Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-05
2001-08-14
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C250S307000
Reexamination Certificate
active
06274323
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the detection of analytes in a sample. In particular, the invention relates to assays that use semiconductor nanocrystals as a detectable label. The invention further relates to assays in which multiple analytes can be detected simultaneously by using more than one semiconductor nanocrystal as a detectable label, each of which emits at a distinct wavelength.
BACKGROUND OF THE INVENTION
A variety of chemical and biological assays exist to identify an analyte of interest in a given sample. For example, immunassays, such as enzyme-linked immunosorbent assays (ELISAs) are used in numerous diagnostic, research and screening applications. In its most common form, an ELISA detects the presence and/or concentration of an analyte in a sample using an antibody which specifically recognizes the analyte. An enzyme label, capable of providing a detectable signal, is conjugated to the antibody. The analyte is either immobilized directly onto a solid support (direct-capture ELISA) or is bound to a different specific antibody which itself is immobilized on a solid support. The presence of the immobilized analyte is detected by binding to it the detectably labeled antibody. A variety of different ELISA formats have been described. See, e.g., U.S. Pat. No. 4,011,308 to Giaever, U.S. Pat. No. 4,722,890 to Sanders et al., Re. 032696 to Schuurs et al., U.S. Pat. No. 4,016,043 to Schuurs et al., U.S. Pat. No. 3,876,504 to Koffler, U.S. Pat. No. 3,770,380 to Smith, and U.S. Pat. No. 4,372,745 to Mandle et al.
Another technique for detecting biological compounds is fluorescence in situ hybridization (FISH). Swiger et al. (1996)
Environ. Mol. Mutagen
. 27:245-254; Raap (1998)
Mut. Res
. 400:287-298; Nath et al. (1997)
Biotechnic. Histol
. 73:6-22. FISH allows detection of a predetermined target oligonucleotide, e.g., DNA or RNA, within a cellular or tissue preparation by, for example, microscopic visualization. Thus, FISH is an important tool in the fields of, for example, molecular cytogenetics, pathology and immunology in both clinical and research laboratories.
This method involves the fluorescent tagging of an oligonucleotide probe to detect a specific complementary DNA or RNA sequence. Specifically, FISH involves incubating an oligonucleotide probe comprising an oligonucleotide that is complementary to at least a portion of the target oligonucleotide with a cellular or tissue preparation containing or suspected of containing the target oligonucleotide. A detectable label, e.g., a fluorescent dye molecule, is bound to the oligonucleotide probe. A fluorescence signal generated at the site of hybridization is typically visualized using an epi fluorescence microscope. An alternative approach is to use an oligonucleotide probe conjugated with an antigen such as biotin or digoxygenin and a fluorescently tagged antibody directed toward that antigen to visualize the hybridization of the probe to its DNA target. A variety of FISH formats are known in the art. See, e.g., Dewald et al. (1993)
Bone Marrow Transplantation
12:149-154; Ward et al. (1993)
Am. J. Hum. Genet
. 52:854-865; Jalal et al. (1998)
Mayo Clin. Proc
. 73:132-137; Zahed et al. (1992)
Prenat. Diagn
. 12:483-493; Kitadai et al. (1995)
Clin. Cancer Res
. 1:1095-1102; Neuhaus et al. (1999)
Human Pathol
. 30:81-86; Hack et al., eds., (1980)
Association of Cytogenetic Technologists Cytogenetics Laboratory Manual
. (Association of Cytogenetic Technologists, San Francisco, Calif.); Buno et al. (1998)
Blood
92:2315-2321; Patterson et al. (1993)
Science
260:976-979; Patterson et al. (1998)
Cytometry
31:265-274; Borzi et al. (1996)
J. Immunol. Meth
. 193:167-176; Wachtel et al. (1998)
Prenat. Diagn
. 18:455-463; Bianchi (1998)
J. Perinat. Med
. 26:175-185; and Munne (1998)
Mol. Hum. Reprod
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FISH provides a powerful tool for the chromosomal localization of genes whose sequences are partially or fully known. Other applications of FISH include in situ localization of mRNA in tissues sample and localization of nongenetic DNA sequences such as telomeres.
Signal amplification is yet another method for sensitive detection of nucleic acids and other receptor/ligand interactions. Direct detection of a target nucleic acid is possible by hybridization of a complementary nucleic acid probe to the target. Detection of the complex can be achieved by numerous means, e.g., a labeled probe or a reagent dye that specifically attaches to the target/probe complex. Such “direct” detection systems are often not sensitive enough to detect a target nucleic acid in a biological sample. One method for overcoming this limitation is to employ signal amplification. Signal amplification can be done, for example, by indirectly binding multiple signal-generating molecules to an analyte through a molecule which is (1) complementary to the analyte and (2) contains multiple signal-generating molecule binding sites and which signalgenerating molecules (i) contain a detectable label, (ii) bind to or otherwise activate a label or (iii) contain sites for binding additional layers of molecules which may in turn facilitate generation of a detectable signal. Thus, rather than a single signal-generating label associated with the target molecule, signal amplification results in the association of multiple signal-generating labels associated with the target molecule and, therefore, enhanced assay sensitivity.
Nucleic acid hybridization assays are described in, for example, U.S. Pat. No. 5,681,697 to Urdea et al., U.S. Pat. No. 5,124,246 to Urdea et al., U.S. Pat. No. 4,868,105 to Urdea et al., and European Patent Publication No. 70.685, inventors Heller et al.
There are many assays designed to obtain the sequence of a DNA sample. Each of these methods shares some or all of a set of common features. These features include: sequence specificity derived from complementary oligonucleotide hybridization or annealing; a solid support or solid phase which allows separation of specifically bound assay reagents; and a label which is used for detecting the presence or absence of the specific, intended assay interaction. Examples of assays designed to detect the sequence of a DNA sample can be found in U.S. Pat. No. 5,888,731 to Yager et al., U.S. Pat. No. 5,830,711 to Barany et al., U.S. Pat. No. 5,800,994 to Martinelli et al., U.S. Pat. No. 5,792,607 to Backman et al., U.S. Pat. No. 5,716,784 to Di Cesare, U.S. Pat. No. 5,578,458 to Caskey et al., U.S. Pat. No. 5,494,810 to Barany et al., U.S. Pat. No. 4,925,785 to Wang et al., U.S. Pat. No. 4,9898,617 to Landegren et al.,
Chemical compounds are typically evaluated for potential therapeutic utility by assaying their ability to affect, for example, enzyme activity, ligand-receptor interactions, protein-protein interactions, or the like. Evaluating the effect of each individual candidate compound on a variety of systems can be tedious and time-consuming. Accordingly, protocols have been developed to evaluate rapidly multiple candidate compounds in a particular system and/or a candidate compound in a plurality of systems. Such protocols for evaluating candidate compounds have been referred to as high throughput screening (HTS).
In one typical protocol, HTS involves the dispersal of a candidate compound into a well of a multiwell cluster plate, for example, a 96-well or higher format plate, e.g., a 384-, 864-, or 1536-well plate. The effect of the compound is evaluated on the system in which it is being tested. The “throughput” of this technique, i.e., the combination of the number of candidate compounds that can be screened and the number of systems against which candidate compounds can be screened, is limited by a number of factors, including, but not limited to: only one assay can be performed per well; if conventional dye molecules are used to monitor the effect of the candidate compound, multiple excitation sources are required if multiple dye molecules are used; and as the well size becomes small (e.g., the 1536-well plate can accept about 5 &mgr;l of to
Bruchez Marcel P.
Daniels R. Hugh
Empedocles Stephen A.
Phillips Vince E.
Wong Edith Y.
Horlick Kenneth R.
Quantum Dot Corporation
Robins & Pasternak LLP
Strzelecka Teresa
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