WO2002099425A2 - Sonde - Google Patents

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Publication number
WO2002099425A2
WO2002099425A2 PCT/GB2002/002590 GB0202590W WO02099425A2 WO 2002099425 A2 WO2002099425 A2 WO 2002099425A2 GB 0202590 W GB0202590 W GB 0202590W WO 02099425 A2 WO02099425 A2 WO 02099425A2
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WIPO (PCT)
Prior art keywords
probe
bead
nanocrystals
beads
coating
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Ceased
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PCT/GB2002/002590
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English (en)
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WO2002099425A3 (fr
Inventor
Lars Korsnes
Geir Fonnum
Grete Irene Modahl
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Life Technologies AS
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Dynal Biotech ASA
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Priority to AU2002302800A priority Critical patent/AU2002302800A1/en
Publication of WO2002099425A2 publication Critical patent/WO2002099425A2/fr
Publication of WO2002099425A3 publication Critical patent/WO2002099425A3/fr
Anticipated expiration legal-status Critical
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    • 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
    • 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

Definitions

  • This invention relates to a process for the preparation of synthetic beads comprising nanocrystals, in particular to a process for the preparation of beads comprising a combination of nanocrystals as well as to the resulting probes themselves and the use thereof as probes in biological applications.
  • a wide variety of chemical and biological assays are known for identifying an analyte of interest in a given sample.
  • immunoassays such as enzyme- linked immunosorbent assays (ELISA) are used in numerous diagnostic, research and screening applications. Assays generally utilise detectable labels to identify the analyte of interest.
  • radiolabelled molecules and compounds are frequently used to detect biological compounds both in vivo and in vi tro .
  • non radioactive methods of detecting biological and chemical compounds are preferable .
  • fluorescent labelling of biological systems has become a widely used and known analytical tool in modern biotechnology.
  • Applications for such fluorescent labelling include technologies such as medical fluorescence microscopy, histology, flow cytometry, DNA sequencing, immunoassays, binding assays, separation etc .
  • Fluorescent molecules are therefore commonly used as tags for detecting an analyte of interest.
  • fluorescent labelling involves the use of an organic dye molecule bonded to a moiety which selectively bonds to a particular biological system, the presence of which is then identified by excitation of the dye molecule to cause it to fluoresce.
  • organic dye molecule bonded to a moiety which selectively bonds to a particular biological system, the presence of which is then identified by excitation of the dye molecule to cause it to fluoresce.
  • the emission of light of visible wavelengths from an excited dye molecule is usually characterised by a broad emission spectrum as well as a broad tail of emissions on the red side of the visible electromagnetic spectrum.
  • a broad emission spectrum as well as a broad tail of emissions on the red side of the visible electromagnetic spectrum.
  • the majority of fluorescent dyes have a relatively narrow absorption spectrum therefore requiring multiple excitation or a broad spectrum excitation source which would not be specific for a particular dye.
  • the narrow absorption spectrum therefore limits usefulness of organic dyes.
  • a yet further limitation is that the fluorescence exhibited by a compound may deteriorate upon prolonged and/or repeated exposure to light and conversion of the dye or decomposition of the dye into non-fluorescent species may occur and may be irreversible . Decomposition products may also interfere with separation, analysis or binding procedures.
  • a final limitation is that certain lower molecular weight dyes in everyday usage may not provide a bright enough signal for ready detection.
  • nanocrystalline particles may be employed and such particles have been shown to be reliable as detectable labels in a variety of biological systems.
  • These nanocrystals are capable of producing luminescence and/or absorption when excited by an electromagnetic source and may be tailored so as to have characteristic spectral emissions.
  • the emissions are of high intensity, narrow linewidth (hence there is much less chance for spectral overlap) and excitation of the nanocrystals can be achieved using electromagnetic radiation in the UV or visible range.
  • an organoluminescent semiconductor probe is suggested formed from a nanocrystal compound linked to an affinity ligand, optionally via a linking group.
  • the affinity ligand is selected for its ability to bind a desired detectable substance in a sample .
  • WO 00/68692 describes the use of semi-conducting nanocrystals for detecting one or more target analytes in a sample.
  • the various methods described involve the use of semi-conductor nanocrystal conjugate, i.e. the nanocrystal is linked to a specific binding molecule, which directly or indirectly attaches to the analyte of interest.
  • the nanocrystal may be made part of a solid support, e.g. by binding the nanocrystal to a styrene or other polymer monomer and polymerising this monomer with others to form, for example, a polystyrene bead.
  • nanocrystal particles can be employed much more readily in an assay procedure if the particles are associated with a polymer bead, e.g. contained within pores present in a polymer bead or deposited on the surface of a preformed bead. Such beads are preferably preswollen, optionally porous beads which can be made using the
  • probes Such nanocrystal containing beads, from hereon referred to as probes, can be readily prepared and utilised in a diverse range of assays. Moreover, since by use of differing combinations of nanocrystals the emission spectra of the probe may be tailored, probes can be provided with characteristic "bar-coded" emissions.
  • the invention provides a process for the preparation of a luminescent probe comprising mixing an optionally porous, polymer bead with at least one type, preferably at least two types, of nanocrystal so that the nanocrystals associate with, e.g. enter the pores of or deposit onto, said bead; and subsequently coating said bead to form said probe.
  • the invention provides a probe comprising an optionally porous, polymer bead with at least one type of nanocrystals associated therewith, said bead and nanocrystals being coated with a polymeric coating.
  • the invention provides the use of a probe as hereinbefore described in an assay.
  • the invention provides a method for detecting an analyte in a sample comprising mixing said sample with a probe as hereinbefore described said probe carrying at least one affinity ligand; allowing the analyte to bind to said probe; and detecting the resulting probe-analyte conjugate, e.g. spectroscopically.
  • a nanocrystal (also referred to as a colour particle) as used herein is an inorganic single crystal particle between approximately 1 and 100 nanometres in diameter.
  • the nanocrystals of use in the probes of the invention must be luminescent, i.e. they must be capable of emitting electromagnetic radiation upon excitation.
  • the nanocrystals used in the invention will be described further below and are commercially available under the Trade Names Biocrystals and Quantum Dots (Qdots) .
  • Qdots Quantum Dots
  • the preparation of the nanocrystals is also described in, amongst others, US 5,251,018, US 5,262,357 and US 5,505,928 which are herein incorporated by reference.
  • the nanocrystals may have any suitable diameter although the nanocrystals should be small enough to be able to enter into the pores of the bead if these are present.
  • nanocrystals should be between 2 and 50 nanometres, especially 2 to 20 nanometres, especially 4 to 10 nanometres in diameter.
  • the nanocrystals should however, have a diameter greater than 0.5 nm.
  • the nanocrystals are capable of emitting electromagnetic radiation upon excitation and are therefore luminescent. Typically, they are spherical in nature and are preferably monodisperse, i.e. all the nanocrystals are within a defined particle size range.
  • the nanocrystals may include a core of one or more first semi-conductor materials which may be surrounded by a shell of a second semi-conducting material. Such a species is referred to as a core/shell nanocrystal and again such species are well-known in the art.
  • the surrounding shell material should preferably have a band gap energy that is larger than the band gap energy of the core material and may be chosen to have an atomic spacing close to that of the core substrate.
  • the core and/or shell is a semi-conductor material and generally comprise elements in groups 11, 12 and II- VI of the periodic table. Particularly preferably, an element from group IIB and VIB, IIIB and VB, or IVB and IVB form the core.
  • suitable materials for the manufacture of the nanocrystals of use in the invention include the sulfides, selenides and tellurides of zinc, cadmium, mercury, magnesium, calcium, strontium, barium.
  • the nanocrystal core is made from CdS, ZnS, CdSe, CdTe, ZnSe, ZnTe, GaP or GaAs.
  • the shell substance if present, is preferably a semiconductor material as well and may therefore be selected from those listed above.
  • the nanocrystals will preferably emit light with a narrow linewidth, e.g. a band of 40 nm or less, preferably 20 nm or less thus permitting the simultaneous use of a plurality of differently coloured organoluminescent nanocrystal species without emission overlap.
  • the nanocrystal may also be coated with an organic capping agent as is well known in the art.
  • the capping agent should have an affinity for the nanocrystal surface and may therefore be, for example an monomer/polymer species.
  • the capping agent may also be present to aid association of the nanocrystal with the optionally porous bead, for example by allowing interaction of the capping agent with a suitable group present on the surface of the bead or within a pore in the bead.
  • a nanocrystal carrying a carboxyl capping agent may deposit readily onto the surface of a bead carrying an amine functionality on its surface.
  • the capping agent may be hydrophobic so as to allow ready diffusion of a nanocrystal into the pore of a bead which has been prefunctionalised so as to provide a similar hydrophobic environment.
  • the probes of the invention may comprise only one type of nanocrystals (e.g. CdSe nanocrystals of a 'particular size and emission spectrum) it is preferred if at least two different types of nanocrystal are employed thus allowing the preparation of probes having an emission spectrum which can act as a kind of "bar code".
  • nanocrystals e.g. different sizes, size distributions, chemical composition etc each probe can be made to have a very characteristic emission spectrum, i.e. a particular linewidth, intensity, luminescence lifetime etc.
  • a probe comprising two types of nanocrystals can be said to exhibit at least two distinguishable emissions in its spectrum.
  • probes When a number of such barcoded probes are used together in an assay, it is easy to determine which probe has bound to which analyte simply by recording the nature of the probe's emission spectrum. The technique therefore allows valuable information to be ascertained.
  • the probes may also be used to label biological entities such as chromosomes for spectral karyotyping or the like.
  • the polymer beads in which the nanocrystals are to be held may be prepared using techniques known in the art, e.g. core/shell beads, but are preferably prepared using the well-known Ugelstad two-step swelling process and the improvements thereon described comprehensively in WO 00/61647. Beads made in this way are, from hereon, called Ugelstad beads.
  • Ugelstad beads Before the advances in bead preparation made by Professor Ugelstad, polymer beads were produced by diffusing a monomer and a polymerisation initiator into polymer seeds in an aqueous dispersion. The seeds swell and following initiation of polymerisation, large polymer particles are produced. The maximum volume increase in such a process is typically x5.
  • styrene oligomer has diffused into it a polymerisation initiator and subsequently a monomer such as a styrene, acrylate, unsaturated chloride, ester, acetate, amide and alcohol monomer. Polymerisation is then initiated.
  • a polymerisation initiator such as a styrene, acrylate, unsaturated chloride, ester, acetate, amide and alcohol monomer.
  • Polymerisation is then initiated.
  • the "swollen" beads i.e.
  • beads produced by swelling may comprise polystyrene (including high density polystyrene latexes such as brominated polystyrene) , polymethylmethacrylate and other polyacrylic acids, polyacrylonitrile, polyacrylamide, polyacrolein, polydi ethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride and polydivinylbenzene .
  • polystyrene including high density polystyrene latexes such as brominated polystyrene
  • polymethylmethacrylate and other polyacrylic acids polyacrylonitrile
  • polyacrylamide polyacrolein
  • polydi ethylsiloxane polybutadiene
  • polyisoprene polyurethan
  • the beads are made from combinations of styrenes, acrylates and/or methacrylates . More preferably the beads are made from styrene, divinylbenzene, acrylates and methacrylates, especially styrene and divinylbenzene .
  • the resulting beads are spherical and monodisperse. This is important since agglomeration of beads may cause signal detection problems. If non- monodisperse particles are employed in the probes of the invention agglomeration may occur and it will then be impossible to tell whether an intense signal is due to a particular probe or simply due to the large number of probes which agglomerate. Light intensity measurements may therefore be of little value. If monodisperse beads are employed then signal strength from the probes of the invention will be dependent on intensity of the nanocrystals incorporated in each probe allowing an accurate concentration dependent analysis to be made. This is an important advantage in a multiplex assay.
  • Beads may be non-porous (compact) or porous.
  • a porogen is employed as is known in the art.
  • the porogen is typically added prior to monomer polymerisation.
  • Suitable porogens are low molecular weight aliphatic or aromatic hydrocarbons or alcohols such as heptane, toluene or cyclohexanol .
  • the use of glycidyl methacrylate in the final swelling stage of the Ugelstad process has been found to yield beads which are more readily functionalised then conventional beads and this forms a further aspect of the invention.
  • Beads may also be cross-linked as is known in the art.
  • the resulting beads may have a diameter of between 0.5 and 100 microns, e.g. 2 to 20 microns, especially 2 to 5 microns. If pores are required, a pore volume of 20 to 60% is preferable. The pores are generally between 5 and 200 nanometres in diameter and may be up to 200 n in depth.
  • the nanocrystals need to become associated with the beads.
  • the nanocrystals need to bind chemically, physically or electrostatically with the bead or simply become associated with the bead via diffusion or suction into the pores of the bead.
  • the nanocrystals associate physically or electrostatically with the beads, e.g. via deposition onto the bead surfaces . This may be achieved using any convenient process .
  • the surfaces of the beads of the invention may be left unfunctionalised, it is preferred if the surfaces are coated with appropriate chemical functionalities to ensure that the nanocrystals are in a suitable chemical environment on the bead surface or within the pores.
  • the surface of the bead/pore may be coated with hydrophobic/hydrophilic groups or may be functionalised so as to be charged depending on the nature of the nanocrystal to be added.
  • the surfaces of the bead and nanocrystal unit are designed so as to readily associate, i.e.
  • the nanocrystal may comprise a corresponding acid functionality so that binding or deposition can occur.
  • a positive charge in the pore surface needs to be associated with a negative charge on the nanocrystal.
  • Functionalisation/coating of the bead surface is readily achieved by known processes.
  • the beads may therefore be aminated so as to allow the binding of a carboxy functionalised nanocrystal.
  • the beads may be functionalised to allow hydrophobically functionalised nanocrystals to become associated with the beads.
  • Nanocrystals may also diffuse into the pores of the beads if a suitable concentration gradient exists between the pores and the medium in which the nanocrystals are in. Nanocrystals may also displace groups present on the bead or pore surfaces. The skilled artisan would readily devise methods for incorporating nanocrystals into the porous beads .
  • nanocrystals An increased absorption of nanocrystals is seen if the bead coating contains functional groups which are known to coordinate with the shell of the nanocrystal, e.g. amines, thiols, phosphines, phosphine oxides, pyridine, furans etc.
  • functional groups which are known to coordinate with the shell of the nanocrystal, e.g. amines, thiols, phosphines, phosphine oxides, pyridine, furans etc.
  • the introduction of the nanocrystals into or onto the beads should take place in a medium that favours association of the nanocrystals with the beads.
  • the nature of the medium clearly depends on the nature of the nanocrystal as well as the nature of the pore surface but where the nanocrytals carries a polar functionality, an alcohol such as ethanol is a convenient medium.
  • suitable solvents for the absorption process include toluene, 1,4-dioxane, butyl acetate, butanol, propanol, chloroform and mixtures thereof.
  • the nanocrystals are found within the pores of a porous bead, e.g. at least 50%, preferably 80%, especially to be found in a pore.
  • the nanocrystals are deposited on the surface of a non-porous bead, e.g. by specific deposition.
  • the amount of nanocrystals required per bead will be readily determined by the person skilled in the art and will depend on the nature of the nanocrystal, bead and the intensity of signal required. Typically, however, to prepare 10 g of probes according to the invention 1 ml to 100 ml of 2.5 ⁇ M nanocrystal dispersion is added. The association procedure may take from 10 minutes to 24 hours, e.g. 2 hours.
  • Ugelstad beads are often made magnetisable by adding a magnetisable material (e.g. iron material) to the porous beads.
  • a magnetisable material e.g. iron material
  • the magnetisable material can be added before, simultaneously with or after addition of the nanocrytals to the beads.
  • iron (II) sulphate or iron (II) oxide particles are incorporated into beads when contact between the beads and the iron compound is made.
  • the beads can be nitrated to allow the association with the magnetisable particles.
  • This chemistry is well-known and will be readily achieved by the person skilled in the art.
  • the magnetisable particles are introduced into the pores of a bead prior to association of the beads with the nanocrystals .
  • the nanocrystals may then be additionally added to the pores or deposited on the bead surfaces, preferably the bead is then coated to ensure that the magnetisable particles do not dissociate from the beads whilst also functionalising the bead surface to allow nanocrystal association. Coating may be achieved using an epoxide polymer for example.
  • Nanocrystals can then be associated with the beads as described in detail above to form the probes of the invention.
  • the nanocrystals associated with the probes of the invention may be "free floating" within the mesh of the bead or deposited on the surface of a bead, it is necessary to coat the probes with a suitable coating material so as to prevent loss of nanocrystals in the reaction medium or sample and to prevent possible degradation of the nanocrystals by chemicals, buffers etc.
  • a coating has proved vital since in the absence of a coating nanocrystals can be lost or degraded by chemicals, buffers etc which the probes contact.
  • the probes are greatly stabilised giving the probes of the invention a longer shelf-life and a reproducible and constant emission allowing a higher degree of multiplexing.
  • the resulting probes are coated using, for example, a polymer again using known processes.
  • the polymer used as for the external coat is an epoxy or polyurethane polymer or a glycidyl ether.
  • probe coating can be achieved by cross- linking a coating already present from an earlier stage of the probe synthesis.
  • a epoxide polymer has been used to coat the beads after the introduction of magnetisable particles into bead pores, this polymer can be crosslinked with, for example, a diisocyanate to produce an external polymer coating.
  • the invention provides process for the preparation of a luminescent probe comprising:
  • the surface chemistry of the bead may be manipulated to provide epoxy, hydroxy, amino, etc. functionalities on the surface to allow binding to affinity ligands.
  • the result of the secondary coating procedure is a bead surface already suitable to bind to affinity ligands, e.g. by using prefunctionalised polymer monomers in the manufacture of the secondary polymer coat. If this cannot be achieved functionalisation of the bead surface is readily achieved as is known in the art, e.g. by reaction with certain bifunctional reagents.
  • the probe can then be bound to an affinity ligand the nature of which will be selected based on its affinity for the particular detectable substance whose presence or absence in a sample is to be ascertained.
  • the affinity molecule may therefore comprise any molecule capable of being linked to a luminescent probe which is also capable of specific recognition of a particular detectable substance.
  • Affinity ligands therefore include monoclonal antibodies, polyclonal antibodies, antibody fragments, nucleic acids, oligonucleotides, proteins, polysaccharides, sugars, peptides, peptide nucleic acid molecules, antigens, drugs and ligands. Lists of suitable affinity ligands are available in the published literature and are universally well known.
  • affinity ligands used previously in assays involving fluorescent dyes are of use in the present invention.
  • the use of further binding partners, secondary affinity ligands and linking groups which is routine in the art will not be discussed further herein although it will be appreciated that the use of such species with the beads of the invention is possible if desired.
  • nucleic acid detection generally involves probing a sample thought to contain target nucleic acids using a nucleic acid probe that contains a nucleic acid sequence that specifically recognises, e.g. hybridises with, the sequence of the target nucleic acids, such that the nucleic acid affinity ligand and the target nucleic acids in combination create a hybridisation layer.
  • the target material is optionally a material of biological or synthetic origin but is present as a molecule or as a group of molecules, including, antibodies, amino acids, proteins, peptides, polypeptides, enzymes, enzyme substrates, hormones, lymphokines, metabolites, antigens, haptens, lectins, avidin, streptavidin, toxins, poisons, environmental pollutants, carbohydrates, oligosaccharides, polysaccharides, glycoproteins, glycolipids, nucleotides, oligonucleotides, nucleic acids and derivatised nucleic acids, DNA, RNA, natural or synthetic drugs, receptors, virus particles, bacterial particles virus components, cells, cellular components, natural or synthetic lipid vesicles, polymer membranes, polymer services and particles and glass and plastic surfaces .
  • the nanocrystals in the probes of the invention may be excited by conventional means.
  • the nanocrystals are preferably excitable over a broad bandwidth but exhibit emission only in a narrow waveband, i.e. in sharp contrast to organic dyes.
  • electromagnetic radiation of wavelength ranging from X-ray to ultraviolet to visible to infrared may be used to excite the luminescence in the nanocrystals.
  • the nanocrytals may also be excited by bombardment by a particle beam, e.g. electron beam.
  • a particle beam e.g. electron beam.
  • one excitation source can be employed to excite several different nanocrystal types, i.e. several nanocrystal types which give off radiation at different frequencies thus permitting simultaneous excitation and detection of the presence of several nanocrystal types and hence several detectable substances from a sample .
  • a laser source of a given frequency may excite a nanocrystal which emits green light and a further nanocrystal which emits red light. Detection of both red and green light would show the presence of the biological target to which the bead in which the nanocrystal resides was designed to bind.
  • Detection of the emission spectra of the excited nanocrystals can be achieved using a commercially available detection system such as a spectrometer. Detection of a particular nanocrystal emission will of course signify detection of the analyte to which the polymer bead/nanocrystal system was designed to bind.
  • the probes of the present invention may be employed in a wide variety of assays. Perhaps in its simplest form probes with a suitable affinity ligand, e.g. an antibody molecule or a general or specific affinity ligand, can be added to a biological sample, e.g. a crude blood or cell lysate, containing a target protein.
  • a specific detecting antibody may be added to bind to the probe/target protein conjugate.
  • the conjugate may then be separated from the remaining sample, conveniently magnetically, and then bound to a suitably functionalised solid substrate, e.g. a streptavidin functionalised substrate via the specific detecting ligand, e.g. a biotinylated antibody, thereby allowing detection of the probes, and hence the target protein, by their luminescence .
  • a microorganism or a specific type of cell could be isolated from a sample, e.g. a crude blood or tissue sample, by attaching to the microorganism or cell the magnetisable probes of the invention via a suitable affinity ligand.
  • a detecting ligand e.g. an antibody
  • a solid substrate such as a streptavidin coated chip or sensor.
  • the invention provides a method for detecting a target in a sample comprising mixing a probe with a suitable affinity ligand with a biological sample, e.g. crude blood or cell lysate, containing said target;
  • Said analyte can of course be a protein, cell, microorganism, etc.
  • a major benefit of the probes of the present invention is the ability to use more than one type of probe in one assay procedure.
  • a mixture of different probes i.e. probes with different spectral emissions, comprising affinity ligands for a variety of targets, may be added to a crude sample whereupon various components of the crude sample can be bound and then separated using the techniques described above.
  • probe detection takes place and due to the different emission spectra of each different type of probe, many analytes can be detected from a single sample . For example, particular proteins within a crude sample or particular cells within a sample may be detected.
  • probes of the invention can detect the presence of a specific nucleic acid.
  • the probes simply need to carry a suitable capture oligonucleotide to bind to a nucleic acid of interest.
  • the resulting conjugate can then be labelled e.g. using a standard polymerase/ biotinylated nucleotide procedure or by hybridisation with a specific biotinylated or other labelled oligonucleotide. After heating/washing (and hence removal of the nucleic acid detected) and magnetic separation, probes can be bound to a solid substrate and those carrying the biotin detected.
  • the invention provides a method for detecting a plurality of targets in a sample comprising mixing a plurality of different types of probes, each type of probe having a different affinity ligand, to a biological sample; allowing the probes to bind to a plurality of targets within the sample to form probe .”target conjugates; mixing said conjugates with a specific detecting ligand; separating the conjugates from the sample, e.g. magnetically; binding the conjugates to a solid substrate via said specific detecting ligand and detecting the probes via their different emission spectra.
  • the affinity ligand is preferably an oligonucleotide and the specific detecting ligand is a label such as a biotinylated or other labelled oligonucleotide.
  • a more sophisticated assay allows the detection of particular genes which may be expressed in a cell or tissue or indeed any other appropriate biological entity, e.g. a microorganism.
  • standard Dynabeads ® are used to isolate a cell, microorganism etc from a sample of interest.
  • the isolated cell/bead conjugate is then placed in lysis buffer, the cells lysed and the beads, cell membranes and other cell debris removed.
  • Other methods of isolating mRNA may be used.
  • the remaining supernatant containing mRNA e.g.
  • a medium comprising a variety of probes of the invention with varying gene-specific oligonucleotide affinity ligands or capture oligonucleotides, e.g. 20 different types .
  • the mRNA binds to certain of the probes having suitable affinity ligands or capture oligonucleotides and after washing to remove unbound mRNA and magnetic separation a label can be attached to those probes which are bound to mRNA.
  • a suitable label for use in the detection of mRNA is a labelled dT oligonucleotide.
  • labelling may be achieved by primer extension, incorporating labelled nucleotides, optionally of the di-deoxy type or primer extension to incorporate a biotinylated nucleotide followed by addition of streptavidin with a strong fluorescent label .
  • the probes can then be transferred to a solid substrate and it is then possible to detect which probes are bound to a label and hence what mRNA sequences (and therefore genes) are present. Detection is conveniently achieved by microscope image analysis and the use of an IR laser. For example, a first laser may be employed to quantify the bound mRNA. A blue laser may then be employed to excite the nanocrystals allowing detection of which nanocrystals are bound to the target. This more sophisticated assay is also suitable for detecting proteins from specific cells, microorganisms etc or detecting other forms of nucleic acid, e.g. DNA. Thus, a variety of different probes are employed and using techniques as described above, it is possible to identify which probes have bound protein, DNA etc and hence which proteins, DNA etc are present.
  • the invention provides a method for detecting a plurality of nucleic acid sequences in a sample comprising mixing a plurality of different types of probes, each type of probe having a different gene-specific oligonucleotide affinity ligands, to a sample comprising free nucleic acid; allowing the probes to bind to a plurality of free nucleic acid sequences within the sample to form probe :nucleic acid conjugates; separating the conjugates from the sample, e.g. magnetically; labelling said conjugates, e.g. with a dT oligonucleotide; binding the conjugates to a solid substrate and detecting the labels/probes.
  • the probes of the invention may be of use in single nucleotide polymorphism (SNP) analysis, e.g. genotyping of disease related SNP's.
  • SNP single nucleotide polymorphism
  • a large number of SNP's will need to be analysed to determine a patient's genotype, either to characterise a disease or to design personalised therapies.
  • the relevant number of SNP's to be analysed from a single sample may be within the range of 10 to 2000.
  • a flexible, multiplexed SNP scoring platform is therefore required and a probe based platform is an example.
  • genomic DNA for genetic typing of a patient may be isolated from any cell e.g. a white blood cell, using conventional techniques, e.g. using an automator as sold by Thermo Labsystems .
  • SNP scoring may then be achieved using DNA sequencing, sequence specific oligonucleotide hybridisation (SSO) or sequence specific priming of a PCR reaction (SSP) .
  • SSO sequence specific oligonucleotide hybridisation
  • SSP sequence specific priming of a PCR reaction
  • the probes of the invention may also have utility in microfluidic devices .
  • the use of such devices for the rapid detection of DNA, proteins, or other molecules associated with a particular disease is described in detail in WOOl/94635 and WO02/29106.
  • Detection of targets is achieved by measuring a signal from a detectable reporter, e.g. a fluorescent reporter.
  • the probes of the present invention are suitable as detectable reporters for use in microfluidic devices and this forms a further aspect of the invention.
  • emulsion 5.0 ml of dioctanoylperoxide, 42 ml of H 2 0 and 0.15 g of sodium laurylsulphate is homogenised to an emulsion.
  • This emulsion is combined with 23.1 ml of a latex consisting of monosized polystyrene latex having a diameter of 0.95 ⁇ m.
  • the amount of latex added contains 2.5 ml of polystyrene particles and 20.6 ml of a solution of 0.15% SDS in water.
  • the mixture is cooled and washed with water and methanol to remove the cyclohexanol.
  • the process gives monosized particles.
  • Porous epoxy-functional acrylic beads with a pore volume of 50% and a diameter of 8.2 ⁇ m were made as described in WO 00/68692 and US 4,336,173
  • TOP trioctylphosphine
  • TOPO triooctylphosphineoxide
  • Fluorescence spectroscopy showed that the emission maximum of the colored bead dispersion was 608 nm.
  • the beads were analysed in a fluorescence microscope (Olympus BX61) .
  • the excitation source was a 100 W mercury lamp with a band pass excitation filter with transmission interval: 425- 475 nm, and a band pass emission filter with transmission interval: 595 - 625 nm.
  • the objective used was a 40x UplanApo.
  • the pictures were taken with a F-view digital camera (4096 grey levels) and analysed with Analysis Pro (Soft Imaging system) to extract the mean grey values of single beads .
  • the Qdot dispersion was added the bead dispersion while vortexing. After 15 hours the beads were washed with 1,4 dioxane .
  • the beads are transferred to water. Fluorescence microscopy shows that the beads are fluorescent .

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  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Nanotechnology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Materials Engineering (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Manufacture Of Porous Articles, And Recovery And Treatment Of Waste Products (AREA)

Abstract

L'invention concerne une sonde comprenant une perle de polymère éventuellement poreuse associée à au moins un type de nanocristaux. Cette perle et ces nanocristaux sont recouverts d'un revêtement polymère.
PCT/GB2002/002590 2001-06-06 2002-06-06 Sonde Ceased WO2002099425A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002302800A AU2002302800A1 (en) 2001-06-06 2002-06-06 Polymeric bead probe with nanocrystal, manufacture and use of the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0113772A GB0113772D0 (en) 2001-06-06 2001-06-06 Process
GB0113772.8 2001-06-06

Publications (2)

Publication Number Publication Date
WO2002099425A2 true WO2002099425A2 (fr) 2002-12-12
WO2002099425A3 WO2002099425A3 (fr) 2003-05-30

Family

ID=9916030

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2002/002590 Ceased WO2002099425A2 (fr) 2001-06-06 2002-06-06 Sonde

Country Status (3)

Country Link
AU (1) AU2002302800A1 (fr)
GB (1) GB0113772D0 (fr)
WO (1) WO2002099425A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1441227A3 (fr) * 2003-01-23 2004-12-29 Hitachi Software Engineering Co., Ltd. Billes fonctionnalisées, méthodes et appareils pour les lire
US8038987B2 (en) * 2003-07-17 2011-10-18 Invitrogen Dynal As Process for the preparation of coated polymer particles

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1116036B1 (fr) * 1998-09-18 2004-08-11 Massachusetts Institute Of Technology Nanocristaux semiconducteurs fluorescents hydrosolubles
US6426513B1 (en) * 1998-09-18 2002-07-30 Massachusetts Institute Of Technology Water-soluble thiol-capped nanocrystals
US6261779B1 (en) * 1998-11-10 2001-07-17 Bio-Pixels Ltd. Nanocrystals having polynucleotide strands and their use to form dendrimers in a signal amplification system
WO2000027365A1 (fr) * 1998-11-10 2000-05-18 Biocrystal Limited Nanocristaux fonctionnalises et leur utilisation dans des systemes de detection
US6235540B1 (en) * 1999-03-30 2001-05-22 Coulter International Corp. Semiconductor nanoparticles for analysis of blood cell populations and methods of making same
AU4701200A (en) * 1999-05-07 2000-11-21 Quantum Dot Corporation A method of detecting an analyte using semiconductor nanocrystals
US6696299B1 (en) * 1999-05-11 2004-02-24 Massachusetts Institute Of Technology Polarization label for measuring 3-dimensional orientation

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1441227A3 (fr) * 2003-01-23 2004-12-29 Hitachi Software Engineering Co., Ltd. Billes fonctionnalisées, méthodes et appareils pour les lire
US7153697B2 (en) 2003-01-23 2006-12-26 Hitachi Software Engineering Co., Ltd. Functional beads, method for reading the same and bead-reading apparatus
US8038987B2 (en) * 2003-07-17 2011-10-18 Invitrogen Dynal As Process for the preparation of coated polymer particles

Also Published As

Publication number Publication date
GB0113772D0 (en) 2001-07-25
WO2002099425A3 (fr) 2003-05-30
AU2002302800A1 (en) 2002-12-16

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