Massachusetts Institute of Technology
`Ex. 2001
`Part 3 of 4
`
`
`
`

`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`lntemalional Bureau
`/£3RNA'l'IONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`/ernatlonal Patent Classification 7 =
`(11) International Publication Number:
`WO 00/17642
`I§01N 33/53
`‘
`(43) International Publication Date:
`30 March 2000 (30.03.00)
`
`(81) Designated Slates: AE. AL, AM. AT. AU. AZ, BA, BB. BG.
`BR. BY. CA, CH, CN. CU. CZ, DE. DK, EE. ES. FI. GB.
`GD. GE. GH. GM. HR. HU. ID. IL. IN. IS. JP. KE. KG.
`KP. KR. KZ. LC. LK. LR. LS. LT. LU. LV. MD. MG. MK,
`MN, MW. MX. NO. NZ, PL. PT, RO, RU, SD, SE, SG, SI.
`SK, SL. TJ, TM, ‘IR, 1'I'. UA, UG, UZ. VN. YU. ZA. ZW.
`ARIPO patent (GH, GM. KE. LS, MW. SI). SL. SZ, TZ,
`UG. ZW), Eurasian patent (AM, AZ. BY. KG. KZ. MD.
`RU, TJ. TM). European patent (AT. BE. CH. CY. DE. DK,
`ES. FI. FR. GB. GR. IE. IT. LU. MC. NL. PT, SE). OAPI
`patent (BF. BJ. CF. CG. CI, CM. GA. GN, GW, ML, MR,
`NE. SN, TD. TG).
`
`Published
`Without international search report and to be republished
`upon receipt of that report.
`
`/) International Application Number:
`
`PCT/US99/2lSS2
`
`.1’
`.22) International Filing Date:
`
`17 September I999 (I7.09.99)
`
`(30) Priority Data:
`09/IS6.863
`60/I01.046
`60/ l00.947
`09/I60.4S4
`09/l60.458
`09/397.436
`09/397,432
`09I‘.'i97.428
`
`_
`
`I8 September I998 (I8.09.98)
`I8 September I998 (l8.09.98)
`I8 September I993 ( 18.09.98)
`24 September 1993 (2409.98)
`24 September I998 (2409.98)
`I7 September 1999 ( 17.09.99)
`I7 September I999 (I7.09.99)
`I7 September I999 (I7.09.99)
`
`(71) Applicant: MASSACHUSETTS INSTITUTE OF TECHNOL-
`OGY [US/US]; 77 Massachusetts Avenue. Cambridge. MA
`02l42 (US).
`
`(72) Inventors: BAWENDI, Moungi. Gs. Apartment 28, 285
`Beacon Street. Boston. MA 02I 16 (US). MIKULEC,
`Frederick. V.; Apartment 2, 96 Willow Avenue, Somervillc,
`MA 02l44 (US). SUNDAR. Viltram, C.; 49 Gorham Street,
`Somerville. MA 02144 (US).
`
`(74) Agent: PRAHL, Eric. L.; Fish & Richardson. 225 Franklin
`Street. Boston. MA 021 I0—2804 (US).
`
`(54) Title: BIOLOGICAL APPLICATIONS OF SEMICONDUCTOR NANOCRYSTALS
`
`_
`Single-Quantum Dot Labeled Immunoassay
`
`(57) Abstract
`
`The present invention provides a com-
`position comprising fluorescent semiconduc-
`tor nanocrystnls associated to a compound,
`wherein the nnnocrystals have a characteris-
`tic spectral emission. wherein said spectral
`emission is tunable to a desired wavelength
`by controlling the size of the nanocryslal. and
`wherein said emission provides information
`about a biological state or event.
`
`MIT901_2001-1067
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`r the PCT.
`
`Codes used to identify Slates pany to the PC? on the from pages
`LS
`ES
`Spain
`Albania
`LT
`Finland
`FI
`l.U
`Armenia
`France
`FR
`Austria
`LV
`Gabon
`GA
`Auslralin
`MC
`GB
`Uniied Kingdom
`MD
`Aurbaijan
`GE
`Georgia
`Bosnia and Hzrugovina
`MG
`Ghana
`GH
`Barbados
`MK
`Guinea
`GN
`Belgium
`Greece
`GR
`Burkina Faso
`HU
`Hungary
`Bulgaria
`IE
`Ireland
`Benin
`Israel
`ll.
`Bnuil
`Iceland
`IS
`Belarus
`IT
`lxaly
`Canada
`JP
`Japan
`Cenxral African Republic
`KE
`Kenya
`Congo
`KG
`Kyrgyulan
`Swilzevlarld
`KP
`Democratic People's
`CM! d'lvuim
`Republic of Korea
`Cameroon
`Republic of Korea
`Chinn
`Kuahian
`Cuba
`Saint Lucia
`Cueh Republic
`Liacmenslein
`Germany
`Sll Lanka
`Denmari
`Liberia
`Blmin
`
`FOR THE PURPOSES OF INFORMATION ONLY
`of pamphlets publishing inlemalional applications unde
`SI
`Slovenia
`Lesotho
`Slovakia
`SK
`Liihuania
`SN
`Senegal
`Luxembourg
`Swaziland
`S'L
`Latvia
`TD
`Chad
`Monaco
`TG
`Togo
`Republic of Moldova
`TJ
`Tajikistan
`Madagascar
`TM
`Turkmenistan
`The former Yugoslav
`TR
`'|\:rltey
`Republic of Maud.-mia
`Tl‘
`Trinidad and Tobago
`Mali
`UA
`Ukraine
`Mongolia
`U G
`Uganda
`Mauritania
`US
`United Slam: of Anleiicu
`Malawi
`Uzoekixlan
`UZ
`Mexico
`Viel Nam
`VN
`Niger
`YU
`Yugoslavia
`Nelherlandx
`Zimbabwe
`7.W
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Fcderaaion
`Sudan
`Sweden
`Singapore
`
`ML
`MN
`MR
`MW
`MX
`NB
`NI.
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`S6
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`KR
`K7.
`LC
`Ll
`LK
`LR
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`9
`
`BIOLOGICAL APPLICATIONS OF
`SEMICONDUCTOR NANOCRYSTALS
`
`This invention was made with U.S. government supportunder Contract Number
`94-00334 awarded by the National Science Foundation. The U.S. government has certain
`
`rights in the invention
`
`This application is related to the following commonly owned applications: U.S.
`' applieation Serial No. 09/160,458 entitled “Inventory Conuol," by Bawendi et al.filed
`September 18, 1998; and US. application Serial No. 09/156,863. entitled “Water-Soluble
`Luminescent Nanocrystals," by Bawendi et al., filed on September 18, 1998. This
`application claims priority under 35 U.S.C. ll9(e) to the provisional U.S. application
`Serial No. 60/100,947 entitled “Detection of Compounds and Interactions in Biological
`Systems Using Quantum Dots," by Bawendi et al., filed September 18, 1998. This
`application is a continuation-in-part of U.S. application Serial No. 09/160,454, filed
`
`September 24, 1998.
`
`Field of the Invention
`
`This invention relates generally to a compositions for use in biological applications.
`
`More specifically, the invention relates to compositions comprising fluorescent
`semiconductor nanocrystals associated with compounds for use in biological applications,
`such as affinity molecules capable of interacting specifically with biological targets, and to
`
`methods of using such compounds.
`
`Background of the Invention
`
`Traditional methods for detecting biological compounds in vivo and in vitro rely on
`
`the use of radioactive markers. For example, these methods commonly use radiolabeled
`probes such as nucleic acids labeled with "P or ”S and proteins labeled with ”S or "51 to
`detect biological molecules. These labels are effective because of the high degree of
`
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`sensitivity for the detection of radioactivity. However, many basic difficulties exist with‘
`the use of radioisotopes. Such problems include the need for specially trained personnel,
`general safety issues when working with radioactivity, inherently short half-lives with
`many commonly used isotopes. and disposal problems due to full landfills and
`governmental regulations. As a result, current efforts have shifted to utilizing
`non-radioactive methods of detecting biological compounds. These methods ofien consist
`
`of the use of fluorescent molecules as tags (e.g. fluorescein, ethidium, methyl coumarin,
`
`rhodarnine, and Texas red), or the use of chemiluminescence as a method of detection.
`Presently however, problems still exist when using these fluorescent and chemiluminescent
`markers. These problems include photobleaching, spectral separation, low fluorescence
`intensity, short half-lives, broad spectral linewidths, and non—gaussian asymmetric emission
`
`spectra having long tails.
`Fluorescence is the emission of light resulting from the absorption of radiation at
`
`one wavelength (excitation) followed by nearly immediate reradiation usually at a different
`wavelength (emission). Fluorescent dyes are frequently used as tags in biological systems.
`For example, compounds such as ethidium bromide, propidium iodide, Hoechst dyes (e.g., ‘
`
`benzoxanthene yellow and bixbenzimide
`((2'-[4-hydroxyphenyl}$-[4-methyl-1-piperazinyl]-2,5 ' -bi-1H-benzimidazol) and
`(2’-[4~ethoxyphenyl]-5-[4—methyl-l-piperazinyl]-2,5’-bi-1H-benzimidazol)). and DAPI
`(4,6-diamidino-2-phenylindole) interact with DNA and fluoresce to visualize DNA. Other
`biological components can be visualized by fluorescence using techniques such as
`immunofluorescence which utilizes antibodies labeled with a fluorescent tag and directed
`
`at a particular cellular target. For. example, monoclonal or polyclonal antibodies tagged
`with fluorescein or rhodamine can be directed to a desired cellular target and observed by
`
`fluorescence microscopy. An alternate method uses secondary antibodies that are tagged
`
`with a fluorescent marker and directed to the primary antibodies to visualize the target.
`
`Another application of fluorescent markers to detect biological compounds is
`fluorescence in situ hybridization (FISH). Swiger et al. (1996) Environ. M01. Muragen.
`
`113245-254; Raap (1998) Mat. Res. 10_Q:287—298; Nath et al. (1997) Biotechnic. Histol.
`
`1_3_:6-22. This method involves the fluorescent tagging of an oligonucleotide probe to
`detect a specific complementary DNA or RNA sequence. An alternative approach is to
`
`use an oligonucleotide probe conjugated with an antigen such as biotin or digoxygenin and
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`a fluorescently tagged antibody directedgtoward that antigen to visualize the hybridization
`of the probe to its DNA target. FISH is 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 samples and localization of non-genetic
`
`DNA sequences such as telomeres. A variety of FISH fonnats are known in the art.
`Dewald et al. (1993) Bone Marrow Transplantation _1_2_:149-154; Ward et al. (1993) Am. J.
`Hum. Genet. £354-865; Jalal et al. (1998) Mayo Clin. Proc. 1§_:l32-137; Zahed et al.
`(1992) Prenat. Diagn. _1g:483-493; Kitadai et al. (1995) Clin. Cancer Res. 1:l095-1102;
`Neuhaus et al. (1999) Human Pathal. 33:81-86; Hack et al., eds., (1980) Association of
`Cy_togenetic Technologists Cytogenetics Laboratory Manual. (Association of Cytogenetic
`Technologists, San Francisco, CA); Buno et al. (1998) Blood 912315-2321; Patterson et
`al. (1993) Science _2£0_:976-979; Patterson et al. (1998) Cytometry Q2265-274; Borzi et al.
`(1996) J. Immunol. Meth. Q3167-176; Wachtel et al. (1998) Prenat. Diagn. 1_8;:455-463;
`Bianchi (1998) J. Perinat. Med. _2_6_:l75-185; and Munne (1998) Mol. Hum. Reprod.
`
`$863-$170.
`Fluorescent dyes also have applications in non-cellular biological systems. For
`example, the advent of fluorescently-labeled nucleotides has facilitated the development of
`new methods of high-throughput DNA sequencing and DNA fragment analysis__(ABI
`system; Perkin-Elmer, Norwalk, CT). DNA sequencing reactions that once occupied four
`lanes on DNA sequencing gels can now be analyzed simultaneously in one lane. Briefly,
`four reactions are performed to detennine the positions of the four nucleotide bases in a
`DNA sequence. The DNA products of the four reactions are resolved by size using
`polyacrylamide gel electrophoresis. With singly radiolabeled (”P or ”S) DNA, each
`reaction is loaded into an individual lane. The resolved products result in a pattern of
`
`bands that indicate the identity of a base at each nucleotide position. This pattern across
`
`four lanes can be read like a simple code corresponding to the nucleotide base sequence of
`
`the DNA template. With fluorescent dideoxynucleotides, samples containing all four
`reactions can be loaded into a single lane. Resolution of the products is possible because
`
`each sample is marked with a different colored fluorescent dideoxynucleotide. For
`example, the adenine sequencing reaction can be marked with a green fluorescent tag and
`the other three reactions marked with different fluorescent colors. When all four reactions
`
`are analyzed in one lane on a DNA sequencing gel, the result is a ladder of bands
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`consisting of four different colors. Each fluorescent color corresponds to the identity of'a
`
`nucleotide base and can be easily analyzed by automated systems.
`
`There are chemical and physical limitations to the use of organic fluorescent dyes.
`
`One of these limitations is the variation of excitation wavelengths of different colored
`
`dyes. As a result, simultaneously using two or more fluorescent tags with different
`excitation wavelengths requires multiple excitation light sources. This requirement thus
`
`adds to the cost and complexity of methods utilizing multiple fluorescent dyes.
`
`Another drawback when using organic dyes is the deterioration of fluorescence
`
`intensity upon prolonged exposure to excitation light. This fading is called photobleaching
`and is dependent on the intensity of the excitation light and the duration of the
`illumination.
`In addition, conversion of the dye into a nonfluoresccnt species is
`
`irreversible. Furthermore, the degradation products of dyes are organic compounds which
`
`may interfere with biological processes being examined.
`Another drawback of organic dyes is the spectral overlap that exists from one dye
`
`to another. This is due in part to the relatively wide emission spectra of organic dyes and
`
`the overlap of the spectra near the tailing region. Few low molecular weight dyes have a
`combination of a large Stokes shifl, which is defined as the separation of the absorption
`and emission maxima, and high fluorescence output.
`In addition, low molecular weight
`dyes may be impractical for some applications because they do not provide a bright
`enough fluorescent signal. The ideal fluorescent label should fulfill many requirements.
`Among the desired qualities are the following: (i) high fluorescent intensity (for detection
`in small quantities), (ii) a separation of at least 50 nm between the absorption and
`fluorescing frequencies, (iii) solubility in water, (iv) ability to be readily linked to other
`molecules, (v) stability towards harsh conditions and high temperatures, (vi) a symmetric,
`nearly gaussian emission lineshape for easy deconvolution of multiple colors, and (vii)
`compatibility with automated analysis. At present, none of the conventional fluorescent
`labels satisfies all these requirements. Furthermore, the differences in the chemical
`properties of standard organic fluorescent dyes make multiple, parallel assays quite
`impractical since different chemical reactions may be involved for each dye used in the
`
`variety of applications of fluorescent labels.
`
`Thus, there is a need in the art for a fluorescent label that satisfies the
`
`above-described criteria for use in biological assay systems
`
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`Summary of the Invention
`
`The present invention provides a composition that can provide information about a
`
`biological state or event. The composition by way of example can detect the presence or
`amounts of a biological moiety, e.g., a biological target analyte; the structure, composition,
`
`and conformation of a biological moiety, e.g., a biological molecule or a portion or
`
`fragment thereof; the localization of a biological moiety, e.g., a biological target analyte in
`an environment; interactions of biological moieties, e.g., a biological molecule or a portion
`
`or fragment thereof; alterations in structures of biological compounds, e.g., a biological
`
`molecule or a portion or fiagnent thereof; and/or alterations in biological processes.
`
`The composition is comprised of a fluorescent semiconductor nanocrystal (also
`
`know as a Quantum Dot” particle) having a characteristic spectral emission, which is
`
`tunable to a desired energy by selection of the particle size, size distribution and
`
`composition of the semiconductor nanocrystal. The composition further comprises a
`
`compound associated with the semiconductor nanocrystal that has an affinity for a
`biological target. The composition interacts or associates with a biological target due to
`the affinity of the compound with the target. Location and nature of the association can
`
`be detected by monitoring the emission of the semiconductor nanocrystal.
`
`In operation, the composition is introduced into an environment containing a
`
`biological target and the composition associates with the target. The compositionztarget
`complex may be spectroscopically view or otherwise detected, for example, by irradiation
`of the complex with an excitation light source. The semiconductor nanocrystal emits a
`
`characteristic emission spectrum which can be observed and measured, for example,
`
`spectroscopically.
`
`As an advantage of the composition of the present invention, the emission spectra
`
`of a population of semiconductor nanocrystals have linewidths as narrow as 25-30 nm,
`
`depending on the size distribution heterogeniety of the sample population, and lineshapes
`
`that are symmetric, gaussian or nearly gaussian with an absence of a tailing region. The
`
`combination of tunability, narrow linewidths, and symmetric emission spectra without a
`
`tailing region provides for high resolution of multiply-sized nanocrystals, e.g., populations
`
`of monodisperse semiconductor nanocrystals having multiple distinct size distributions,
`
`within a system and enables researchers to examine simultaneously a variety of biological
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`moieties, e.g., target analytes, tagged with nanocrystals.
`
`In addition, the range of excitation wavelengths of the nanocrystals is broad and
`
`can be higher in energy than the emission wavelengths of all available semiconductor
`nanocrystals. Consequently, this allows the simultaneous excitation of all populations of
`semiconductor nanocrystals in a system having distinct emission spectra with a single light
`
`source, usually in the ultraviolet or blue region of the spectrum. Semiconductor
`
`nanocrystals are also more robust than conventional organic fluorescent dyes and are more
`resistant to photobleaching than the organic dyes. The robustness of the nanocrystal also
`alleviates the problem of contamination of the degradation products of the organic‘dyes‘ln
`the system being examined. Therefore, the present invention provides uniquely valuable
`
`tags for detection of biological molecules and the interactions they undergo.
`
`In one preferred embodiment, the composition comprises semiconductor
`nanocrystals associated with molecules that can physically interact with biological
`compounds. Without limiting the scope of the invention, molecules include ones that can
`bind to proteins, nucleic acids, cells, subcellular organelles, and other biological molecules.
`The compound used in the composition of the present invention preferably has an affinity
`for a biological target.
`In some preferred embodiments, the compound has a specific
`affinity for a biological target. The affinity may be based upon any inherent properties of
`the compound, such as without limitation, van der Waals attraction, hydrophilic attractions,
`ionic, covalent, electrostatic or magnetic attraction of the compound to a biological target.
`
`As used herein, “biological target" is meant any moiety, compound, cellular or sub—cellular
`
`component which is associated with biological functions. The biological targetincludes
`without limitation proteins, nucleic acids, cells, subcellular organelles and other biological
`moieties.
`
`In another preferred embodiment, the composition comprises semiconductor
`
`nanocrystals associated with proteins. Without limiting the scope of the invention, the
`
`proteins may be antibodies that are directed towards specific antigens, for example,
`biological antigens such as other proteins, nucleic acids, subcellular organelles, and small
`molecules that are conjugated to biological compounds. The proteins may also be proteins
`
`that interact specifically or non-specifically with other biological compounds.
`
`In another preferred embodiment, the composition comprises semiconductor
`
`nanocrystals associated with nucleic acids. Without limiting the scope of the invention,
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`the nucleic acids may be oligonucleotides or deoxyribooligonucleotides thatvhybridize to’
`nucleic acid polymers in vivo or in vitro. The nucleic acids may also be nucleotides,
`
`deoxynucleotides, dideoxynucleotides, or derivatives and combinations thereof that are
`
`used for the synthesis of DNA or RNA.
`
`In yet another preferred embodiment of the invention, a method of detecting
`
`biological compounds using semiconductor nanocrystals is provided.
`
`One aspect of the invention includes use of a semiconductor nanocrystal as a tag
`
`for at least one member of a biological binding pair. The tagging can be achieved by
`
`covalent, noncovalent, hydrophobic, hydrophilic, electrostatic or magnetic association, or
`
`by coordination through a metal complex. Preferably, the semiconductor nanocrystal is
`water-soluble.
`
`Another aspect of the invention includes use of a semiconductor nanocrystal,
`
`preferably a water-soluble semiconductor nanocrystal, for tagging a biological molecule or
`event.
`
`Yet another aspect of the invention includes a method of labelling a biological
`
`molecule or event with a fluorescent label, wherein said label is a semiconductor
`
`nanocrystal in which the emission spectrum of the fluorescence is dependent upon the
`
`nanocrystal size.
`
`Still another aspect of the invention includes a method of controlling the
`
`fluorescence emission spectrum of a fluorescent moiety in use in a biological system,
`
`comprising selecting a semiconductor nanocrystal having a desired fluorescence emission
`
`spectrum and using the selected nanocrystal as said fluorescent moiety.
`
`A further aspect of the invention includes use of a semiconductor nanocrystal as a
`
`fluorescent label in immunochemistiy, optionally in immunocytochemistry or in an
`
`immuno assay, in DNA sequence analysis, as a fluorescent label in fluorsecence resonance
`
`energy transfer in assessing the proximity of two or more biological compouds to each
`
`other, as a fluorescent label in flow cytometry or in a fluorescence activated cell sorter, as
`
`a fluorescnet label in a diagnostic method or as a fluorescent label in biological imaging.
`
`Yet a further aspect of the invention includes the aforementioned uses and methods
`
`in which two or more semiconductor nanocrystals, preferably up to 20 different-sized
`
`nanocrystals, are employed.
`
`These and other embodiments of the present invention will readily occur to those of
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`ordinary skill in the an in view of the disclosure herein.
`
`Brief Description of the Drawing
`
`Figure 1 is a pictorial depiction of the single-sized semiconductor nanocrystal
`
`preparation labeled immunoassay.
`
`Figure 2 is a pictorial depiction of the multicolored semiconductor nanocrystal
`labeled, parallel immunoassay.
`i'»
`
`Figure 3 is a pictorial depiction of the use of two differently colored nanocrystals
`
`or one color nanocrystal and one organic dye to detect proximity of compounds.
`
`In this
`
`example, two oligonucleotide probes are hybridized to DNA sequences in close proximity
`
`and detected by fluorescence resonance energy transfer
`
`Figure 4 is a pictorial depiction of the formation of water-soluble semiconductor
`
`nanocrystals by cap exchange
`
`Figure 5 and Figure 6 illustrate an outline of the reaction between biotin and
`
`hexane dithiol to form the biotin—hexane dithiol (Bl-IDT) derivative.
`
`Figure 7 is an outline of the reaction between biotin and a diarnine to form
`biotin-amine derivative.
`
`Figure 8 depicts the formation of the biotin—thiol-nanocrystal complex for a
`
`water-soluble nanocrystal.
`
`Figure 9 depicts the formation of the biotin-amine-nanocrystal complex where the
`
`amine is adsorbed to the outer layer of the nanocrystal.
`
`Figure 10 depicts the formation of the biotin-amine-nanocrystal complex where the
`
`amine is conjugated to the carboxylic acid group of the water-solubilizing layer.
`
`Detailed Description of the Invention
`
`Definitions and nomenclature:
`
`Before the present invention is disclosed and described in detail, it is to be
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`understood that this invention is not limited to specific assay formats, materials or
`
`reagents, as such may, of course, vary.
`
`It is also to be understood that the terminology
`
`used herein is for the purpose of describing particular embodiments only and is not
`
`intended to be limiting.
`
`It must be noted that, as used in the specification and the appended claims, the
`
`singular forms "a," "an" and "the" include plural referents unless the context clearly
`
`dictates otherwise. Thus, for example, reference to "a nanocrystal" includes more than one
`
`nanocrystal, reference to "a target analyte" includes more than one such analyte, and the
`like.
`‘~
`‘ *
`
`In this specification and in the claims which follow, reference will be made to a
`
`number of terms which shall be defined to have the following meanings:
`
`"Quantum dot” particles" are a semiconductor nanocrystal with size-dependent
`
`optical and electronic properties.
`
`In particular, the band gap energy of a semiconductor
`
`nanocrystal varies with the diameter of the crystal.
`
`"Semiconductor nanocrystal" includes, for example, inorganic crystallites between
`
`about 1 nm and about 1000 run in diameter, preferably between about 2 nm and about 50
`
`nm, more preferably about 5 nm to about 20 nm (such as about 6, 7, 8, 9, 10, ll,_ 12, 13,
`
`I4, 15, 16, I7, 18, 19, or 20 nm) that includes a “core" of one or more first semiconductor
`
`materials. and which may be surrounded by a “shell" of a second semiconductor material.
`
`A semiconductor nanocrystal core surrounded by a semiconductor shell is referred to as a
`
`“core/shell” semiconductor nanocrystal. The surrounding “shell" material will preferably
`
`have a bandgap greater than the bandgap of the core material and can be chosen so 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 Il—VI (ZnS, ZnSe,
`
`ZnTe, CdS, CdSe, CdTe, I-lgS, I-lgSe, HgTe, MgTe and the like) and IH—V (GaN, GaP,
`
`GaAs, GaSb, lnN, InP, In.As, InSb, AlAs, All’, AlSb, AIS, and the like) and IV (Ge, Si,
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`Pb and the like) materials, and an alloy thereof, or a mixture thereof.
`
`A semiconductor nanocrystal is, optionally, surrounded by a "coat" of an organic
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`capping agent. The organic capping agent may be any number of materials, but has an
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`affinity for the semiconductor nanocrystal surface.
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`In general, the capping agent can be an
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`isolated organic molecule, a polymer (or a monomer for a polymerization reaction), an
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`inorganic complex, and an extended crystalline structure. The coat is used to convey
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`-9.
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`MIT901_2001-1077
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`

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`W0 00/I7642
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`PCT/US99/21552
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`solubility, e.g., the ability to disperse a coated semiconductor nanocrystal homogeneously.
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`into a chosen solvent, functionality, binding properties, or the like.
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`In addition, the coat
`
`can be used to tailor the optical properties of the semiconductor nanocrystal.
`
`As used herein, the term "binding pair" refers first and second molecules that
`
`specifically bind to each other. "Specific binding" of the first member of the binding pair
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`to the second member of the binding pair in a sample is evidenced by the binding of the
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`first member to the second member, or vice versa, with greater affinity and specificity than
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`to other components in the sample. The binding between the members of the binding pair.
`
`is typically no‘n,-covalent. The terms "affinity molecule" and "target analyte" are used
`herein to refer to first and second members of a. binding pair, respectively.
`
`Exemplary binding pairs include any haptenic or antigenic compound in
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`combination with a corresponding antibody or binding portion or fragment thereof (e.g.,
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`digoxigeninarid anti-digoxigenin; mouse immunoglobulin and goat anti-mouse
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`immunoglobulin) and nonimmunological binding pairs (e.g., biotin-avidin,
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`biotin-strepavidin, hormone [e.g., thyroxine and cortisol]-hormone binding protein,
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`receptor-receptor agonist or antagonist (e.g., acetylcholine receptor-acetylcholine or an
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`analog thereof) IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,
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`enzyme-enzyme-inhibitor, and complementary polynucleotide pairs capable of forming
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`nucleic acid duplexes) and the like.
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`“Semiconductor nanocrystal conjugate" or “nanocrystal conjugate” includes, for
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`example, a semiconductor nanocrystal linked, through the coat, to a member of a "binding
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`pair" that will selectively bind to a detectable substance present in a sample, e.g.,
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`biological sample as defined herein. The first member of the binding pair linked to the
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`semiconductor nanocrystal can comprise any molecule, or portion of any molecule, that is
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`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.
`
`"Monodisperse particles" include a population of particles wherein at least 60% of
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`the particles in the population fall within a specified particle size range. A population of
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`monodispersed particles deviate less than 10% rrns (root-mean-square) in diameter and
`
`preferably less than 5% ms.
`"Quantum yield" yield is defined as the ratio of photons erriitted to that absorbed.
`The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid
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`-10-
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`MIT901_2001-1078
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`

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`wo 'oo/17642
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`PCT/US99I2l552
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`molecule" as used herein to include a polymeric form of nucleotides of any length, either
`
`tibonucleotides or deoxyribonucleotides. This term refers only to the primary structure of
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`the molecule. Thus, the term includes triple-, double- and single—stranded DNA, as well as
`
`It also includes modifications, such as by
`tn'ple-. double- and single-stranded RNA.
`methylation and/or by capping, and unmodified forms of the polynucleotide.More
`particularly, the terms "polynucleotide," "oligonucleotidc," "nucleic acid" and "nucleic acid
`molecule" 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-Vitals, lnc., 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. There is no intended distinction in
`length between the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic
`acid molecule," and these terms will be used interchangeably. These terms refer only to
`
`the primary structure of the molecule. Thus, these terms include, for example,
`
`3'-deoxy-2',5’—DNA, oligodeoxyribonucleotide N3’ PS’ 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 which are lcnown in the
`
`art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides
`with an analog, intemucleotide 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, etc.), and
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`with positively charged linkages (e.g., aminoalklyphosphoramidates,
`
`arninoalkylphosphotxiesters), those containing pendant moieties, such as, for example,
`
`proteins (including nucleases, toxins, antibodies, signal peptides, po1y—L-lysine, etc.), those
`with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals,
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`radioactive metals, boron, oxidative metals, etc.), those containing allcylators, those with
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`modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of
`
`the polynucleotide or oligonuclcotide.
`
`In particular, DNA is deoxyribonucleic _acid.
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`-11.
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`MIT901_2001-1079
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`

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`wo (Jun 7642
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`rcr/us99mss2
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`The terms "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 fiom a variety of
`
`sources. e.g., biological fluids or solids, food stuffs, environmental materials, etc., and may
`
`be prepared for the hybridization analysis by a variety of means, eg., proteinase K/SDS,
`
`_
`chaotropic salts, or the like.
`As used herein, the term "target nucleic acid region" or "target nucleotide
`
`sequence" includes a probe-hybridizing region contained within the target molecule. The
`
`term "target nucleic acid sequence" includes a sequence with which a probe will form a
`
`stable hybrid under desired conditions.
`
`As used herein, the term "nucleic acid probe" includes reference to a structure
`
`g 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
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`polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic
`
`nucleotide analogs.
`
`It will be appreciated that the hybridizing sequences need not have perfect
`complementarity to provide stable hyb