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WO2000027365A1 - Nanocristaux fonctionnalises et leur utilisation dans des systemes de detection - Google Patents

Nanocristaux fonctionnalises et leur utilisation dans des systemes de detection Download PDF

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Publication number
WO2000027365A1
WO2000027365A1 PCT/US1999/026487 US9926487W WO0027365A1 WO 2000027365 A1 WO2000027365 A1 WO 2000027365A1 US 9926487 W US9926487 W US 9926487W WO 0027365 A1 WO0027365 A1 WO 0027365A1
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WIPO (PCT)
Prior art keywords
acid
soluble
functionalized
water
substrate
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PCT/US1999/026487
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English (en)
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WO2000027365A8 (fr
WO2000027365A9 (fr
Inventor
Emilio Barbera-Guillem
Stephanie Castro
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Biocrystal Limited
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Priority claimed from US09/372,729 external-priority patent/US6114038A/en
Application filed by Biocrystal Limited filed Critical Biocrystal Limited
Priority to EP99965776A priority Critical patent/EP1128818A4/fr
Priority to AU21470/00A priority patent/AU2147000A/en
Publication of WO2000027365A1 publication Critical patent/WO2000027365A1/fr
Publication of WO2000027365A8 publication Critical patent/WO2000027365A8/fr
Publication of WO2000027365A9 publication Critical patent/WO2000027365A9/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/223—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material by irradiating the sample with X-rays or gamma-rays and by measuring X-ray fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2223/00—Investigating materials by wave or particle radiation
    • G01N2223/07—Investigating materials by wave or particle radiation secondary emission
    • G01N2223/076—X-ray fluorescence

Definitions

  • This invention relates to novel compositions comprising functionalized nanocrystals. More particularly, the present invention relates to. water-soluble nanocrystals which have a coat comprising a capping compound, and one or more additional compounds successively overlayered onto the capped nanocrystal . The present invention also relates to the use of the functionalized nanocrystals for providing a detectable signal in detection systems in which the nanocrystals are employed.
  • Nonisotopic detection systems have become a preferred mode in scientific research and clinical diagnostics for the detection of biomolecules using various assays including flow cytometry, nucleic acid hybridization, DNA sequencing, nucleic acid amplification, immunoassays, histochemistry, and functional assays involving living cells.
  • fluorescent organic molecules such as fluoroscein and phycoerythrin are used frequently in detection systems
  • there are disadvantages in using these molecules in combination For example, each type of fluorescent molecule typically requires excitation with photons of a different wavelength as compared to that required for another type of fluorescent molecule.
  • a single light source is used to provide a single excitation wavelength (in view of the spectral line width) , often there is insufficient spectral spacing between the emission optima of different fluorescent molecules to permit individual and quantitative detection without substantial spectral overlap.
  • nonisotopic detection systems typically are limited in sensitivity due to the finite number of nonisotopic molecules which can be used to label a biomolecule to be detected.
  • quantum dots Semiconductor nanocrystals
  • Examples of quantum dots are known in the art to have a core selected from the group consisting of CdSe, CdS, and CdTe (collectively referred to as "CdX”) .
  • CdX quantum dots have been passivated with an inorganic coating ("shell") uniformly deposited thereon.
  • the shell which is used to passivate the quantum dot is preferably comprised of YZ wherein Y is Cd or Zn, and Z is S, or Se .
  • Quantum dots having a CdX core and a YZ shell have been described in the art. However, the above described quantum dots, passivated using an inorganic shell, have only been soluble in organic, non-polar (or weakly polar) solvents.
  • the quantum dots are water- soluble.
  • Water-soluble is used herein to mean sufficiently soluble or suspendable in a aqueous-based solution, such as in water or water-based solutions or buffer solutions, including those used in biological or molecular detection systems as known by those skilled in the art.
  • CdX core/YZ shell quantum dots are over-coated with trialkylphosphine oxide, with the alkyl groups most commonly used being butyl and octyl .
  • One method to make the CdX core/YZ shell quantum dots water-soluble is to exchange this overcoating layer with a coating which will make the quantum dots water-soluble.
  • a mercaptocar- boxylic acid may be used to exchange with the trialkylphosphine oxide coat. Exchange of the coating group is accomplished by treating the water-insoluble quantum dots with a large excess of neat mercaptocarboxylic acid.
  • exchange of the coating group is accomplished by treating the water- insoluble quantum dots with a large excess of mercaptocarboxylic acid in CHC1 3 solution.
  • the thiol group of the new coating molecule forms Cd (or Zn) -S bonds, creating a coating which is not easily displaced in solution.
  • Another method to make the CdX core/YZ shell quantum dots water-soluble is by the formation of a coating of silica around the dots.
  • An extensively polymerized poly- silane shell imparts water solubility to nanocrystalline materials, as well as allowing further chemical modifications of the silica surface.
  • quantum dots which have been reported as water-soluble may have limited stability in an aqueous solution, particularly when exposed to air (oxygen) and/or light. More particularly, oxygen and light can cause the molecules comprising the coating to become oxidized, thereby forming disulfides which destabilize the attachment of the coating molecules to the shell. Thus, oxidation may cause the coating molecules to migrate away from the surface of the nanocrystals, thereby exposing the surface of the nanocrystals in resulting in "destabilized nanocrystals" .
  • Destabilized nanocrystals form aggregates when they interact together, and the formation of such aggregates eventually leads to irreversible flocculation of the nanocrystals (e.g. , see FIG. 1A) .
  • a semiconductor nanocrystal which (a) is water-soluble; (b) is functionalized to enhance stability in aqueous solutions; (c) is a class of semiconductor nanocrystals that may be excited with a single wavelength of light resulting in detectable luminescence emissions of high quantum yield and with discrete luminescence peaks; and (d) is functionalized so as to be both water-soluble, and able to bind ligands, molecules, or probes of various types for use in an aqueous-based environment.
  • the present invention provides a composition comprising functionalized nanocrystals for use in non- isotopic detection systems.
  • the composition comprises quantum dots (capped with a layer of a capping compound) that are water-soluble and functionalized by operably linking, in a successive manner, one or more additional compounds.
  • the one or more additional compounds form successive layers over the nanocrystal.
  • the functionalized nanocrystals comprise quantum dots capped with the capping compound, and comprise a coating (a plurality of molecules comprising) diaminocarboxylic acid which is operatively linked to the capping compound.
  • the functionalized nanocrystals may have a first layer comprising the capping compound, and a second layer comprising diaminocarboxylic acid; and may further comprise one or more successive layers including a layer of amino acid, a layer of affinity ligand, or multiple layers comprising a combination thereof.
  • the composition comprises a class of quantum dots that can be excited with a single wavelength of light resulting in a detectable luminescence emissions of high quantum yield and with discrete luminescence peaks.
  • the functionalized nanocrystals are further functionalized by binding an affinity ligand thereto.
  • the resultant functionalized nanocrystals are placed in contact with a sample being analyzed for the presence or absence of a substrate for which the affinity ligand has binding specificity.
  • FIG. 1A is a bar graph comparing the stability of capped quantum dots ("W-SN") to the stability of functionalized nanocrystals ("FN") under oxidizing conditions.
  • FIG. IB is a bar graph comparing the non-specific binding of capped quantum dots ("W-SN") to the non-specific binding of functionalized nanocrystals ("FN") .
  • FIG. 2 is a schematic illustrating chemically modifying a water-soluble quantum containing a layer of a capping compound to further comprise a layer of a diaminocarboxylic acid, and a layer of an affinity ligand (e.g., avidin) .
  • an affinity ligand e.g., avidin
  • FIG. 3 is a schematic illustrating chemically modifying a water soluble quantum dot containing a layer of a capping compound to further comprise a layer of a diaminocarboxylic acid, an additional layer of a diaminocarboxylic acid, and a layer of an affinity ligand.
  • substrate is meant, for the purposes of the specification and claims to refer to a molecule of an organic or inorganic nature, the presence and/or quantity of which is being tested for; and which contains a molecular component (domain or sequence or epitope or portion or chemical group or determinant) for which the affinity ligand has binding specificity.
  • the molecule may include, but is not limited to, a nucleic acid molecule, protein, glyco- protein, eukaryotic or prokaryotic cell, lipoprotein, peptide, carbohydrate, lipid, phospholipid, aminoglycans, chemical messenger, biological receptor, structural component, metabolic product, enzyme, antigen, drug, therapeutic, toxin, inorganic chemical, organic chemical, and the like.
  • the substrate may be in vivo, in vi tro, in si tu, or ex vivo .
  • a preferred substrate may be used to the exclusion of a substrate other than the preferred substrate.
  • affinity ligand is meant, for purposes of the specification and claims, to mean a molecule which has binding specificity and avidity for a molecular component of, or associated with, a substrate.
  • affinity ligands are known to those skilled in the art to include, but are not limited to, lectins or fragments (or derivatives) thereof which retain binding function; monoclonal antibodies ( "mAb” , including chimeric or genetically modified monoclonal antibodies (e.g., "humanized”)); peptides; aptamers; nucleic acid molecules (including, but not limited to, single stranded RNA or single-stranded DNA, or single- stranded nucleic acid hybrids) ; avidin, or streptavidin, or avidin derivatives; and the like.
  • the invention may be practiced using a preferred affinity ligand (e.g., a lectin) to the exclusion of affinity ligands other than the preferred affinity ligand.
  • a preferred affinity ligand e.g., a lectin
  • the term "monoclonal antibody” is also used herein, for purposes of the specification and claims, to include immunoreactive fragments or derivatives derived from a mAb molecule, which fragments or derivatives retain all or a portion of the binding function of the whole mAb molecule.
  • immunoreactive fragments or derivatives are known to those skilled in the art to include F(ab') 2 , Fab', Fab, Fv, scFV, Fd 1 and Fd fragments. Methods for producing the various fragments or derivatives from mAbs are well known in the art.
  • F(ab') 2 can be produced by pepsin digestion of the monoclonal antibody, and Fab' may be produced by reducing the disulfide bridges of F(ab') 2 fragments.
  • Fab fragments can be produced by papain digestion of the monoclonal antibody, whereas Fv can be prepared according to methods described in U.S. Patent No. 4,642,334.
  • Single chain antibodies can be produced as described in U.S. Patent No. 4,946,778.
  • the construction of chimeric antibodies is now a straightforward procedure in which the chi- meric antibody is made by joining the murine variable region to a human constant region.
  • "humanized” antibodies may be made by joining the hypervariable regions of the murine monoclonal antibody to a constant region and portions of variable region (light chain and heavy chain) sequences of human immunoglobulins using one of several techniques known in the art . Methods for making a chimeric non-human/human mAb in general are known in the art (see, e.g., U.S. Patent No. 5,736,137). Aptamers can be made using methods described in U.S. Patent No. 5,789,157.
  • Lectins and fragments thereof, are commercially available. Lectins are known to those skilled in the art to include, but are not limited to, one or more of Aleuria aurantia leetin, Amaranthus caudatus lectin, Concanavalin A, Datura stramonium lectin, Dolichos biflorus agglutinin, soybean agglutinin, Erythrina cristagalli lectin, Galanthus nivalis lectin, Griffonia simplicifolia lectins, Jacalin, Macckia amurensis lectins, Maclura pomifera agglutinin, Phaeolepiota aurea lectins 1 and 2, Phaseolus vulgaris lectins, Ricin A, Moluccella laevis lectin, peanut agglutinin, Bauhinia purpurea agglutinin, Ricinus communis a
  • a preferred affinity ligand may be used to the exclusion of an affinity ligand other than the preferred affinity ligand.
  • operably linked is meant, for purposes of the specification and claims to refer to fusion or bond or an association of sufficient stability to withstand conditions encountered in a method of detection, between a combination of different molecules such as, but not limited to, between the quantum dot and a capping compound, between a capping compound and a diaminocarboxylic acid, between a diaminocarboxylic acid and a diaminocarboxylic acid, between a diaminocarboxylic acid and an affinity ligand, between a diaminocarboxylic acid and an amino acid, and between an amino acid and an affinity ligand, and a combination thereof.
  • Reactive functionalities include, but are not limited to, bifunctional reagents/linker molecules, biotin, avidin, free chemical groups (e.g., thiol , or carboxyl , hydroxyl, amino, amine, sulfo, etc.), and reactive chemical groups (reactive with free chemical groups) .
  • a preferred reactive functionality may be used to the exclusion of a reactive functionality other than the preferred reactive functionality.
  • linker is meant, for purposes of the specification and claims to refer to a compound or moiety that acts as a molecular bridge to operably link two different molecules, wherein one portion of the linker is operably linked to a first molecule, and wherein another portion of the linker is operably linked to a second molecule.
  • the two different molecules may be linked to the linker in a step-wise manner.
  • Linkers are known to those skilled in the art to include, but are not limited to, chemical chains, chemical compounds, carbohydrate chains, peptides, haptens, and the like.
  • the linkers may include, but are not limited to, homobifunctional linkers and heterobifunctional linkers.
  • Heterobifunctional linkers well known to those skilled in the art, contain one end having a first reactive functionality to specifically link a first molecule, and an opposite end having a second reactive functionality to specifically link to a second molecule.
  • the linker may have: a carboxyl group to form a bond with the polynucleotide, and a carboxyl group to form a bond with the diaminocarboxylic acid.
  • Heterobifunctional photo-reactive linkers e.g., phenylazides containing a cleavable disulfide bond
  • a sulfosuccinimidyl-2- (p-azido salicylamido) ethyl- 1, 3 ' -dithiopropionate contains a N-hydroxy-succinimidyl group reactive with primary amino groups, and the phenyl - azide (upon photolysis) reacts with any amino acids.
  • the linker may further comprise a protective group which blocks reactivity with a functional group on the linker which is used to react with and bind to a molecule to be linked.
  • a deprotection reaction may involve contacting the linker to one or more conditions and/or reagents which removes the protective group, thereby exposing the functional group to interact with the molecule to be linked.
  • deprotection can be achieved by various methods known in the art, including, but not limited to photolysis, acidolysis, hydrolysis, and the like.
  • the linker may vary in length and composition for opti- mizing such properties as flexibility, stability, and resistance to certain chemical and/or temperature parameters .
  • short linkers of sufficient flexibility include, but are not limited to, linkers having from 2 to 10 carbon atoms.
  • a preferred linker may be used to the exclusion of a linker other than the preferred linker.
  • diaminocarboxylic acid is meant, for purposes of the specification and claims to refer to an amino acid that has two free amine groups.
  • the amino acid may be a naturally occurring amino acid, a synthetic amino acid, a modified amino acid, an amino acid derivative, and an amino acid precursor (e.g., citrulline and ornithine are intermediates in the synthesis of arginine) .
  • the diaminocarboxylic acid contains neutral (uncharged) polar functional groups which can hydrogen bond with water, thereby making the diaminocarboxylic acid (and the quantum dot to which it is made a part of) relatively more soluble in aqueous solutions containing water than those with nonpolar functional groups.
  • Exemplary diaminocarboxylic acids include, but are not limited to, lysine, asparagine, glutamine, arginine, citrulline, ornithine, 5- hydroxylysine, djenkolic acid, ⁇ -cyanoalanine, and synthetic diaminocarboxylic acids such as 3 , 4-diaminobenzoic acid, 2 , 3-diaminopropionic acid, 2 , 4-diaminobutyric acid, 2,5- diaminopentanoic acid, and 2 , 6-diaminopimelic acid.
  • a preferred diaminocarboxylic acid may be used to the exclu- sion of a diaminocarboxylic acid other than the preferred diaminocarboxylic acid.
  • amino acid is meant, for purposes of the specification and claims to refer to a molecule that has at least one free amine group and at least one free carboxyl group.
  • the amino acid may have more than one free amine group, or more than one free carboxyl group, or may further comprise one or more free chemical reactive groups other than an amine or a carboxyl group (e.g., a hydroxyl, a sulfhydryl, etc.) .
  • the amino acid may be a naturally occurring amino acid, a synthetic amino acid, a modified amino acid, an amino acid derivative, and an amino acid precursor.
  • the amino acid may further be selected from the group consisting of a monoaminocarboxylic acid, and a diaminocarboxylic acid.
  • the monoaminocarboxylic acid contains one or more neutral (uncharged) polar functional groups which can hydrogen bond with water, thereby making the monoaminocarboxylic acid (and the quantum dot to which it is made a part of) relatively more soluble in aqueous solutions containing water than those with non-polar functional groups.
  • exemplary monoaminocarboxylic acids include, but are not limited to, glycine, serine, threonine, cysteine, ⁇ -alanine, homoserine, and ⁇ -aminobutyric acid.
  • a preferred amino acid may be used to the exclusion of an amino acid other than the preferred amino acid.
  • capping compound is meant, for purposes of the specification and claims to refer to a compound having the formula HS(CH 2 ) n X, wherein X is a carboxylate (carboxylic moiety); or the formula HS(CH 2 ) n YX, wherein X is a carboxylate and Y is an amine; as will be more apparent from the following descriptions, "n” is a number in the range of from 1 to about 20, and preferably greater than 4.
  • the thiol group of the capping compound forms Cd (or Zn) -S bonds (depending on whether the shell is Cd or Zn) , creating a layer which is not easily displaced in solution.
  • the carboxylic acid moiety of the capping compound imparts some water solubility to the quantum dots.
  • Exemplary capping compounds according to the present invention include, but are not limited to, mercaptocarboxylic acid, or mercaptofunctionalized amines (e.g., aminoethanethiol-HCl , homocysteine, or 1-amino-2 -methyl -2 -propanethiol -HC1) .
  • a preferred capping compound may be used to the exclusion of a capping compound other than the preferred capping compound.
  • the present invention provides compositions which can be used to generate a detectable signal comprising a light emission (e.g., fluorescence emission) of high quantum yield, thereby considerably improving the sensitivity of a non-isotopic detection system.
  • functionalized nanocrystals comprise quantum dots (core and shell) which comprises a first additional layer or coating comprising a capping compound, and a second layer or coating comprising diaminocarboxylic acid.
  • functionalized nanocrystals comprise quantum dots which comprise a first layer comprising the capping compound, a second layer comprising diaminocarboxylic acid, and an addition comprising affinity ligand (one or more molecules of affinity ligand) .
  • functionalized nanocrystals comprise quantum dots which comprising a first layer comprising the capping compound, a second layer comprising diaminocarboxylic acid, and a third layer comprising amino acid.
  • functionalized nanocrystals comprise quantum dots (core and shell) which comprise a first layer or coating comprising the capping compound, a second layer comprising diaminocarboxylic acid, a third layer comprising amino acid, and wherein the third layer has operably linked thereto one or more molecules of affinity ligand.
  • the component of each successive layer is operably linked to the component of any contacting layer, as will be more apparent from the figures and following description.
  • the functionalized nanocrystal comprises quantum dots, the capping compound, diaminocarboxylic acid, and operably linked to diaminocarboxylic acid is one or more molecules of affinity ligand.
  • the functionalized nanocrys- tals are first contacted with a sample under conditions suitable for the nanocrystals to contact and bind, via the affinity ligand portion, the substrate, if present, in the sample being analyzed for the presence or absence of the substrate.
  • the functionalized nanocrystals may comprise quantum dots, the capping compound, diaminocarboxylic acid, amino acid, and affinity ligand operably Inked to the amino acid.
  • the functionalized nanocrystals comprise quantum dots, a coating of capping compound, and a coating comprising diaminocarboxylic acid.
  • the user may then operably link the desired affinity ligand to the diaminocarboxylic acid of the functionalized nanocrystal using methods known in the art.
  • the functionalized nanocrystals may comprise quantum dots, a coating comprising the capping compound, a coating comprising diaminocarboxylic acid, and a coating comprising an amino acid; and the user may then operably link the desired affinity ligand to the amino acid of the functionalized nanocrystal using methods known in the art.
  • the composition according to the present invention comprises quantum dots which are capped by the addition of a layer comprising a capping compound, and more preferably a capping compound having the formula HS(CH 2 ) n X, (wherein X is a carboxylic moiety) , and comproises one or more successive layers comprising diaminocarboxylic acid, amino acid, or a combination thereof.
  • Desirable features of the functional - ized nanocrystals according to the present invention are that (a) can be excited with a single excitation light source, (b) when excited, emit a detectable light emission (e.g., fluorescence emission) of high quantum yield (e.g., a single quantum dot having at a fluorescence intensity at least a log greater than that of conventional fluorescent dye molecules) , (c) have a light emission having a discrete fluorescence peak, and (d) are water-soluble.
  • a detectable light emission e.g., fluorescence emission
  • high quantum yield e.g., a single quantum dot having at a fluorescence intensity at least a log greater than that of conventional fluorescent dye molecules
  • the functionalized nanocrystals typically should comprise a quantum dot particle of substantially uniform size of less than 100 Angstroms, and preferably have a substantially uniform size in the range of sizes of from about 2 nm to about 10 nm (diameter) .
  • Preferred quantum dots used in the production of functionalized nanocrystals are comprised of a core of CdSe passivated with ZnS .
  • Exemplary quantum dots comprise a CdSe core, and a ZnS shell, " (CdSe) ZnS” .
  • TOPO capped CdSe were produced by placing TOPO (5g) in a vessel, and dried at 150°C for 1 hour under vacuum. The vessel was then backfilled with argon and heated to 300°C.
  • CdMe 2 (7.2 ⁇ l , 0.1 mmol) and 1 M tri- octylphosphine-Se solution (90 ⁇ l , 0.09 mmol) and trioctyl- phosphine (5 ml) were mixed, and then placed into an injector.
  • This mixture was added to the TOPO in a reaction vessel, previously removed from the heat, in a single continu- ous injection with vigorous stirring, thereby resulting in the temperature decreasing to about 180°C.
  • the reaction vessel was then subjected to heat to raise the temperature 5°C every 10 minutes. Aliquots may be removed from the reaction vessel at various time intervals (5 to 10 minutes) to monitor the increase in size of nanocrystals over time, by the observation of the absorption spectra.
  • the temperature may be changed, or the reaction halted, upon reaching nanocrystals of the desired characteristics. For example, the reaction vessel was cooled to about 60°C, 40 ml of methanol was added to cause the nanocrystals to flocculate.
  • the pyridine overcoating of the (CdX) core/YZ shell nanocrystals were exchanged with a capping compound which contributes to the water-solubility of the resultant nanocrystals.
  • a capping compound comprising mercaptocarboxylic acid may be used to exchange with the pyridine overcoat.
  • Exchange of the coating group is accomplished by treating the water- insoluble, pyridine-capped quantum dots with a large excess of neat mercapto-carboxylic acid.
  • the pyridine-capped (CdSe) ZnS quantum dots were precipitated with hexanes, and then isolated by centri- fugation.
  • the residue was dissolved in neat mercaptoacetic acid, with a few drops of pyridine added, if necessary, to form a transparent solution.
  • the solution is allowed to stand at room temperature for at least six hours. Longer incubation times lead to increased substitution by the thiol . Overnight incubations are ideal.
  • Chloroform is added to precipitate the nanocrystals and wash away excess thiol.
  • the nanocrystals were isolated by centrifugation, washed once more with chloroform, and then washed with hexanes.
  • the residue was briefly dried with a stream of argon.
  • the resultant nanocrystals, coated with the capping compound, showed some solubility in water or other aqueous solutions.
  • the nanocrystals, in an aqueous solution were centrifuged once more, filtered through a 0.2 ⁇ m filter, degassed with argon, and stored in an amber vial. Failure to protect the nanocrystals, in solution, from air and light leads to rapid, irreversible flocculation.
  • the capping compound a mercaptocarboxylic acid; e.g., mercaptoacetic acid, mercaptopropionic acid, mercaptoundecanoic acid, etc.
  • a mercaptocarboxylic acid e.g., mercaptoacetic acid, mercaptopropionic acid, mercaptoundecanoic acid, etc.
  • the functionalized nanocrystals comprising a coat of diaminocarboxylic acid (“FN") unexpectedly show a significant increase in stability in an aqueous environment compared to quantum dots having an outer layer of just the capping compound ("W-SN) , when exposed over time to identical conditions of an oxidizing environment (e.g., light and air) . Additionally, as shown in FIG.
  • IB functionalized nanocrystals containing a coat of diaminocar- boxylic acid ("FN") unexpectedly result in a significant decrease in non-specific binding compared to quantum dots having an outer layer of just the capping compound ("W-SN) , when each were contacted with a surface that is both hydrophilic and hydrophobic (e.g., as may be encountered in a detection system) , followed by washing of the surface, followed by detection of residual nanocrystals (as measured by number of events of fluorescence versus the intensity of fluorescence; using a fluorescence microscope with a video camera attachment, time of exposure- l/30 th of a second) .
  • W-SN just the capping compound
  • the diaminocarboxylic acid (a) enhances the water-solubility of the functionalized nanocrystal; (b) has at least two free functional groups which are carboxyl -reactive, thereby enabling the diaminocarboxylic acid molecule to operably link to and crosslink carboxyl groups extending from the capping compound on the capped quantum dots; and (c) once operably linked to the capping compound, has one or more free functional groups which can be used for operably linking affinity ligand thereto. Additionally, a free carboxylic acid group on the diaminocarboxylic acid will remain as a site for attachment (operably linking) of other molecules to the diaminocarboxylic acid layer.
  • the diaminocarboxylic acid comprises lysine (2 , 6-diaminohexanoic acid).
  • lysine (2 , 6-diaminohexanoic acid) for operably linking diaminocarboxylic acid to the capping compound of capped quantum dots.
  • commercially avail - able crosslinking agents and methods known to those skilled in the art may be used.
  • mercaptoacetic acid-capped nanocrystals were dissolved in an aqueous buffer system (pH of about 7) .
  • the buffer may comprise such buffers as PBS or HEPES; however, the presence of phosphate may dramatically decrease the lifetime of the crosslinking agent.
  • EDC l-ethyl-3- [3-dimethylaminopropyl] carbdiimide
  • sulfoNHS sulfo-N-hydroxysuccinimide
  • the resulting solution was stirred at room temperature for 30 minutes.
  • Mercaptoethanol was added to neutralize unreacted EDC at 20 mM concentration and stirred for 15 minutes.
  • the entire solution was then added dropwise, with stirring, to a solution of lysine (large excess) in the same buffer; and the mixture was stirred for 2 hours at room temperature.
  • Ethanolamine (30 mM) was added to quench the reaction; and the mixture was stirred for 30 minutes at room temperature or left overnight at 4°C.
  • the solution was centrifuged to remove any precipitated solids, and then ultrafiltered through a 30kD MW centrifugal filter.
  • the resultant concentrated, functionalized nanocrystals can be solubilized in an aqueous solution of choice. Once solubilized, the resulting solution can be stored in an amber vial under an inert gas to prevent flocculation.
  • the resulting solution can be stored in an amber vial under an inert gas to prevent flocculation.
  • the functionalized nanocrystals comprised of a first layer comprising capping compound and a second layer comprising diaminocarboxylic acid is further functionalized by the addition of affinity ligand.
  • a protein glycoprotein, peptide, lipoprotein, etc.
  • a free carboxyl -reactive group e.g., an amine group
  • an amine group can be operably linked to the free carboxyl group of the diaminocarboxylic acid of the functionalized nanocrystals using methods known in the art.
  • an affinity ligand selected from the group consisting of avidin, a monoclonal antibody, an F'ab fragment, or a lectin may be operably linked using EDC and sulfo-NHS using the general methods as previously described herein. More particularly, EDC functions to activate at least one reactive functionality (e.g., a carboxylate) to catalyze its reaction with another reactive functionality such as the amine group of a protein.
  • EDC functions to activate at least one reactive functionality (e.g., a carboxylate) to catalyze its reaction with another reactive functionality such as the amine group of a protein.
  • the functionalized nanocrystals (1 ml, 8.1 x 10 ⁇ 9 mol) were esterified by treatment with EDC (8.1 x 10 ⁇ 6 mol), followed by treatment with sulfo-NHS (8.9 x 10 "6 mol) at ambient temperature in buffered aqueous solution (at about pH 7.4) for 30 minutes. 2-mercaptoethanol was added to the solution at a concentration of 20 mM, and the mixture was stirred for 15 minutes to quench any unreacted EDC.
  • WGA wheat germ agglutinin
  • the nanocrys- tals were then contacted with WGA (8.1 x 10 "9 mol in PBS, 1 mg/ml) with vigorous stirring, and the reaction mixture was stirred for 2 hours (e.g., conditions sufficient to form an amide bond between the EDC-activated carboxylates of the diaminocarboxylate layer and the amine groups on WGA in forming functionalized nanocrystals which are water-soluble and have lectin operably linked thereto) .
  • Ethanolamine was added at a concentration of 30 mM to quench the coupling reaction, and the reaction mixture was stirred for 30 minutes.
  • the resulting solution was then filtered through a 30 kD molecular weight cutoff centrifugal filter to remove excess reagents.
  • the concentrated material was then diluted to 1 ml in buffer (e.g., PBS) or other suitable aqueous solution.
  • buffer e.g., PBS
  • the same procedure can be used to operably link avidin, an antibody, or other affinity ligand having at least one free carboxyl -reactive group.
  • the functionalized nanocrystals comprise avidinylated, functionalized nanocrystals (e.g., (CdX) core/YZ shell, capped with the capping compound, coated with diaminocarboxylic acid that is operably linked to the capping compound, followed by addition of avidin which is operably linked to the diaminocarboxylic acid) which are then contacted with, and operably linked to, a plurality of molecules of the desired oligonucleotide, each of which contains one or more biotin molecules (including native biotin or a biotin derivative having avidin-binding activity; e.g., biotin dimers, biotin multimers, carbo-biotin, and the like) .
  • biotin molecules including native biotin or a biotin derivative having avidin-binding activity; e.g., biotin dimers, biotin multimers, carbo-biotin, and the like
  • the oligonucleotides are biotinylated at a single terminus of the strand.
  • biotin molecules can be added to or incorporated in a nucleotide strand, and even localized to one terminus, such as by directing synthesis of the nucleotide strands with nucleotides and biotin-nucleotides, or by biotinylating the 5' aminogroup of the nucleotide with sulfo-NHS-biotin.
  • a functionalized nanocrystal having a plurality of oligonucleotides extending therefrom e.g., through the biotin-avidin binding, the plurality of oligonucleotides become operably linked to the functionalized nanocrystals.
  • These functionalized nanocrystals may then be used as probes in a nucleic acid probe hybridization detection system using standard methods known to those skilled in the art.
  • the functionalized nanocrystals comprise quantum dots with a first layer comprising the capping compound, a second layer comprising diaminocarboxylic acid, and a third layer comprising an amino acid.
  • Functionalized nanocrystals comprising capping compound, and diaminocarboxylic acid may be produced using the methods outlined in Example 1, and FIG. 2 herein.
  • These functionalized nanocrystals are further functionalized by the addition of another layer comprising an amino acid, such as illustrated in FIG. 3.
  • FIG. 3 illustrates the addition of an additional layer of an amino acid wherein the amino acid comprises a diaminocarboxylic acid.
  • the diaminocarboxylic acid molecules of the third layer can operably link, and crosslink, the free carboxyl groups of the diaminocarboxylic acid molecules of the second layer.
  • the number of free functional groups for reaction to operably link with a subsequent carboxylic acid layer or affinity ligand is reduced.
  • an affinity ligand is to be operably coupled to diaminocarboxylic acid comprising a third layer
  • a reduction in the number of free functional groups for reaction with the affinity ligand may be desira- ble, particularly if it is desired to operably link relatively fewer molecules of the affinity ligand to the functionalized nanocrystals (e.g., because of one or more of the size, chemical characteristics, and specificity of the affinity ligand, or substrate to which the affinity lignd binds) .
  • affinity ligands are desired to be operably linked to the functionalized nanocrystals, it may be disadvantageous to use a third layer comprising an amino acid comprising a diaminocarboxylic acid.
  • alternative embodiments include: (a) operably linking the affinity ligand to functionalized nanocrystals comprising quantum dots, the capping compound, and the diaminocarboxylic acid; or (b) operably linking a third layer (com- prising an amino acid comprising monoaminocarboxylic acid operably linked to the diaminocarboxylic acid) , and then operably link the affinity ligand to the functionalized nanocrystals via the free carboxyl group of the monoaminocarboxylic acid.
  • various factors such as the nature of the affinity ligand to be operably linked, may guide the choice of a carboxylic acid for a third layer in further functionalizing the nanocrystals according to
  • Ethanolamine (30 mM) is added to quench the reaction; and the mixture is stirred for 30 minutes at room temperature or left overnight at 4°C .
  • the solution is centrifuged to remove and precipitate solids, and then ultrafiltered through a 30kD MW centrifugal filter.
  • the resultant concentrated, functionalized nanocrystals can be solubilized in an aqueous solution of choice.
  • This process can also be used to add a third layer comprising an amino acid comprising a monoaminocarboxylic acid rather than a diaminocarboxylic acid.
  • functionalized nanocrystals comprising a third layer comprising an amino acid may be further functionalized by operably linking affinity ligand to the free amine reactive group (s) (or other free reactive groups) of the amino acid comprising the third layer using methods previously described herein.
  • diaminocarboxylic acid may be operably linked to a capping compound comprising mercapto-functionalized amine, and more particularly, by the use of a linker.
  • the functionalized nanocrystals are placed in contact with a sample being analyzed for the presence or absence of a substrate for which the affinity ligand of the functionalized nanocrystals has binding speci- ficity.
  • Contact, and subsequent binding, between the affinity ligand of the functionalized nanocrystal and the sub- strate, if present in the sample, in a detection system results in complexes comprising the functionalized nano- crystal-substrate which can emit a detectable signal for quantitation, visualization, or other form of detection.
  • the detectable signal emitted therefrom may be detected by first exposing the complexes formed in the detection system to a wavelength spectrum of light (visible, or UV, or a combination thereof) that is suitable for exciting the functionalized nanocrystals to emit a fluorescence peak.
  • a wavelength spectrum of light visible, or UV, or a combination thereof
  • the peak is then detected, or detected and quantitated, by appropriate detection means (e.g., photodetector, filters, fluorescence microscope, and the like) . Quantitation of the amount of substrate present is directly related to the intensity of the emitted fluorescence peak.
  • the absorbance peak and fluorescence peak emissions depend on such factors which include, but are not limited to, the chemical nature, and size, of the func- tionalized nanocrystals.
  • functionalized include, but are not limited to, the chemical nature, and size, of the func- tionalized nanocrystals.
  • CdSe/ZnS nanocrystals having a substantially uniform core size comprising a diameter of about 68.4 angstroms (A) may be excited with light in the spectral range of from about 400nm to 500nm, and emit a fluorescence peak (corresponding to the color orange) at 609nm which may be detected using appropriate detection means.
  • Functionalized CdSe/ZnS nanocrystals having a substantially uniform core size comprising a diameter of about 53.2 A may be excited with light in the spectral range of from about 400nm to 500nm, and emit a fluorescence peak (corresponding to the color yellow) at 545 nm which may be detected using appropriate detection means.
  • Functionalized CdSe/ZnS nanocrystals having a substantially uniform core size comprising a diameter of about 46.6 A may be excited with light in the spectral range of from about 400nm to 500nm, and emit a fluorescence peak (corresponding to the color green) at 522 nm which may be detected using appropriate detection means.
  • Detection may be by detection means comprising a scanner or reader or other analytical instrument which can detect fluorescence peaks in the range of about 410 nm to about 750 nm; and, optionally (when more than one color is used in the detection system) , distinguish between discrete fluorescence peaks within that range.
  • nanocrystals used in the present invention many sizes of which can be excited with a single excitation light source, resulting in many emissions of colors that can be detected simultaneously and distinctly.
  • more than one target substrate may be detected in a detection system simultaneously by using more than one uniform size of functionalized nanocrystals; with each uniform size having an affinity ligand operably linked thereto which has a different binding specificity (hence can detect a different target substrate) than the affinity ligand operably linked to functionalized nanocrystals of a different uniform size.
  • the detection system may include, but is not limited to, one or more of an affinity assay (e.g, immunoassay such as an ELISA) , fluorescent staining (e.g., immunofluorescence staining on a glass slide), flow cytometry, nucleic acid hybridization assay, molecular sorting (e.g., cell sorting by flow cytometry), and the like.
  • an affinity assay e.g, immunoassay such as an ELISA
  • fluorescent staining e.g., immunofluorescence staining on a glass slide
  • flow cytometry e.g., flow cytometry
  • nucleic acid hybridization assay e.g., cell sorting by flow cytometry
  • functionalized nanocrystals comprising diaminocarboxylic acid which is operably linked to the capping compound
  • affinity ligand comprising lectin WGA (wheat germ agglutinin) which is operably linked to the diaminocarboxylic acid
  • WGA-labeled, functionalized nanocrystals To a tube containing approximately 70,000 cells of Met-129 cancer cell line (chemically induced murine mammary carci- noma) was added 200 ⁇ l of the WGA-labeled, functionalized nanocrystals, and the mixture was then rotated gently on a platform mixer.
  • Met-129 cells have one or more cell surface glycoproteins with either terminal N-acetylglucosamine residues or with terminal sialic acid residues (e.g., mucin) which may be reactive with WGA.
  • a drop of the mixture was placed on a microscope slide, and covered with a coverslip. Examination of the sample with a fluorescence microscope revealed that the Met-129 cells aggregated together, with the outlines of the cells clearly visible by fluorescent staining with the WGA-labeled, functionalized nanocrystals. There was very little background fluorescence remaining in the reaction media.
  • another sample was examined, and again at 2 hours. Both of the latter samples showed agglutination of the cells, with fluorescent staining of the outside cell walls by the WGA- labeled, functionalized nanocrystals.

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Abstract

L'invention concerne des compositions renfermant des nanocristaux hydrosolubles fonctionnalisés. Ces nanocristaux hydrosolubles fonctionnalisés comprennent des points quantiques coiffés d'une couche d'un composé de coiffage, ainsi qu'un ou plusieurs composés additionnels, liés par la suite de manière opérationnelle. Un composé additionnel contient de préférence de l'acide diaminocarboxylique lié de manière opérationnelle audit composé de coiffage, ainsi qu'un acide aminé, un ligand d'affinité, ou une combinaison de ceux-ci. Cette invention concerne par ailleurs des procédés d'utilisation des nanocristaux fonctionnalisés susmentionnés comprenant un ligand d'affinité, ces procédés étant destinés à détecter la présence ou l'absence d'un substrat cible dans un échantillon. Ces procédés consistent tout d'abord à mettre lesdits nanocristaux fonctionnalisés en présence de cet échantillon, afin de former des complexes entre les nanocristaux et le substrat, si ce dernier est présent, puis à exposer ces complexes du système de détection à une source de lumière d'excitation, et enfin à détecter le pic de fluorescence émise.
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