified by a displacement reaction, based upon the desired use therefor.
• Selection criteria important in determining the minimum number of semiconductor nanocrystals needed to observe a distinct and unique spectral emission of sufficient intensity include: (1) providing a sufficient number of semiconductor nanocrystals that are bright (i.e., that emit light versus those that are dark) and, (2) providing a sufficient number of semiconductor nanocrystals to average out over the blinking effect observed in single semiconductor nanocrystal emissions.
• eight or more semiconductor nanocrystals of a particular composition and particle size distribution can be provided. If, for example, the desired method of use utilizes three different particle size distributions of a particular composition, eight of each of the tliree different particle size distributions of a semiconductor nanocrystal is used, in order to observe sufficiently intense spectral emissions from each to provide reliable information regarding the location or identity of a particular analyte of interest. Fewer than eight semiconductor nanocrystals of a particular composition and particle size distribution can be utilized provided that a unique spectral emission of sufficient intensity is observed, as determined by the selection criteria set forth above.
• the above method can be used to prepare separate populations of semiconductor nanocrystals, wherein each population exhibits a different characteristic photoluminescence spectrum.
• Each of a plurality of populations of semiconductor nanocrystals can be conjugated to distinct first members of binding pairs for use in a multiplexed assay or analytical method in which each of a plurality of co ⁇ esponding second members of the binding pairs can be detected simultaneously.
• the narrow spectral linewidths and nearly gaussian symmetrical lineshapes lacking a tailing region observed for the emission spectra of nanocrystals combined with the tunability of the emission wavelengths of nanocrystals allows high spectral resolution in a system with multiple nanocrystals.
• up to 10-20 or more different-sized nanocrystals or different size distributions of monodisperse populations of nanocrystals from different preparations of nanocrystals, with each sample having a different emission spectrum can be used simultaneously in one system, i.e., multiplexing, with the overlapping spectra easily resolved using techniques well known in the art, e.g., optically with or without the use of deconvolution software.
• the ability of the semiconductor nanocrystals to produce discrete optical transitions, along with the ability to vary the intensity of these optical transitions, enables the development of a versatile and dense encoding scheme.
• the characteristic emissions produced by one or more sizes of semiconductor nanocrystals attached to, associated with, or embedded within a particular support, compound or matter enables the identification of the analyte of interest and/or its location. For example, by providing N sizes of semiconductor nanocrystals (each having a discrete optical transition), each having M distinguishable states resulting from the absence of the semiconductor nanocrystal, or from different intensities resulting from a particular discrete optical transition, Mn different states can be uniquely defined.
• the encoding scheme would thus be defined by a base 2 or binary code.
• M in which the two states could be the presence or absence of the semiconductor nanocrystal
• the encoding scheme would be defined by a base 3 code.
• base M codes wherein M is greater than 2 are termed higher order codes. The advantage of higher order codes over a binary order code is that fewer identifiers are required to encode the same quantity of information.
• each discrete emission or color is capable of being detectable at two to twenty different intensities.
• a base 11 code comprising the absence of the semiconductor nanocrystal, or the detection of the semiconductor nanocrystal at 10 different intensities.
• one or more semiconductor nanocrystals may act as a barcode, wherein each of the one or more semiconductor nanocrystals produces a distinct emissions spectrum. These characteristic emissions can be observed as colors, if in the visible region of the spectrum, or may also be decoded to provide information about the particular wavelength at which the discrete transition is observed.
• the characteristic wavelengths that the discrete optical transitions occur at provide information about the identity of the particular semiconductor nanocrystal, and hence about the identity of or location of the analyte of interest.
• the color of light produced by a particular size, size distribution and/or composition of a semiconductor nanocrystal can be readily calculated or measured by methods which will be apparent to those skilled in the art.
• the bandgaps for nanocrystals of CdSe of sizes ranging from 12A to 115A are given in Mu ⁇ ay et al. (1993) J. Am. Chem. Soc. 115:8706.
• These techniques allow ready calculation of an appropriate size, size distribution and/or composition of semiconductor nanocrystals and choice of excitation light source to produce a nanocrystal capable of emitting light device of any desired wavelength.
• An example of a specific system for automated detection for use with the present methods includes, but is not limited to, an imaging scheme comprising an excitation source, a monochromator (or any device capable of spectrally resolving the image, or a set of na ⁇ ow band filters) and a detector anay.
• the apparatus consists of a blue or UN source of light, of a wavelength shorter than that of the luminescence detected.
• This may be a broadband UN light source, such as a deuterium lamp with a filter in front; the output of a white light source such as a xenon lamp or a deuterium lamp after passing through a monochromator to extract out the desired wavelengths; or any of a number of continuous wave (cw) gas lasers, including but not limited to any of the Argon Ion laser lines (457, 488, 514, etc. mn), a HeCd laser; solid state diode lasers in the blue such as Ga ⁇ and GaAs (doubled) based lasers or the doubled or tripled output of YAG or YLF based lasers; or any of the pulsed lasers with output in the blue, to name a few.
• cw continuous wave
• the luminescence from the dots may be passed through an imaging subtracting double monochromator (or two single monochromators with the second one reversed from the first), for example, consisting of two gratings or prisms and a slit between the two gratings or prisms.
• the monochromators or gratings or prisms can also be replaced with a computer controlled color filter wheel where each filter is a na ⁇ ow band filter centered at the wavelength of emission of one of the dots.
• the monochromator assembly has more flexibility because any color can be chosen as the center wavelength.
• a CCD camera or some other two dimensional detector records the images, and software color codes that image to the wavelength chosen above. The system then moves the gratings to a new color and repeats the process. As a result of this process, a set of images of the same spatial region is obtained and each is color-coded to a particular wavelength that is needed to analyze the data rapidly.
• the apparatus is a scanning system as opposed to the above imaging scheme.
• a scanning scheme the sample to be analyzed is scanned with respect to a microscope objective.
• the luminescence is put through a single monochromator or a grating or prism to spectrally resolve the colors.
• the detector is a diode a ⁇ ay that then records the colors that are emitted at a particular spatial position.
• the software then ultimately recreates the scanned image and decodes it.
• the present invention utilizes various conjugates that generally comprise a biological molecule and one or more semiconductor nanocrystals, such that the conjugate can detect the presence, absence and/or amounts of various complexes formed on addressable arrays.
• semiconductor nanocrystal conjugates comprise any molecule or molecular complex, linked to a semiconductor nanocrystal, that can interact with a biological target, to detect biological processes, or reactions, as well as alter biological molecules or processes.
• the molecules or molecular complexes or conjugates physically interact with a biological compound.
• the interactions are specific.
• the interactions can be, but are not limited to, covalent, noncovalent, hydrophobic, hydrophilic, electrostatic, van der Waals, or magnetic.
• these molecules are small molecules, proteins, or nucleic acids or combinations thereof.
• Semiconductor nanocrystal conjugates can be made using techniques known in the art.
• moieties such as TOPO and TOP, generally used in the production of semiconductor nanocrystals, as well as other moieties, can be readily displaced and replaced with other functional moieties, including, but not limited to carboxylic acids, amines, aldehydes, and styrene to name a few.
• factors relevant to the success of a particular displacement reaction include the concentration of the replacement moiety, temperature and reactivity.
• any functional moiety may be utilized that is capable of displacing an existing functional moiety to provide a semiconductor nanocrystal with a modified functionality for a specific use.
• the ability to utilize a general displacement reaction to modify selectively the surface functionality of the semiconductor nanocrystals enables functionalization for specific uses.
• the present invention utilizes semiconductor nanocrystals that are solubilized in water.
• the outer layer includes a compound having at least one linking moiety that attaches to the surface of the particle and that terminates in at least one hydrophilic moiety.
• the linking and hydrophilic moieties are spanned by a hydrophobic region sufficient to prevent charge transfer across the region.
• the hydrophobic region also provides a "pseudo-hydrophobic" environment for the nanocrystal and thereby shields it from aqueous su ⁇ oundings.
• the hydrophilic moiety can be a polar or charged (positive or negative) group. The polarity or charge of the group provides the necessary hydrophilic interactions with water to provide stable solutions or suspensions of the semiconductor nanocrystal.
• hydrophilic groups include polar groups such as hydroxides (-OH), amines, polyethers, such as polyethylene glycol and the like, as well as charged groups, such as carboxylates (-CO “ ), sulfonates (SO 3 " ), phosphates (-PO 4 2" and - PO 3 " ), nitrates, ammonium salts (-NH 4 ), and the like.
• a water-solubilizing layer is found at the outer surface of the overcoating layer. Methods for rendering semiconductor nanocrystals water-soluble are known in the art and described in, e.g., PCT Publication No. WO 00/17655, published March 30, 2000.
• the affinity for the nanocrystal surface promotes coordination of the linking moiety to the semiconductor nanocrystal outer surface and the moiety with affinity for the aqueous medium stabilizes the semiconductor nanocrystal suspension.
• a displacement reaction can be employed to modify the semiconductor nanocrystal to improve the solubility in a particular organic solvent. For example, if it is desired to associate the semiconductor nanocrystals with a particular solvent or liquid, such as pyridine, the surface can be specifically modified with pyridine or pyridine-like moieties to ensure solvation.
• the surface layer can also be modified by displacement to render the semiconductor nanocrystal reactive for a particular coupling reaction.
• displacement of TOPO moieties with a group containing a carboxylic acid moiety enables the reaction of the modified semiconductor nanocrystals with amine containing moieties (commonly found on solid support units) to provide an amide linkage. Additional modifications can also be made such that the semiconductor nanocrystal can be associated with almost any solid support such as those describe supra.
• the semiconductor nanocrystals of the present invention can readily be functionalized to create styrene or acrylate moieties, thus enabling the inco ⁇ oration of the semiconductor nanocrystals onto polystyrene, polyacrylate or other polymers such as polyimide, polyacrylamide, polyethylene, polyvinyl, polydiacetylene, polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone, polypy ⁇ ole, polyimidazole, polythiophene, polyether, epoxies, silica glass, silica gel, siloxane, polyphosphate, hydrogel, agarose, cellulose, and the like.
• Nanocrystal Colloids Manganese Doped Cadmium Selenide, (Core)Shell Composites for Biological Labeling, and Highly Fluorescent Cadmium Telluride" (1999) Doctoral dissertation, Massachusetts Institute of Technology.
• cDNA microa ⁇ ay slides were prepared as described in the Fabrication section of www.nhgri.iiih. gov/DIR/microarray. Further guidance on fabrication, sample labeling and conditions for hybridization using microa ⁇ ays is provided, for example, by Bittner M., et al. (2000) Nature 406:536-540; Khan J., et al. (1999) Electrophoresis
• cDNA was labeled with biotin.
• SCNC semiconductor nanocrystals
• Cy®3 dUTP was replaced with biotin- 16- dUTP (Roche Molecular Biochemicals, Indianapolis, IN). After hybridization, the slide was incubated in 4X SSC, 0.1% Tween® 20,
• bovine serum albumin (BSA) at room temperature for a minimal of 30 minutes and rinsed in IX phosphate-buffered saline (PBS), 1% BSA, 1 mM MgCl 2 .
• PBS IX phosphate-buffered saline
• BSA 1 mM MgCl 2
• the slide was incubated in 25 nM 630 nm SCNC-streptavidin in IX PBS, 1% BSA, 10 mM MgCl 2 for 1 hour at room temperature.
• the slide was rinsed in IX PBS, 1% BSA, 1 mM MgCl 2 followed by 10 mM Phosphate buffer pH 7.4 and spin-dried in centrifuge at 500 ⁇ m for 5 minutes.
• the spots on the microa ⁇ ay were viewed under a fluorescence microscope and scanner optimized for SCNCs.
• RNA sample labeling by reverse transcription and hybridization were performed using methods described in Khan et al. (1999) Biochim. Biophys. Ada 1423:17-28 or from www.nhgri.nih.gov/DIR/microanay.
• One cDNA was labeled with biotin and the other with Cy3 or Cy5.
• Cy5- or Cy3-dUTP was replaced with biotin- 16-dUTP (Roche Molecular Biochemicals, Indianapolis, IN).
• the microa ⁇ ay slide was incubated in 4X SSC, 0.1% Tween 20, 1% BSA for 30-60 minutes at room temperature, rinsed in 0.06X SSC, and spun dry at 500 ⁇ m for 5 minutes in a centrifuge with a horizontal rotor for microplates.
• SCNC-streptavidin were added to 6X SSPE, 1% BSA, 10 mM MgCl 2 to a final concentration of 25 nM.
• 40-80 ⁇ L of the SCNC- streptavidin was applied on the a ⁇ ay area, a coverslip was added and the covered microa ⁇ ay was incubated in a humidified container for 1 hour at room temperature.
• the slide was rinsed in IX SSPE followed by 0.06X SSPE and spin-dried in centrifuge at 500 ⁇ m for 5 minutes.
• microa ⁇ ay was viewed on a fluorescence microscope. SCNC-labeled cDNA hybridized to the microa ⁇ ay was easily detected under fluorescence microscopy.
• RNA Ribonucleic acid
• sample labeling by reverse transcription and hybridization are performed by using method as described in Khan et al. (1999) Biochim. Biophys. Acta 1423:17-28 or from web site www.nhgri.nih.gov/DIR/microanay, or by any suitable method known to those skilled in the art.
• One cDNA is labeled with biotin and the other with a hapten (e.g., fluorescein, digoxygenin, or estradiol).
• hapten e.g., fluorescein, digoxygenin, or estradiol
• Cy5- and Cy3-dUTP is replaced with biotin- 16-dUTP (Roche Molecular Biochemicals, Indianapolis, IN) and dUTP -hapten (e.g., fluorescein-12-dUTP, DIG 11-dUTP, estradiol- 15 -dUTP).
• biotin- 16-dUTP Roche Molecular Biochemicals, Indianapolis, IN
• dUTP -hapten e.g., fluorescein-12-dUTP, DIG 11-dUTP, estradiol- 15 -dUTP.
• the slide is incubated in blocking solution 4X SSC, 0.1% Tween 20,1% BSA for 30-60 minutes at room temperature, rinsed in 0.06X SSC, and dried by spinning at 500 ⁇ m for 5 minutes in a centrifuge with a horizontal rotor for microplates.
• SCNC-streptavidin and SCNC-anti-hapten are added to 6X SSPE, 1% BSA, 10 mM MgCl 2 to a final concentration of 25 nM each.
• a coverslip is added over the a ⁇ ay area and the covered a ⁇ ay is incubated in a humidified container for 1 hour at room temperature.
• the slide is rinsed in IX SSPE followed by 0.06X SSPE and dried by spinning in centrifuge at 500 ⁇ m for 5 minutes.
• the microa ⁇ ay is read on a scanner optimized for SCNC emission.
• RNA sample labeling by reverse transcription and hybridization are performed by using method as described in Khan et al. (1999) Biochim. Biophys. Acta 1423:17-28 or from web site www.iiligri.mh.gov/DIR microa ⁇ ay or by other methods well known in the art.
• SCNC1 SCNC
• SCNC2 SCNC2
• Cy5- or Cy3-dUTP is replaced with SCNCl-dUTP and SCNC2-dUTP, respectively.
• the dried slide is read on a seamier optimized for SCNCs. It is anticipated that each distinctly labeled cDNA will be easily distinguished.
• Oligonucleotide microarray chips can purchased from Operon
• RNA and sample labeling by reverse transcription are performed using method described in Khan et al. (1999) Biochim. Biophys. Acta 1423:17- 28, from web site www.nhgri.nih. gov/DIR microa ⁇ av. or using methods known to those of skill in the art.
• preparation of cDNA and hybridization can be performed using protocols described in web site www.pangloss.com seidel/Protocols.
• One cDNA is labeled with biotin and the other with Cy3 or Cy5.
• Cy5- or Cy3-dUTP is replaced with biotin- 16-dUTP (Roche Molecular Biochemicals, Indianapolis, IN).
• the hybridization buffer contains the labeled cDNAs in 4X SSC and 1 mg/ml poly(dA) (Pharmacia) and 0.2 mg /ml yeast tRNA (Sigma).
• the probe mixture is denatured at 98 °C for 2 minutes, cooled to 45 °C and a small volume of 10% SDS solution is added to a final concentration of 0.2% SDS.
• the volume of 15-30 ⁇ L depending on the size of the a ⁇ ay is applied onto microa ⁇ ay area and the microa ⁇ ay area is covered with a glass cover-slip. The covered microa ⁇ ay is placed in a humidified chamber and incubated overnight at 65 °C.
• the slide is sequentially rinsed in IX SSC with 0.03 % SDS, 0.2X SSC and 0.05X SSC.
• the slide is dried by spinning in a centrifuge with horizontal rotor at 500 ⁇ m for 5 minutes.
• the slide is incubated in blocking solution 4X SSC, 0.1% Tween 20, 1% BSA for 30-60 minutes at room temperature, rinsed in 0.05X SSC, and dried by spinning in centrifuge.
• SCNC- streptavidin is added to 6X SSPE, 1% BSA, 10 mM MgCl 2 to a final concentration of 25 nM.
• a coverslip is applied over the a ⁇ ay area and the cover a ⁇ ay is incubated in a humidified container for 1 hour at room temperature.
• the slide is rinsed in IX SSPE followed by 0.06X SSPE and dried by spinning in centrifuge at 500 ⁇ m for 5 minutes.
• the microarray is read in a scanner optimized for SCNCs and Cy dyes.
• RNA and sample labeling by reverse transcription are performed using method described in Khan et al. (1999) Biochim. Biophys. Acta 1423:17- 28 or from web site www.nhgri.mh. gov/DIR/microanay .
• hybridization can be performed using protocols described in web site www.pangloss.com/seidel/Protocols.
• One cDNA is labeled with biotin and the other with a hapten such as digoxygenin, fluorescein, estradiol.
• Cy5- or Cy3-dUTP is replaced with biotin- 16-dUTP (Roche Molecular Biochemicals, Indianapolis, IN) and hapten-dUTP (e.g., fluorescein- 12-dUTP, DIG 11-dUTP, estradiol- 15-dUTP).
• the hybridization buffer contains the labeled cDNAs in 4X SSC and 1 mg/ml poly(dA) (Pharmacia), 0.2 mg /ml yeast tRNA (Sigma), the probe mixture is denatured at 98 °C for 2 minutes, cool to 45 °C and a small volume of 10% SDS solution is added to a final concentration of 0.2% SDS.
• the volume 15-30 ⁇ L depending on the size of the a ⁇ ay is applied on microa ⁇ ay, a cover slip is placed over the array area, and the covered a ⁇ ay is placed in a humidified chamber and incubated overnight at 65 °C.
• the slide is sequentially rinsed in IX SSC with 0.03 % SDS, 0.2X SSC and 0.05X SSC.
• the slide is dried by spinning in centrifuge with horizontal rotor for microplates at 500 ⁇ m for 5 minutes.
• the slide is incubated in blocking solution 4X SSC, 0.1% Tween 20, 1% BSA for 30-60 minutes at room temperature, rinsed in 0.05X SSC, and dried by spinning in centrifuge.
• SCNC-streptavidin and SCNC-anti-hapten is added to 6X SSPE, 1% BSA, 10 mM MgCl 2 to a final concentration of 25 nM each.
• 40-80 ⁇ L of the mixture is applied on the a ⁇ ay area, a cover slip is applied over the a ⁇ ay area and the covered a ⁇ ay is incubated in a humidified container for 1 hour at room temperature.
• the slide is rinsed in IX SSPE followed by 0.06X SSPE and dried by spinning in centrifuge at 500 ⁇ m for 5 minutes.
• the microa ⁇ ay is read on a scanner optimized for SCNCs.
• RNA and sample labeling by reverse transcription are performed by using method as described in Khan et al. (1999) Biochim. Biophys. Acta 1423:17-28 or from web site www.nhgri ,nih. gov/DIR microa ⁇ ay .
• hybridization can be performed using protocols described in web site www.pangloss.com/seidel/Protocols.
• SCNC1 and SCNC2 Cy5- or Cy3-dUTP is replaced with SCNCl-dUTP and SCNC2-dUTP.
• the hybridization buffer contains the labeled cDNAs in 4X SSC and 1 mg/ml poly(dA) (Pharmacia), yeast tRNA 0.2 mg /ml, the probe mixture is denatured at 98 °C for 2 minutes, cooled to 45 °C and a small volume of 10% SDS solution is added to a final concentration of 0.2% SDS.
• the volume of hybridization mixes various from 15- 30 ⁇ L depending on the size of the a ⁇ ay. The mixture is applied on microa ⁇ ay, a cover slip is applied over the a ⁇ ay area, the covered microa ⁇ ay is placed in a humidified chamber and incubated overnight at 65 °C.
• the slide is sequentially rinsed in IX SSC with 0.03% SDS, 0.2X SSC and 0.05X SSC.
• the slide is dried by spinning in centrifuge with horizontal rotor for microplates at 500 ⁇ m for 5 minutes.
• the microa ⁇ ay is scanned on a scanner optimized for SCNCs.
• Oligonucleotide microa ⁇ ays were purchased from Operon Technologies, Inc.
• test a ⁇ ays contained forty spots each, ten spots from each of the four 70 mers selected from Caspase 9-Genbank U56390, Laminin gamma 3 chain precusor, LAMC3- Genbank AF041835, Alpha-rubulin-Genbank K00558 and Ribosomal protein S9- Genbank Ul 4971. 50-mer complementary oligonucleotides biotinylated at the 3' end were made from each of the four 70 mers.
• the hybridization buffer contained the biotin-labeled 50 mer complementary sequences, 4 mg/ml herring sperm DNA as carrier in 4X SSC and 1 mg/ml poly(dA) (Pha ⁇ nacia), 0.2 mg /ml yeast tRNA (Sigma).
• the probe mixture was denatured at 98 °C for 2 minutes, cooled to 45 °C and a small volume of 10% SDS solution is added to a final concentration of 0.2% SDS.
• the volume of 15-30 ⁇ L depending on the size of the array was applied on the microa ⁇ ay area and a cover slip was applied over the microa ⁇ ay area.
• the covered microa ⁇ ay slide was placed in a humidified chamber and incubated overnight at 65 °C.
• the slide was sequentially rinsed in IX SSC with 0.03% SDS, 0.2X SSC and 0.05X SSC.
• the slide was dried by spinning in centrifuge with horizontal rotor at 500 ⁇ m for 5 minutes.
• the slide was incubated in blocking solution 4X SSC, 0.1% Tween 20, 1% BSA for 30-60 minutes at room temperature, rinsed in 0.05X SSC, and dried by spinning in centrifuge.
• SCNC-streptavidin is added to 6X SSPE, 1% BSA, 10 mM MgCl 2 to a final concentration of 25 nM.
• a ⁇ ay 40-80 ⁇ L of the SCNC-streptavidin was applied on the a ⁇ ay area and the a ⁇ ay area was covered with a glass coverslip.
• the cover a ⁇ ay slide was incubated in a humidified container for 1 hour at room temperature.
• the slide was rinsed in IX SSPE followed by 0.06X SSPE and dried by spinning in centrifuge at 500 rpm for 5 minutes.
• the microa ⁇ ay was scanned in a scanner optimized for SCNCs.
• a protein anay was prepared to intenogate protein species on a spatially addressed a ⁇ ay.
• Protein generated from any source e.g., a recombinant expression system, a differentially treated cell supernatants, or the like
• the spot size can vary from micrometer to millimeter diameter dependent on the assay substrate.
• 1 ⁇ L of rabbit IgG or mouse IgG was spotted onto nitrocellulose and allowed to dry; 50 spots of each different IgG dilution were addressed in a 5 x 10 a ⁇ ay.
• nitrocellulose was then blocked by incubation in phosphate- buffered saline (PBS)/1% bovine serum albumin (BSA) for 30 minutes at room temperature.
• PBS phosphate- buffered saline
• BSA bovine serum albumin
• a 1 ⁇ g/ml solution of biotinylated anti-rabbit IgG (Vector) was then applied for 30 minutes and the membrane subsequently washed in excess PBS.
• the a ⁇ ay was then exposed to 5 mis of a 25 nM solution of streptavidin-conjugated 630 nm emitting semiconductor nanocrystals in PBS/1% BSA for 30 minutes at room temperature.
• the membrane was then washed in excess PBS and the luminescence from the semiconductor nanocrystals was detected using an ultraviolet transilluminator (Stratagene Eagle Eye®) and a microa ⁇ ay scanner set to excite at 488 nm with an argon ion laser.
• an ultraviolet transilluminator Stratagene Eagle Eye®
• a microa ⁇ ay scanner set to excite at 488 nm with an argon ion laser.
• tissue samples can be simultaneously analyzed with semiconductor nanocrystal-labeled ligands. This, coupled with a spatial a ⁇ aying of tissue samples in a defined area, allows a further increase in the throughput of analyzing cellular markers.
• Small sections of tissue can be immobilized on a microscope slides or some other support as is well known in the art.
• the tissue source can be derived from a living organism, from a population of cultured cells treated in various ways, or the like.
• a specific intracellular antigen has been detected on a tissue section attached to a microscope slide.
• the tissue section was mouse stomach and kidney and was purchased from InovaDX (San Diego, CA).
• the goal of this Example was to detect the presence or absence of auto-immune markers, antinuclear antibodies (ANA), that recognize nuclear antigens.
• ANA antinuclear antibodies
• the anti-nuclear antibodies can be specifically detected using a biotinylated anti-human antibody followed by a binding thereto of a streptavidin-conjugated semiconductor nanocrystal.
• the tissue section was incubated for one hour with a positive control containing ANA (InovaDX) or with a human serum sample diluted in phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA).
• a negative control sample was also provided by InovaDX and this is also incubated with a section to provide background or non-specific binding information.
• the section was then washed by repeated immersion in PBS.
• the section was then incubated with 3 ⁇ g/ml biotinylated anti-human antibody (Vector) for a further 30 minutes at room temperature. The slide was washed in PBS.
• nuclei were clearly observed as brightly stained whereas the cytosol and surrounding tissue were not stained. No nuclei were observed in the negative control section.
• results were quantified by counting analyte molecules in a defined area of the assay surface (a circular region of about 60 ⁇ m in diameter defined by the illumination pattern of our single molecule microscope).
• the results were linear with concentration of biotinylated rabbit IgG and the sensitivity extended to densities of about 0.001 molecules/ ⁇ m 2 .

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