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WO2008084247A1 - Microsphère codée - Google Patents

Microsphère codée Download PDF

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Publication number
WO2008084247A1
WO2008084247A1 PCT/GB2008/000097 GB2008000097W WO2008084247A1 WO 2008084247 A1 WO2008084247 A1 WO 2008084247A1 GB 2008000097 W GB2008000097 W GB 2008000097W WO 2008084247 A1 WO2008084247 A1 WO 2008084247A1
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WIPO (PCT)
Prior art keywords
microsphere
microspheres
quantum dots
polymer
encoded
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PCT/GB2008/000097
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English (en)
Inventor
Robert Wilson
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UK Secretary of State for Defence
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UK Secretary of State for Defence
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Publication of WO2008084247A1 publication Critical patent/WO2008084247A1/fr
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic

Definitions

  • the present invention is directed to a method for encoding a microsphere, an encoded microsphere and uses thereof. It is particularly, although not exclusively, concerned with use of the encoded microsphere for multiplexed assays, especially in relation to explosive mixtures.
  • a suspension (three-dimensional) array can avoid many of these problems and offer reliable quantitative assay where specifically functionalised substrates can be manufactured in large quantity with high reproducibility.
  • the freedom of movement available to substrates in suspension means that they must contain some kind of code which enables them to be uniquely identified.
  • Commercial substrates for suspension array are based on polystyrene microspheres in which one or more fluorescent dyes have been trapped by a swell-shrink cycle.
  • the present invention generally aims to provide a method for the production of encoded microspheres which overcomes the above-mentioned limitations.
  • Quantum dots are known to give rise to size-dependent photoluminescent emission spectra of narrow bandwidth (20 to 30 nm) and have been used to label a wide variety of solid substrates.
  • polystyrene microspheres including a paramagnetic material can be labelled by trapping quantum dots (S. Mulvaney et al., BioTechniques, 2004, 36, 602- 607). Encoded non-paramagnetic polystyrene microspheres can be similarly prepared (X. Gao and S. Nie, Anal. Chem., 2004, 76, 2406 - 2410; M. Han, et al., Nature, 2001, 631-635).
  • a quantum dot labelled polystyrene microsphere has been prepared using layer-by- layer (LbL) chemistry (D. Wang et al., Nano Lett., 2002, 2(8), 857-861). This self- assembly chemistry has also been used for quantum dot labelling of negatively charged ferric oxide nanoparticles (X. Hong et al., Chem. Mater., 2004, 16, 4022- 4027).
  • LbL layer-by- layer
  • LbL chemistry can provide microspheres encoded by quantum dots on a large scale and with good reproducibility.
  • the present invention provides a method for encoding a microsphere comprising the steps of i) providing a layer of a polyionic polymer to the microsphere, ii) coating the layer with quantum dots and, optionally, repeating these steps, iii) providing one or more protective layers of a transparent polyionic polymer and iv) providing to the microsphere so obtained an overcoat of a transparent material which includes a moiety capable of recognising a target molecule or is, or can be, adapted for the attachment of a molecule capable of recognising a target molecule.
  • Quantum dot will be understood to refer to a particle of a semiconductor material having a dimension smaller than the exciton Bohr radius corresponding to the bulk material.
  • references to different quantum dots herein are references to the same and/or other such materials which have particle dimension such that they emit light at a predetermined wavelength of incident light which can be resolved from light emitted by any other. References herein to the same quantum dots will be construed accordingly.
  • microsphere will be understood to refer to a particle, particularly, although not essentially, to a particle of circular cross-section, which has largest dimension or mean diameter ranging from 1 to 10 ⁇ m.
  • the term "transparent" as used in relation to a polyionic polymer or a material will be understood to mean that the polymer or material permits the propagation of light capable of exciting the quantum dots as well as the propagation of light emitted from them.
  • the transparent polymer or material may, in particular, permit the propagation of wavelengths of light ranging from 250 to 1400 nm.
  • the microsphere is functionalised by an uncharged organic moiety capable of forming a covalent bond with a polyionic polymer.
  • the method provides a base, polyionic polymer layer which is covalently bound to the microsphere.
  • the covalently bound polymer layer is suitable for direct coating with quantum dots by step ii) and the method avoids the need for one or more priming layers.
  • the method may nonetheless include the preliminary step of providing one or more priming layers to the microsphere.
  • the preliminary step may use one or more polyionic polymers such that the final priming layer is of opposite charge to the polyionic polymer of step i).
  • the preliminary step may also result in a covalently bound base polymer layer - but embodiments in which the polymer is provided by an electrostatic interaction with the microsphere are also possible.
  • the method may use polyionic polymers of high molecular weight (> 10 kDa) for these steps. It may, in particular, use a high molecular weight polyamine (> 10 kDa) such as poly(allylamine), poly(ethyleneimine), poly(lysine) or chitin and/or negatively charged polymers such as poly(sodium 4-styrenesulphonate).
  • a high molecular weight polyamine > 10 kDa
  • negatively charged polymers such as poly(sodium 4-styrenesulphonate).
  • the method may additionally use high molecular weight (> 10 kDa) polyelectrolytic salts such as those based on poly(diallyldimethylammonium) or poly(4-vinylpryridine) for these steps.
  • high molecular weight polyelectrolytic salts such as those based on poly(diallyldimethylammonium) or poly(4-vinylpryridine)
  • suitable polyionic polymers will be known to those skilled in the art. It will be understood, however, that the method need not use a transparent polyionic polymer for these steps.
  • a transparent polyionic polymer is always required.
  • the method may use any one of the aforementioned polymers for repeat step i).
  • the microsphere may comprise any suitable material, for example, a functionalised latex or silica microsphere.
  • the microsphere is paramagnetic - so as to permit convenient handling by magnetic precipitation during the separation and re- suspension steps accompanying the method.
  • the microsphere may, in particular, comprise an epoxy-functionalised, paramagnetic or non-paramagnetic, polystyrene microsphere ( ⁇ 4.5 ⁇ m) - available from Dynal (UK) and Spherotech (US) respectively.
  • Paramagnetic microsheres of smaller diameter may also be suitable provided that they can be separated from suspension within a reasonable time period.
  • a suitable paramagnetic microsphere of diameter 300 run (Estapor®) has recently become commercially available from Merck.
  • the method may use quantum dots which comprise compounds of Group II-VI elements, for example CdSe, or Group III-V elements, for example InP. It may use quantum dots having an overcoat of a material of larger band gap but limited mismatch in crystal structure (for example, ZnS) - in order to increase photoluminescent quantum yield.
  • quantum dots which comprise compounds of Group II-VI elements, for example CdSe, or Group III-V elements, for example InP. It may use quantum dots having an overcoat of a material of larger band gap but limited mismatch in crystal structure (for example, ZnS) - in order to increase photoluminescent quantum yield.
  • the amount of quantum dots deposited to a polyionic layer is determined largely by surface area.
  • the method therefore, reproducibly loads a precise amount of quantum dots to the microsphere. ⁇
  • the intensity of the photoemission spectrum of a microsphere on which steps i) and ii) have been repeated once is roughly twice the intensity of that of a microsphere on which these steps have been performed only once.
  • the method may, therefore, repeat these steps a predetermined number of times whereby to load the microsphere with a desired amount of one or more quantum dots.
  • the method encodes the microsphere by the wavelength and/or intensity of its photoemission spectrum on excitation at a predetermined wavelength of incident light.
  • the wavelength of incident light is chosen to be lower than the wavelength of the first exciton peak of the quantum dots.
  • the incident light may, for example, comprise a wavelength ranging from 350 to 400 run.
  • the method may use an excess of quantum dots for all steps ii) and repeat steps i) and ii) from 1 to 20 times (for example, at least once, twice or three times) using the same and/or different quantum dots to those used in any one preceding step ii).
  • the method may, in particular, repeat these steps using different quantum dots for each step ii) to those used in any preceding step ii).
  • the amount of quantum dot encoding the microsphere is to a lesser extent also controlled by the number of priming layers and/or the inclusion of one or more intervening layers.
  • the method comprises the additional step of providing one or more intervening layers of a transparent polyionic polymer following at least one step ii).
  • the method may use the high molecular weight polymers mentioned in relation to repeat step i) for the additional step.
  • steps i) and ii) can be repeated with or without the additional step appears limited only by practical considerations of time and economy.
  • a paramagnetic microsphere is particularly advantageous - in that it permits rapid separation of the microsphere from solution by magnetic precipitation.
  • the method may, therefore, avoid time consuming and complicated separation techniques such as filtration and centrifugation.
  • steps i) and ii) of the method are performed in a protic solvent and step ii) uses an excess of quantum dots (opposite in charge to the polymer).
  • the quantum dots may be capped by a negatively charged moiety - for example, thioalkyl carboxylate.
  • Such quantum dots can be obtained by treatment of commercially available tri-n-octylphosphine oxide (TOPO)- or tri-n- octylphosphine (TOP)-capped quantum dots with mercaptoacetic acid.
  • TOPO tri-n-octylphosphine oxide
  • TOP tri-n- octylphosphine
  • the method may provide for encoding by consecutive layers of polymer coated in the same quantum dots.
  • the method may comprise five consecutive steps ii) using green quantum dots followed by five consecutive steps ii) using red quantum dots.
  • step i) of the method is performed in a protic solvent and step ii) is performed in an aprotic solvent using an excess of quantum dots.
  • the method may use TOPO- or TOP-capped quantum dots.
  • quantum dots capped by other hydrophobic ligand may be suitable - especially if they are monovalent and/or can be displaced by, or interact with, the polyionic polymers used for step i).
  • step i) is followed by a drying step and that step ii) is followed by a wetting step.
  • the drying step may comprise washing with a suitable organic solvent - but other means for removing the protic solvent may be employed.
  • the wetting step may comprise washing with an organic solvent whereby to substantially remove the aprotic solvent.
  • the aprotic solvent may, in particular, comprise chloroform - in which case both the drying and the wetting step may comprise washing with methanol.
  • a single coating of quantum dots is sufficient for high intensity photoemission spectra according to this embodiment - especially where the polyionic polymer used for steps i) is poly(ethyleneimine).
  • the method may perform all steps ii) in an aprotic solvent. Alternatively, it may perform at least one step ii) in protic solvent and at least one step ii) in protic solvent.
  • step iii) of the method comprises providing one or more layers of a transparent polyionic polymer. It may, in particular, comprise repeating step i) or repeating the preliminary or additional step. The method may use any of the above- mentioned polymers for this step.
  • the protective layer encapsulates the QD assembly and provides an outer layer to the microsphere for the attachment of a transparent polymer or for coating with transparent nanoparticles.
  • step iv) of the method comprises coating the protective layer with transparent nanoparticles.
  • Step iv) may, in particular use silica or germanium oxide nanoparticles - but other transparent nanoparticles, comprising for example chalcogenides, are envisaged. Negatively charged silica nanoparticles are available under the trade name Ludox® (Grace & Co, USA).
  • step iv) may comprise the additional step of providing one or more layers of a low molecular weight ( ⁇ 10 kDa) transparent polyionic polymer, such as those mentioned above, to the glass coated 'microsphere and coating the polymer with transparent nanoparticles.
  • a low molecular weight ( ⁇ 10 kDa) transparent polyionic polymer such as those mentioned above
  • the resultant (outer) vitreous coating can be functionalised by a convenient and short protocol which permits the covalent attachment of a wide variety of molecules.
  • silanization of the vitreous coating and chemical modification of the silanized surface whereby to covalently attach or to permit covalent attachment of a molecule capable of recognising the target molecule.
  • the method may include the further step of functionalising the vitreous coating and covalently attaching to the functionalised microsphere a molecule capable of recognising the target molecule.
  • step iv) of the method may comprise providing a layer of a transparent polymer to the (outer) protective layer which polymer includes a moiety capable of recognising the target molecule.
  • the polymer may electrostatically anchor or covalently attach to the protective layer.
  • target molecule - suitable target molecules comprise chemical compounds, antibodies, anti-antibodies, receptors or nucleic acids sequences.
  • the molecule or moiety capable of recognising the target molecule may, therefore, comprise any conventional probe - such as antibody, aptamer, hapten, oligonucleotide sequence or a ligand recognising a particular receptor.
  • step iv) of the method uses a transparent polymer (for example, a dextran) comprising an analogue or hapten for one of the explosives RDX, PETN or TNT (see G.M. Blackburn et al., J. Chem. Soc, Perkin Trans. I, 2000, 225-230; R. Wilson et al., Anal. Chem., 2003, 75, 4244-4249).
  • a transparent polymer for example, a dextran
  • an analogue or hapten for one of the explosives RDX, PETN or TNT see G.M. Blackburn et al., J. Chem. Soc, Perkin Trans. I, 2000, 225-230; R. Wilson et al., Anal. Chem., 2003, 75, 4244-4249.
  • the present invention provides for an encoded microsphere obtainable according to the first aspect.
  • the present invention provides an encoded microsphere comprising a core-shell structure in which the core is comprised by the microsphere and the shell by one or more quantum dot coated polyionic polymer layers.
  • the shell comprises a first layer of quantum dot coated polymer which is covalently bound to the core microsphere.
  • the shell comprises a base, priming layer of polyionic polymer which is covalently bound to the core microsphere.
  • the shell may comprise one or more additional priming layers of polyionic polymer provided to the base priming layer.
  • the priming layer(s) and the first layer of quantum dot coated polymer need not comprise a transparent polymer - but the second (and any subsequent) polymer layer should comprise a transparent polyionic polymer.
  • the priming layer(s) and the first quantum dot coated polymer layer may, in particular, comprise high molecular weight (> 10 kDa) polyamines - for example, poly(allylamine), poly(ethyleneimine), poly(lysine) and chitin and poly(sodium 4- styrenesulphonate).
  • the second (and any subsequent) transparent polyionic layer may be comprised from the above-mentioned polymers as well as polyionic polymers based on quaternary salts - such as poly(diallyldimethylammonium) and poly(4-vinylpyridine).
  • the shell may comprise any arrangement of quantum dot coated polymer layers. It may, in particular, comprise at least two, three, four or five overlying polymer layers each coated with one type of quantum dots.
  • the shell may alternatively or additionally comprise at least two, three, four or five overlying polymer layers each coated with different quantum dots from one another.
  • It may, in particular, comprise multiple layers of polymer coated with one type of quantum dots in which each layer is separated from the other by one or more layers of a polymer coated with a different type of quantum dots.
  • the shell may also comprise one or more intervening layers of a transparent polyionic polymer which separate one or more quantum dot coated polymer layers.
  • the polyionic polymer of the intervening layer(s) may comprise one or other of the above- mentioned polymers.
  • the shell includes one or more protective layers of a transparent polyionic polymer which encapsulates the QD assembly.
  • the protective layer(s) may, in particular, comprise any of the high molecular weight polymers mentioned in relation to repeat step i).
  • the shell includes an overcoat of a transparent material which includes a moiety capable of recognising a target molecule or is or can be adapted to recognise a target molecule.
  • the overcoat may, in particular, comprise a vitreous coating formed by treating the protective polymer layer with silica or germanium oxide nanoparticles. It may comprise additional layers of a low molecular weight ( ⁇ 10 kDa) transparent polyionic polymer, for example polyamine, each of which is coated with silica or germanium oxide nanoparticles.
  • a low molecular weight ( ⁇ 10 kDa) transparent polyionic polymer for example polyamine, each of which is coated with silica or germanium oxide nanoparticles.
  • the vitreous coating is functionalised by covalent attachment of a molecule capable of recognising the target molecule.
  • the overcoat may comprise a layer of a transparent polymer including a moiety capable of recognising the target molecule which is electrostatically anchored or covalently attached to the (outer) protective layer.
  • the overcoat comprises a polymer (for example, a dextran) including an analogue or hapten for one of the explosives RDX, PETN or TNT (see G.M. Blackburn et al., J. Chem. Soc, Perkin Trans. I, 2000, 225-230; R. Wilson et al., Anal. Chem., 2003, 75, 4244-4249).
  • a polymer for example, a dextran
  • an analogue or hapten for one of the explosives RDX, PETN or TNT
  • the core microsphere may comprise any suitable material, for example functionalised latex or silica.
  • the microsphere is paramagnetic.
  • the present invention provides for a library comprising a plurality of encoded microspheres according to the second aspect of the invention.
  • the library may, in particular, be obtained according to the first aspect of the invention.
  • the method is used to produce a plurality of microspheres each encoded by the wavelength and/or intensity of its photoemission spectrum on excitation at a predetermined wavelength of incident light.
  • the library may, in particular, comprise encoded microspheres including a protective vitreous overcoat - for functionalising by the end user.
  • the library may comprise encoded microspheres which have already been adapted for a specific use.
  • the library may comprise microspheres encoded by different colours or by the same colour or colours at different intensities.
  • the encoding may rely on relative as well as absolute intensities.
  • the present invention provides for use of the encoded microsphere or the library for multiplexed assays, multiplexed screening or for combinatorial synthesis.
  • Such use may provide for the detection of a variety of materials including drug, pesticide, explosive and biological materials.
  • a competitive (reagent limited) immunoassay comprises exposing a sample of suspected explosive material to a mixture comprising two or more suitable antibodies and two or more encoded microspheres, each of which codes for a different hapten for one or other of the explosives RDX, PETN or TNT.
  • Suitable antibodies comprise antibodies for RDX, PETN or TNT and may, for example, carry a label or a moiety to which a label can be attached following the exposure, hi the example below, biotinylated antibodies for RDX, PETN or TNT are labelled after the exposure by sequential treatment with an excess of polystreptavidin and AlexaFluor 660 biotinylated dextran.
  • the use may also provide for the detection of bacteria, spores and viruses, especially pathogenic types, by targeting certain antigen or nucleic acids sequences.
  • the multiplexed assays may comprise, for example, a sandwich (reagent excess) assay. It may, in particular, comprise exposing a suitable sample of the suspected material to a mixture comprising two or more suitable (detector) antibodies and two or more encoded microspheres, each of which codes for a (capture) antibody for a different target molecule.
  • the detector and capture antibodies may comprise antibodies for the material raised in the same animal species (X) - except where it is desired to label the detector antibodies by anti-species antibody. In that case, the capture antibodies and the anti- species antibody must be raised in a different animal species (Y).
  • the assay can comprise exposing a suitable sample of the suspected material to two or more encoded microspheres, each of which is conjugated to an oligonucleotide capable of hybridising to a different target oligonucleotide.
  • the assay may, in particular, target a RNA or DNA sequence - and if appropriate include a preliminary amplification (PCR or ligase chain reaction) step.
  • the label may be incorporated during the amplification step.
  • it may be incorporated via a detector oligonucleotide or simply bind to the hybridised molecule.
  • the label may comprise a fluorescent label - but other types of label, are also contemplated.
  • the fluorescent label should emit at a wavelength of light which is resolvable from the light emitted by the various quantum dots at the predetermined wavelength of incident light.
  • the fluorescent label emits in the red region. It may, for example, comprise AlexaFluor 660 or Cy-5.
  • the detection instrument may, in particular, comprise one or more optical detectors arranged in combination with a number of filters for fixed imaging or high throughput detection.
  • the detection instrument comprises a flow cytometer.
  • the detection instrument may be associated with a computer programme that decodes the encoded microsphere.
  • the library or encoded microspheres may be included in a test strip (lateral flow) device allowing use with, for example, a fixed imager, in the field and/or by low trained personnel.
  • the encoded microspheres typically have mean diameter about a tenth of the medium through which they are to be transported. Suitable diameters are 300 nm or less and in particular, are lower than 150 nm and preferably lower than 100 nm.
  • the present invention provides a reliable method for large scale production of encoded microsphere with good reproducibility.
  • the method permits very precise loading with quantum dots of one or colours and in controlled amounts.
  • the method offers high levels of encoding and multiplexed assays for many more analytes than is possible with dye encoded microspheres.
  • the encoded microsphere may be loaded with additional colours of , quantum dot and that each colour may be loaded to a specific intensity.
  • N C v - 1 where C is the number of colours and v is the number of intensities. Loading with just five different colours of quantum dot in five different amounts may provide 3124 resolvable codes - many more in practice than with fluorescent dyes.
  • each quantum dot may be excited at the same (predetermined) wavelength of incident light use of the encoded microsphere is not limited by a requirement for decoding instruments having a large number of excitation sources.
  • a paramagnetic core provides for easy and rapid separation of the microsphere from aqueous and aprotic solution. It avoids the need for costly and time consuming filtration and/or centrifugation steps and may permit automation of the method as well as automation in use.
  • Another advantage of the method is that it can avoid the need to prepare and/or use hazardous mercapto-capped quantum dots.
  • the method also enables production of an encoded microsphere including a vitreous protective layer which can be functionalised according to short and convenient protocols - so permitting versatile use.
  • the method provides an encoded microsphere which may be rapidly adapted to different target molecules for multiplexed assays, multiplexed screening or combinatorial synthesis.
  • Figure 1 is a scheme showing an encoded microsphere according to a first embodiment of the present invention
  • Figure 2 is a graph plotting zeta ( ⁇ ) potentials against number of layers and coatings obtained by microelectrophoresis during the production of the encoded microsphere of Figure 1 according to a first embodiment of the method of the present invention
  • Figure 3 a shows UV/visible absorption spectra obtained by LbL assembly of quantum dots on a quartz cuvette in accordance with the first embodiment of the method of the present invention
  • Figures 3 b) and c) shows graphs plotting UV/visible absorbance obtained by
  • Figure 4 shows photoemission spectra of a library of encoded microspheres produced according to the first embodiment of the method of the present invention
  • Figure 5 shows a UV/visible difference absorption spectrum reporting the attachment of a haptenylated dextran to an LbL assembly of quantum dots on a quartz cuvette in accordance with the first embodiment of the method of the present invention
  • Figure 6 a) to d) shows atomic force microscopy (AFM) images obtained during LbL assembly of quantum dots on a glass microslide in accordance with the first embodiment of the method of the present invention
  • Figures 7 a) to c) shows transmission electron microscopy (TEM) images obtained during the production of the encoded microsphere of Figure 1 in accordance with the first embodiment of the method of the present invention
  • Figure 8 is a scheme showing the production of an encoded microsphere according to a second embodiment of the method of the present invention.
  • Figure 9 is a graph plotting ⁇ -potentials against number of layers and coatings obtained by microelectrophoresis during production of an encoded microsphere in accordance with the second embodiment of the method of the present invention.
  • Figures 10 a) and b) shows UV/visible absorption spectra and photoemission spectra obtained by LbL assembly of quantum dots on a glass cuvette in accordance with the second embodiment of the method of the present invention
  • Figure 10 c) shows photoemission spectra of a library of encoded microspheres produced in accordance with the second embodiment of the method of the present invention
  • Figures ll a) and c) are respectively scanning electron microscopy (SEM) and
  • Figure 11 b shows high magnification SEM images obtained during the production of an encoded microsphere according to the second embodiment of the present invention
  • Figure 12 is a graph plotting the intensities of photoemission spectra obtained in microspheres encoded with 1 to 5 coatings of the same quantum dot;
  • Figures 13 a) and b) and 14 are schemes illustrating use of a library of encoded microspheres according to the present invention
  • Figure 15 shows photographs of the photoluminescence of certain microspheres coated with streptavidin and conjugated to a biotinylated oligonucleotide probe sequence
  • Figure 16 shows photographs of agarose gel chromatograms obtained following PCR of a template DNA and imaged by fluorescence of ethidium bromide (A) and of Cy-5 dye.
  • an encoded microsphere comprises a core-shell structure, in which a paramagnetic polystyrene microsphere 11 is surrounded by a shell 12 comprising a series a), b), c) of five concentric layers of a transparent polyionic polymer coated with the same quantum dots (green (G), red (R) or yellow (Y)).
  • the shell includes protective layers of transparent polyionic polymers 12 d) and an outer layer of a haptenylated dextran 12 e).
  • the encoded microsphere 10 was produced in accordance with a first embodiment of the method of the present invention - all steps in aqueous solution:
  • step i Paramagnetic, epoxy-functionalised microspheres (200 ⁇ l; ⁇ 4 x 10 microsphere/ml water; mean diameter 4.5 ⁇ m; CV. ⁇ 5%; Dynal, UK) were washed (5 x 1 ml) and re- suspended in water (0.5 ml). To the vortexed suspension was added a solution of polyallylamine hydrochloride (PAH; Sigma; MW 70 kDa; 100 mg/ml; pH 8.0) which was prepared by dissolving in 1 M NaCl solution and diluting (1:1) with a solution of 0.1 M sodium tetraborate.
  • PAH polyallylamine hydrochloride
  • PAA polyallylamine
  • microelectrophoresis measurements (Brookhaven ZetaPlus potential analyser) made in air-equilibrated HPLC grade water (pH 6.5; 0.005 w% microspheres) show a change in ⁇ -potential of the microspheres.
  • the change from - 10.9 mV to + 40.12 mV confirms the deposition of a PAA layer to the microsphere.
  • Brightfield images show that there was no cross-linking of microspheres.
  • TEM images (JEOL TEM 2000 FX microscope operating at 200 V; not shown) show a thin halo surrounding the PAA polymer microsphere which was not present in similar images of the epoxy-functionalised microsphere.
  • TOPO coated CdSe/ZnS core shell quantum dots (Evident Technologies, USA; 100 ⁇ l in toluene) were centrifugally precipitated with methanol (4 x 1 ml) at 900Og (15 min.) in a sealable polypropylene vial.
  • the pellet was suspended in chloroform (50 ⁇ l) and to the suspension was added thioglycolic (mercaptoacetic) acid (MA; 25 ⁇ l; Sigma) followed by a solution (25% in methanol) of tetramethylammonium hydroxide (TMA; 25 ⁇ l; Sigma).
  • TMA thioglycolic
  • TMA tetramethylammonium hydroxide
  • Microelectrophoresis measurements confirm the deposition of the green quantum dots to the PAA layer.
  • the ⁇ -potential of the microspheres became less positive but not negative (+ 9.93 mV) - suggesting either that the microspheres is not completely covered by quantum dots or that the PAA layer partly envelops them.
  • Epifluorescence imaging (Leica DMBL fluorescence microscope with SPOT 2 camera (using 10Ox objective lens at a magnification of 100Ox) from SPOT Diagnostics, USA) shows that the microspheres were uniformly photoluminescent.
  • the coated microspheres were re-suspended in a solution of branched polyethylene- imine (PEI; MW 750 kDa; Sigma; 1 mg/ml) in 0.5 M sodium chloride (pH 8.0) and rotated (15 min.).
  • the PEI covered microspheres were magnetically precipitated and washed with water (4 x 1 ml).
  • the PEI covered microsphere shows greater photoluminescence than the coated microsphere - which is surprising given that UV/visible measurements of these steps applied to a quartz cuvette show that about 30% of the quantum dots are displaced by PEL
  • AFM images show that the surface of the PEI polymer slide (d) is smoother than the surface of the coated PAA slide (c) and that the latter is more uneven than the surface of the PAA polymer slide (b).
  • step H To a suspension of (PEI) polymer microspheres in water (1 ml) is added MA functionalised, green quantum dot and the mixture slow tilt rotated (15 min). The coated microspheres were magnetically precipitated and washed with water (4 x 1 ml; HPLC grade, pH 6.5).
  • Steps i) and ii) were repeated three times to give (5G) microspheres - five polymer layers each coated with green quantum dot.
  • Steps i) and ii) were repeated a further five times with MA-functionalised red quantum dot to give (5G-5R) microspheres.
  • Microelectrophoresis measurements confirm the successive deposition of PEI layer and quantum dot coating. As may be seen, the ⁇ -potential of the microspheres oscillated between + 38.65 mV and + 10.46 mV.
  • Figure 3 a shows the UV/visible spectra obtained when these steps (5G-5R) are applied to a quartz cuvette.
  • Figures 3 b) and c) show a linear increase (upper part) in the absorbance of the first exciton peak of quantum dots with number of coatings on the cuvette and that the intensity of luminescence (lower part) of microspheres similarly , increased in line with the number of coatings (1-5-G; 1-5-R) on the microsphere.
  • Steps i) and ii) were repeated a further five times with MA-functionalised yellow quantum dot to give (5G-5R-5Y) microspheres.
  • the library comprises 5G, 5G-5R and 5G-5R-5Y encoded microspheres.
  • the intensity of luminescence from the green quantum dots decreases when the microsphere also includes quantum dot that emits at longer wavelength. The effect may be due to radiative and/or non-radiative energy transfer.
  • Step i) was used to obtain PEI covered (5G-5R-5Y) encoded microspheres. These microspheres (60 ⁇ g) were slow tilt rotated (15 min.) with 0.5M NaCl solution containing poly(sodium 4-styrenesulphonate) (PSS: MW 70 kDa; Sigma; 1 mg/ml). The PSS covered microspheres were magnetically precipitated and washed with water (4 x 1 ml). These steps were repeated to give (PEIZPSS) 3 covered microspheres.
  • PSS poly(sodium 4-styrenesulphonate)
  • PEI (5G-5R-5Y) encoded microspheres (60 ⁇ g) including protective (PEI/PSS) 3 layers obtained above were re-suspended in a solution of 0.1 M sodium bicarbonate.
  • SPDP dimethylformamide
  • DMF dimethylformamide
  • the addition was repeated and after further rotation (30 min.) the microspheres were washed with 0.1M sodium bicarbonate solution (4 x 1 ml) and phosphate buffer solution (PBS; 15 mM sodium phosphate, 0.15 M NaCl; pH 7.4; 4 x 1 ml).
  • the microspheres were re-suspended in a solution of DTT (10 mM) in 0.1 M sodium bicarbonate solution and slow rotated (15 min.).
  • the microspheres were immediately re-suspended in buffer solution (0.33 ml; 3 x PBS; 45 mM sodium phosphate, 0.45 NaCl; pH 7.4). To the suspension was added a solution (0.66 ml) of haptenylated PDP-dextran in water and the mixture slow tilt rotated at 4 0 C overnight. The haptenylated microspheres were washed with PBS (4 x 1 ml) and water (4 x 1 ml) and stored in water at 4 0 C in darkness.
  • microelectrophoresis measurements confirm attachment of the haptenylated-PDP-dextran.
  • the ⁇ -potential of the microspheres becomes negative (-20.95 mV).
  • the difference UV/visible spectrum obtained by subtracting the underlying spectrum of the LbL assembly from the spectrum acquired for the haptenylated LbL assembly on a quartz cuvette shows a peak at 362 nm corresponding to DNP-PDP-dextran.
  • a competitive assay for TNT employed a suspension array of encoded microspheres produced according to Example 1.
  • a sample solution was prepared by dissolving TNT in acetonitrile and diluting to suitable concentration with PBS solution containing BSA (PBS-BSA; 10 mg/ml).
  • the suspension array (as follows 5G-PETN, 5G-5R-TNT and 5G-5R-5Y-RDX) was suspended in the sample solution (0.5 ml) and the mixture diluted (1:2) with a solution of biotinylated antibodies to PETN, TNT and RDX in PBS-BSA to 25 nM each antibody.
  • the suspension array was slow tilt rotated (30 min.), washed with PBS and re- suspended in PBS-BSA containing an excess of polystreptavidin (DakoCytomation, DK, supplied as 5.9 ⁇ M dextran solution, mean 19 polystreptavidin molecules per molecule dextran). After further rotation (15 min.), the array was washed with PBS and re-suspended in PBS-BSA containing an excess of AlexaFluor 660 biotinylated dextran. After further rotation (15 min) the array was washed with PBS (3 x 1 ml) and re-suspended in PBS (25 ⁇ l) for epifluorescence imaging.
  • DK polystreptavidin
  • biotinylated antibodies to PETN and RDX bind to the corresponding microspheres - but antibodies to TNT do not bind. Consequently, there is no binding of polystreptavidin to the microsphere encoding TNT and no labelling by binding to biotinylated AlexaFluor 660.
  • the microspheres specific for TNT are not visible through the AlexaFluor window (far right of Table I).
  • Example 3 Multiplexed Assays A competitive assay similar to Example 2 was used for sample solutions that contained PETN and RDX in ratios found in Semtex A and Semtex H. The assay reports the presence of these explosive materials at concentrations of PETN and RDX as low as 1000 ppb and 50 ppb respectively.
  • step i) is performed in aqueous solution and steps ii) are performed in chloroform (HCCl 3 ):
  • step i) is performed in aqueous solution and steps ii) are performed in chloroform (HCCl 3 ):
  • step i) is performed in aqueous solution and steps ii) are performed in chloroform (HCCl 3 ):
  • paramagnetic epoxy-functionalised microspheres (6 mg) in water (500 ⁇ l) was added a solution (500 ⁇ l) of branched chain PEI (MW 750 kDa, 100 mg/ml) of 1 M NaCl.
  • the (PEI) polymer microspheres were washed sequentially with 0.5 M NaCl solution (4 x 1 ml), 0.1 M sodium borate solution (pH 9.5; 4 x 1 ml), 0.1M sodium acetate solution (pH 4.5; 4 x 1 ml) and 0.5 M sodium chloride solution (6 x 1 ml).
  • the PEI covered microspheres were re-suspended in water and stored at 4°C.
  • Microelectrophoresis measurements confirm the deposition of PEI layer - the negative ⁇ -potential of the epoxy-functionalised microspheres becomes positive.
  • PEI polymer microspheres were magnetically precipitated from a suspension (60 ⁇ g) in water (20 ⁇ l) and washed with HPLC grade water (pH 6.5; 4 x 1 ml) and then methanol (4 x 1 ml). The microspheres were re-suspended in chloroform (1 ml) and to the suspension was added an excess of TOPO-capped green quantum dot. The mixture was slow tilt rotated (Ih). The coated microspheres were magnetically precipitated and washed with chloroform (1 ml), methanol (4 x 1 ml) and then water (1 ml).
  • Microelectrophoresis measurements confirm the deposition of the quantum dot coating - the ⁇ -potential of the microspheres again becomes less positive.
  • the coated microspheres were slow tilt rotated (1 h) with a solution (1 ml, pH 8.0) of 0.5 M NaCl containing PEI (1 mg/ml).
  • the PEI covered microspheres were magnetically precipitated, washed with water (4 x 1 ml) and then slow tilt rotated (Ih) with a solution (1 ml, pH 6.0) of 0.5 M NaCl containing PSS (1 mg/ml).
  • the PSS covered microspheres were washed with water (4 x 1 ml) and slow tilt rotated (1 h) with a solution (1 ml, pH 8.0) of 0.5 M NaCl containing PEI (1 mg/ml).
  • the PEI covered microspheres were washed with HPLC grade water (4 x 1 ml) and methanol (4 x 1 ml) and re-suspended in chloroform (1 ml). To the suspension was added an excess of TOPO-capped quantum dot and the mixture was slow tilt rotated (Ih). The coated microspheres were magnetically precipitated and washed with chloroform (1 ml), methanol (4 x 1 ml) and then water (1 ml). Subsequent layers and coatings
  • PEI covered (IG) microspheres were obtained and coated with TOPO-capped red quantum dots according to the steps described above.
  • the PEI (IG- IR) microspheres so obtained were similarly coated with TOPO-capped yellow quantum dots to give PEI covered (IG- IR- IY) microspheres.
  • Figure 10 a shows the UV/visible spectra obtained when these steps (5G-5R) are applied to a quartz cuvette. Again a linear increase in the absorbance of the first exciton peak when multiple coatings of the same quantum dot are deposited is found. Vitreous Overcoat (step iv)
  • the coating step was repeated together with the step providing PEI to give (IG- IR- IY) encoded microspheres having an overcoat of SiNP/PEI/SiNP/PEI/SiNP layers.
  • the successive deposition of PEI layer and SiNP coating is confirmed by microelectrophoresis measurements ( Figure 2) - the ⁇ -potential of the microspheres oscillates between positive and negative values.
  • UV/visible measurements show that the overcoat does not increase the absorbance of the microspheres in the range 200 - 700 run or decrease the intensity of the photoemission spectrum.
  • the size of the microspheres after the application of the overcoat (b- IV) is similar to the size of the unloaded microspheres (b-I).
  • the core-shell structure is again confirmed by confocal imaging of individual microspheres (not shown).
  • the photoemission spectra obtained from a library of IG (I), 1G-1R (II) and 1G-1R-1Y (III) encoded microspheres again show that the intensity of luminescence from green quantum dots decreases when the microsphere includes quantum dots emitting at longer wavelength (red). The effect may be due to radiative and/or non-radiative energy transfer.
  • the photoluminescence intensities of microspheres including 1 to 5 layers of the same quantum dot show four distinctly resolvable groups of microsphere.
  • the molar ratio of PDP to albumin was determined as ⁇ 2:1 by UV absorbance measurements (PDP at 343 nm after reduction with dithiothreitol (DTT); albumin at 280 nm corrected for PDP).
  • Albumin Microspheres (5G-5R-5Y) Encoded microspheres (60 ⁇ g) including the protective SiNP/PEI/SiNP/PEI/SiNP layers mentioned above were washed with ethanol (4 x 1 ml) and slow tilt rotated (overnight) with a mixture (1 ml) of (95/3/2) ethanol/water/aminopropyltriethoxysilane (APTS). The microspheres were washed with ethanol (4 x 1 ml) and re-suspended in 0.1 M sodium bicarbonate solution (0.5 ml). The microspheres were added to a solution (0.5 ml) of 0.1 M sodium bicarbonate solution containing 2-iminothiolane, hydrochloride salt (1 mg) and the mixture slow tilt rotated (30 min.).
  • the microspheres were re-suspended in PBS (1 ml) containing PDP-albumin (0.5 mg). The mixture was slow tilt rotated (overnight) and the microspheres washed with PBS (4 x 1 ml). The microspheres were then slow tilt rotated (1 h) with blocking solution (PBS containing gelatin (cold water fish skin; 10 mg/ml; 1 ml) and washed with PBS (4 x 1 ml) and stored in darkness.
  • PBS containing gelatin (cold water fish skin; 10 mg/ml; 1 ml)
  • a competitive assay for BSA employed a suspension array of encoded microspheres produced according to Example 4:
  • a suspension array was prepared by mixing equal amounts of Example 3 encoded microspheres as follows 3G-O V A, 2G- IR-BSA and 5G-5R-5Y-HSA in antibody diluent.
  • a sample solution (10 ⁇ g/ml) was prepared by dissolving BSA albumin in antibody diluent containing anti-albumin (anti-OVA, anti-BSA (mouse ascites fluid), anti-HSA; Sigma; IgG 5 ⁇ g/ml).
  • the suspension array was incubated with the sample solution (1:1 v/v) and the mixture slow tilt rotated (15 min.).
  • the microspheres were magnetically precipitated and washed with PBS containing 0.05% Tween-20® (1 x 1 ml).
  • the microspheres were then slow tilt rotated in antibody diluent containing Cy-5- labelled antimouse antibodies (AbCam, UK); IgG 10 ⁇ g/ml), washed with PBS containing Tween® and imaged with an epifluorescence microscope.
  • the table shows that no anti-BSA antibody binds to the 2G- IR encoded microsphere because they are bound to BSA in solution. Consequently, when the array is incubated with antibody specific to the antibodies there is no binding to the 2G- IR encoded microspheres. Microspheres specific for BSA are not visible when imaged through the Cy-5 window (far right of Table II).
  • a multiplex assay for detection of target bacteria can rely on amplicification of the 16s ribosomal RNA gene. This gene is present in all bacteria but incorporates specific sequences which vary according to bacterium (A).
  • Sequences in the gene which are conserved allow for amplification using a single pair of primers, which can bind adjacent the specific sequences, with incorporation of fluorescent dye Cy-5 (B).
  • the single strand oligonucleotide sequences obtained by treatment of the amplified genes with 5 ⁇ 3 exodeoxyribonuclease ( ⁇ -exonuclease) are characteristic of the target bacteria.
  • the presence of the target bacteria can be revealed by fluorescence of the dye following hybridisation with a library of encoded microspheres each coated with streptavidin and conjugated to a doubly biotinylated oligonucleotide probe for one or other of the strands.
  • Figure 15 confirms binding of Cy-5 labelled oligonucleotides to commercially available paramagnetic microspheres (A, Dynal) and encoded microspheres according to the present invention (B) - each coated with streptavidin and conjugated to a biotinylated oligonucleotide probe.
  • both microspheres could detect single stranded DNA - but the encoded microspheres according to the present invention showed stronger images at all concentrations than the commercial microspheres whilst maintaining a low background signal.
  • an agarose gel chromatogram of the products of a polymerase chain reaction (PCR) on a substrate deoxyribonucleic acid confirms that amplification of the substrate can be obtained with conservation of primers incorporating Cy-5 dye and that the products are converted to single strand oligonucleotides by ⁇ -exonuclease (cf: A4 with B4 and B5; 5 includes enzyme).
  • Encoded microspheres according to the present invention were prepared using TOPO- capped quantum dots as follows:
  • IM NaCl containing 100 mg ml "1 high molecular weight PEI was mixed 1:1 (v/v) with a vortexed suspension containing 6 mg of washed epoxy microspheres in 500 ⁇ l of water, and rotated overnight at room temperature. The microspheres were then washed with 1) 4 x 1 ml of 0.5 M NaCl; 2) 4 x 1 ml of 0.1 M sodium borate; 3) 4 x 1 ml of 0.1 M sodium acetate, pH 4.5; 4) 6 x 1 ml of 0.5 M NaCl and re-suspended in water.
  • microspheres were magnetically precipitated and washed with 1 ml chloroform, 4 x 1 ml of methanol and 1 ml of water and then rotated for 1 hour in 1 ml of an aqueous solution containing 1 mg ml "1 PEI and in 0.5 M NaCl (pH 8.0).
  • microspheres were then washed with 4 x 1 ml of HPLC grade water and 4 x 1 ml of methanol and again rotated in 1 ml of chloroform containing an excess of TOPO capped quantum dots for one hour.

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Abstract

La présente invention concerne un procédé permettant de coder une microsphère qui comprend les étapes consistant à : i) fournir une couche de polymère polyionique à la microsphère, ii) enduire la couche avec des points quantiques, iii) éventuellement, répéter les étapes i) et ii), iv) fournir une ou plusieurs couches protectrices d'un polymère polyionique transparent et v) fournir à la microsphère ainsi obtenue une couche de revêtement d'une substance transparente qui est ou qui peut être adaptée en vue de la fixation d'une molécule ou d'un groupe capable de reconnaître une molécule cible.
PCT/GB2008/000097 2007-01-11 2008-01-11 Microsphère codée Ceased WO2008084247A1 (fr)

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CN101333436B (zh) * 2008-08-06 2011-03-16 湖南大学 多色光学编码硅壳纳米棒及其制备方法
EP2905304A4 (fr) * 2012-11-16 2016-03-02 Snu R&Db Foundation Microparticules de polymère codées
EP3159391A4 (fr) * 2014-06-05 2019-11-13 Joinstar Biomedical Technology Co., Ltd. Particule porteuse et procédé de préparation associé

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EP3116645B1 (fr) * 2014-03-14 2019-04-10 GE Healthcare BioProcess R&D AB Matrices de séparation pour la purification de particules biologiques

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WO2005065081A2 (fr) * 2003-12-22 2005-07-21 Emory University Structures bioconjuguees, procedes de fabrication associes et procedes d'utilisation associes
WO2006044660A2 (fr) * 2004-10-14 2006-04-27 Vanderbilt University Nanoparticules lipidiques solides fonctionnalisees et procedes de fabrication et d'utilisation associes

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WO2005065081A2 (fr) * 2003-12-22 2005-07-21 Emory University Structures bioconjuguees, procedes de fabrication associes et procedes d'utilisation associes
WO2006044660A2 (fr) * 2004-10-14 2006-04-27 Vanderbilt University Nanoparticules lipidiques solides fonctionnalisees et procedes de fabrication et d'utilisation associes

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101333436B (zh) * 2008-08-06 2011-03-16 湖南大学 多色光学编码硅壳纳米棒及其制备方法
EP2905304A4 (fr) * 2012-11-16 2016-03-02 Snu R&Db Foundation Microparticules de polymère codées
US10557846B2 (en) 2012-11-16 2020-02-11 Quantamatrix Inc. Encoded polymeric microparticles
EP3159391A4 (fr) * 2014-06-05 2019-11-13 Joinstar Biomedical Technology Co., Ltd. Particule porteuse et procédé de préparation associé

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