WO2013076159A1 - Nanoparticules biarsénicales pour le marquage in vivo - Google Patents

Nanoparticules biarsénicales pour le marquage in vivo Download PDF

Info

Publication number
WO2013076159A1
WO2013076159A1 PCT/EP2012/073265 EP2012073265W WO2013076159A1 WO 2013076159 A1 WO2013076159 A1 WO 2013076159A1 EP 2012073265 W EP2012073265 W EP 2012073265W WO 2013076159 A1 WO2013076159 A1 WO 2013076159A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
biarsenical
label
protein
nanoparticle
Prior art date
Application number
PCT/EP2012/073265
Other languages
English (en)
Inventor
Deepak Balaji THIMIRI GOVINDA RAJ
Wim Annaert
Gustaaf Borghs
Original Assignee
Imec
Katholieke Universiteit Leuven, K.U.Leuven R&D
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Imec, Katholieke Universiteit Leuven, K.U.Leuven R&D filed Critical Imec
Publication of WO2013076159A1 publication Critical patent/WO2013076159A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • 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

Definitions

  • the present invention relates to the in vivo labeling of proteins.
  • the present invention further describes functionalized nanoparticles with fluorescent labels.
  • the present invention further describes the use of biarsenical labels and their binding to tetracysteine peptide tags.
  • Fluorescent probes/labels are hence been most sought after as imaging tool and several developments have been made to enhance its applicability and functional properties for scientific research.
  • fluorescent probes have used for imaging, particularly small organic fluorescent dyes, nanocrystals ('quantum dots'), and combinations of these probes.
  • These probes are suitable protein detection in live vs. fixed cells such as quantification of protein expression, trafficking, localization, activity state, protein dynamics and potential combination of confocal imaging (live and fixed) with electron microscopy
  • Qdots Quantum dots
  • conjugated biomolecules generally get disintegrated or separated from the probe during harsh environmental conditions like acidic pH or are directed to another signaling pathway in the cell.
  • linker-adapter technology do not offer fluorescent based tracking as the fluorescent dye is coupled to nanoparticles independent of biomolecule conjugation. Hence there is no existing tool which could monitor both protein and nanoparticle localization during the imaging of living cells.
  • biarsenical labels which can bind to a tetracysteine tag (CCXXCC) SEQ ID NO:1 and become fluorescent upon binding [reviewed in e.g. Tsien et al. (2005) FEBS Lett. 579, 927-932]. Certain types of these labels are cell permeable. Labeled protein complexes can be isolated via affinity purification using biarsenical labels coupled to large magnetic beads (W 005040197).
  • the first aspect of the invention relates to methods of generating a detectable protein into a cell comprising the steps of:
  • a cell comprising a protein with a tetracysteine peptide tag with amino acid sequence CCXXCC [SEQ ID NO:1 ].
  • nanoparticle with a diameter between 2 and 200 nm, comprising a biarsenical detectable label
  • Embodiments of these methods further comprise the step of detecting the binding of the label to the tetracysteine peptide tag in the cell.
  • Further embodiments of these methods comprise the step of detecting the disappearance of the label from the tetracysteine peptide tag in the cell.
  • the nanoparticle has a diameter between 1 and 100 nm.
  • the particle further comprises a biocompatible coating.
  • the coating is selected from the group consisting of a phospholipid, carbohydrate, dimercaptosuccinic acid and silane.
  • the nanoparticle further comprises a moiety which targets the particle to a specific cell compartment.
  • the tetracysteine protein tag has the sequence CCPGCC [SEQ ID NO: 2].
  • the detectable label is a fluorescent label.
  • a second aspect of the present invention relates to nanoparticles with a diameter of between 2 and 100 nm comprising :
  • biocompatible coating selected from the group consisting of a phospholipid, carbohydrate, dimercaptosuccinic acid and silane.
  • these particle are magnetic particles.
  • these particles have a diameter between 2 and 50 nm.
  • a further aspect of the present invention relates to the use of the above particles in in vivo labeling methods.
  • kits comprising :
  • a polynucleotide encoding a fusion protein comprising a tetracysteine sequence a nanoparticle comprising a biarsenical detectable label.
  • the nanoparticle has a diameter between 2 and 100 nm.
  • the present invention describes the design of nanoparticles with a reversible fluorescent ON-OFF system for monitoring the particles themselves as well as their binding to a protein.
  • the use of the functionalized nanoparticles of the present invention has the following advantages: -major reduction in perturbation of host protein function compared to other methods of introducing a detectable label into a cell,
  • nanoparticles allow protein trafficking in subcellular compartments
  • the methods and particles of the present invention allow single molecule tracking of nanoparticle-biomolecule complexes
  • the methods and particles of the present invention allow visualization of receptor activation at the cell surface.
  • the present invention further allows evaluating target protein expression, and nanoparticle pharmacokinetics.
  • the particles are suitable for uptake by a cell and for intracellular uptake.
  • the present invention allows to detect particle transport within the cell, and eventual binding to a protein. Both phenomena can be observed independently from each other. Indeed the particle itself, or a dye incorporated therein, or attached therewith, can be visualized. Upon binding with a protein, the flash label appears and will disappears again when the protein-particle complex is degraded in the cell under reducing conditions.
  • Figure 1 illustrates the generation of a fluorescent signal upon binding of a biarsenical label with a tetracysteine peptide tag.
  • Figure 2 illustrates an embodiment of the coating of a nanoparticle and the attachment of a biarsenical label
  • FIAsH-EDT2 Design of FIAsH-EDT2 with a maleamide end group for coupling with thiol- functionalized DMSA-SPMNPs.
  • FIAsH-EDT2-SPMNPs-Peptide complex in vitro/ in vivo conditions for live imaging.
  • nanoparticle relates to a particle of any shape with a size between 2 and 100 nm
  • “tetracysteine peptide tag” relates to hexapeptide sequence with general structure Cys-Cys-Xaa-Xaa-Cys-Cys (CCXXCC) [SEQ ID NO:1].
  • biasenical detectable label refers to As comprising molecules as disclosed in EP1032837 and have the following formula:
  • each X 1 or X 2 independently, is CI, Br, I, OR a , or SR a , or
  • R a is H, C 1 -C4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;
  • Z is 1 ,2-ethanediyl, 1 ,2-propanediyl, 2,3-butanediyl, 1 ,3-propanediyl, 1 ,2 benzenediyl, 4-methyl-1 ,2-benzenediyl, 1 ,2-cyclopentanediyl, 1 ,2-cyclohexanediyl, 3-hydroxy-1 ,2-propanediyl, 3-sulfo-1 ,2-propanediyl, or 1 ,2-bis(carboxy)-1 ,2- ethanediyl;
  • Y 1 and Y 2 independently, are H or CH 3 ;
  • M is O, S, CH 2 , C(CH 3 ) 2 , or NH;
  • R 1 and R 2 independently, are OR a , OAc, NR a R b , or H;
  • R 3 and R 4 independently, are H, F, CI, Br, I, OR a , or R a ; or R 1 together with R 3 , or R 2 together with R 4 , or both, form a ring in which
  • R 1 or R 3 is C 2 -C 3 alkyl and the other is NR a and (ii). one of R 2 and R 4 is C 2 -C 3 alkyl and the other is NR a ;
  • R b is H, C 1 -C4 alkyl, CH 2 CH 2 OH, CH 2 COOH, or CN;
  • X 1 and X 2 together with the arsenic atom, form a ring, with formula
  • Q is chosen from one of the following spirolactones:
  • a first aspect of the invention relates to methods of generating a detectable protein into a cell.
  • This method provides a significant improvement over the existing in vivo labeling methods using biarsenical labels.
  • These biarsenical labels exist in cell impermeable versions for extracellular labeling and cell permeable versions for intracellular labeling. These cell permeable labels enter the cell in an aspecific way.
  • the label is attached to a nanoparticle which allows the particle to enter the cell via an active process. Particles with a diameter above 100 nm typically enter the cell via phagocytosis. Particles with a diameter below 100 nm 2-100 nm typically enter the cell via endocytosis.
  • the methods of the present invention are applicable to animal cells (comprising as well cells of invertebrates as vertebrates (in particular mammalian cells such human or murine cells)).
  • the methods are equally suitable for plant cells, fungi and bacteria. It is noted in this context that the so-called cell permeable versions of the biarsenical labels do not readily enter bacterial or yeast cells.
  • the use of nanoparticles to introduce a biarsenical label into a bacterial or yeast cell provides an additional advantaged compared to the state of the art.
  • Typical diameters of nanoparticles suitable for the uptake by endocytosis are particles with a diameter between 2 and 60 nm, between 5 and 15 nm or between 8 and 12 nm.
  • the particle can be made of, or can comprise, glass, ceramics, metal (e.g. a noble metal such as gold, or a metal with magnetic properties), plastics such as polystyrene, methylstyrene, acrylic polymers.
  • the particle is made of a compound which is degradable in the cell (e.g. protein, carbohydrate).
  • suitable materials used in the manufacture of nanoparticles include agarose beads, latex, cross-linked dextrans, cellulose, and nylon.
  • the particle further comprises functional groups for the coupling of the label to the particle.
  • the particle comprises further a coating to make the particle cell compatible such that the particle can be internalized via the cell.
  • the coating is selected form the group consisting of pospholipids, carbohydrates, dimercaptosuccinic acid (DMSA), and silane.
  • Particular phospholipids in the context of the present invention can comprise amino end-groups, PEG end-groups, carboxy end-groups, thiol end-grouped or combination of these different end-groups.
  • Particular carbohydrates in the context of the present invention are lectins, sucrose and galactose.
  • silanese in the context of the present invention can comprise amino end- groups, PEG end-groups, carboxy end-groups, thiol end-groups or combination of these end-groups.
  • Nanoparticles can be composed of a variety of materials including noble metals (e.g. Au , Ag , Pt , Pd), semiconducting materials (e.g. CdSe, CdS, ZnS , Ti0 2 , PbS, InP, Si), magnetic compounds (e.g. Fe 3 0 4 , Co, CoFe 2 0 4 , FePt , CoPt) and mixtures thereof.
  • noble metals e.g. Au , Ag , Pt , Pd
  • semiconducting materials e.g. CdSe, CdS, ZnS , Ti0 2 , PbS, InP, Si
  • magnetic compounds e.g. Fe 3 0 4 , Co, CoFe 2 0 4 , FePt , CoPt
  • Typical biocompatible coatings further include Poly(amidoamine), Poly(D-glucosamido ethylmethacrylate), Poly(2-lactobionamido ethylmethacrylate), Alkyl thioether end- functionalised poly(methacrylic acid), Polyvinyl pyrollidone), PAA modified with cysteamine and ethylene diamine, Poly(maleic anhydride-alt-1 -octadecene), Poly(butylacrylate), Poly(ethylacrylate), Pyrene-poly(dimethylaminoethyl methacrylate),Poly[2-(methacryloyloxy) ethylphosphorylcholine]-block-(glycerol monomethacrylate), alpha-Acetylene-poly(tert-butyl acrylate), Poly(N- isopropylacrylamide), Poly(L,L-lactic acid), Poly(epsilon)-caprolactone), Poly(2- me
  • the detectable label which is used in the methods and compounds of the present invention is a biarsenical label such as disclosed in the art in various publication of Tsien et al., wherein As'" atoms can bind to vicinal dithiols in peptides and proteins.
  • the spacing of two As atoms (biarsenic) in the label allows the binding of the label to a peptide sequence with general structure CCXXCC [SEQ ID NO:1 ].
  • a particular sequence providing a conformation which minimizes aspecific binding is the peptide with aminoacid sequence CCPGCC [SEQ ID NO: 2].
  • the fluorescence of the label increases dramatically. In other words the label only becomes detectable upon binding of the label to its target protein.
  • the binding of the biarsenical label on the particle to a protein is determined by the presence of a recombinant protein comprising the tetracysteine tag.
  • a polynucleotide construct encoding a fusion protein of a protein of interest with a tetracysteine is transfected or transformed into a cell (viral proteins such as structural proteins - matrix, capsid and nucleocaposid, viral enzymes and viral envelope protein (Pereira C.F.
  • Additional targeting moieties can be provide on the nanoparticles to optimize the trafficking to certain organelles, or subcellular compartments of a cell, for example TOM 22 antibody for targeting to the mitochondrion, transferrin ligand for cell membrane, heparan sulfate proteoglycans for endocytotic compartments, lamp-1 antibody for targeting lysosomal compartments and protein G coated beads for targeting nucleus
  • the nanoparticle and/or the biarsenical label can be further modified to include a detectable group in addition to the detectable label that is generated upon binding of the biarsenical label to a tetracysteine tag.
  • detectable groups are known in the art and include for example a fluorescent group, luminescent group, phosphorescent group, spin label, photosensitizer, photocleavable moiety, chelating center, heavy atom, radioactive isotope, isotope detectable by nuclear magnetic resonance, paramagnetic atom, and combinations thereof.
  • a tetracysteine tagged protein can be introduced into a cell by e.g. electroporation.
  • a fluorescent signal is generated upon binding of the biarsenical label with the tetracysteine peptide tag, whereby the signal remains visible as long as the label remains attached to the tag.
  • This label allows its detection from the binding of the label to the protein until the degradation of the protein.
  • the protein Upon degradation of a protein in the lysosome, the protein is in a strongly reducing environment, which results in the release of the biarsenical label from the protein, thereby loosing its fluorescence.
  • Another aspect of the present invention relates to a composition
  • a composition comprising nanoparticles with a diameter of between 2 and 200 nm, particularly between 2 and 100 nm, more particularly between 2 and 50 nm, with attached thereto a biarsenical label.
  • Larger particles with biarsenical labels have been described in the art, however the particles have diameters above 100 nm, to allow a convenient manipulation in affinity purification methods.
  • Smaller nanoparticles with a biarsenical label and their use in in vivo labeling methods have not been described earlier. Since these prior art particles have not been used in the context of cellular uptake they neither have a coating such as a phospholipid, carbohydrate, dimercaptosuccinic acid or a silane.
  • a further aspect of the present invention relates to a kit comprising a polynucleotide encoding a fusion protein comprising a tetracysteine sequence and a nanoparticle comprising a biarsenical detectable label as disclosed above.
  • a polynucleotide will be provided in the form of a vector for delivery into a cell.
  • the current application is applicable in wide range of biological applications, protein trafficking, stem cell tracking, gene delivery and drug delivery.
  • FIAsH-EDT2 is a small fluorescent probe that has been established for site-specific protein labeling of proteins in intact live cells [Griffin et al., 1998].
  • FIAsH is nonfluorescent by itself but becomes highly fluorescent when bound to a specific sequence consisting of at least six amino acids (tetracysteine peptide tag).
  • the biarsenical labeling technology works through the high-affinity interaction of arsenic for thiols wherein FIAsH is a fluorescein derivative and are virtually non-fluorescent when bound to ethane dithiol (EDT2).
  • EDT2 ethane dithiol
  • FIAsH-EDT2 are coupled to nanoparticles as illustrated in figure 2. Uptake of the nanoparticle by a cell allows to decipher the protein pathway to which the biarsenical label is coupled and allows to the detect the protein activity in subcellular compartments at the single cell level. In addition, FIAsH is much smaller than GFP and therefore there is lower risk that FIAsH-EDT2 will disturb the overall structure of a protein than other fluorescent probes.
  • SPMNPs superparamagnetic nanoparticles
  • DMSA biocompatible dimercaptosuccinic acid
  • FIAsH-EDT2 labels are coupled to the nanoparticles via maleamide end group for coupling with thiol-functionalized DMSA-SPMNPs.
  • the particle Upon entry into a cell the particle will be processed by the cell. Upon binding of the particle to the tagged protein, the a fluorescent signal is generated.
  • a pulse-chase methodology allows monitoring of the dynamics and life cycle of a protein with the use of a fluorescent label.
  • the pre-existing pool of tetracysteine tagged connexin-43 is labeled with FIAsH-EDT 2 , then after different times of incubation the newly expressed protein pool is labeled with ReAsH-EDT 2 (a red-emitting dye).
  • ReAsH-EDT 2 a red-emitting dye.
  • these label moiety can be used to photoconvert DAB (diaminobenzidine) into an osmophilic precipitate.
  • the sample After osmium(VIII) oxide treatment the sample can be viewed in an electron microscope (EM) to gain ultrastructural information (Pomorski A., et al. 2010). As the FIAsH moiety cannot photoconvert DAB, the old or new pool can be visualized by electron microscopy. Labeling in the endoplasmic reticulum and Golgi apparatus is hampered by the oxidizing environment, although it is possible to reduce the cysteines in the TC-tag by the addition of tributylphosphine (TBP) or triethylphosphine (TEP).
  • TBP tributylphosphine
  • TEP triethylphosphine

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des procédés de génération d'une protéine détectable dans une cellule comprenant les étapes de fourniture d'une cellule comprenant une protéine avec un tag de peptide de tétracystéine avec la séquence des acides aminés CCXXCC, de mise en contact de ladite cellule avec une nanoparticule avec un diamètre entre 2 et 200 nm, comprenant un marqueur biarsénical détectable, et de fourniture de conditions permettant l'accumulation de ladite nanoparticule par ladite cellule.
PCT/EP2012/073265 2011-11-22 2012-11-21 Nanoparticules biarsénicales pour le marquage in vivo WO2013076159A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP11190116.1 2011-11-22
EP11190116 2011-11-22

Publications (1)

Publication Number Publication Date
WO2013076159A1 true WO2013076159A1 (fr) 2013-05-30

Family

ID=47226154

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/073265 WO2013076159A1 (fr) 2011-11-22 2012-11-21 Nanoparticules biarsénicales pour le marquage in vivo

Country Status (1)

Country Link
WO (1) WO2013076159A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054271A (en) * 1997-10-21 2000-04-25 The Regents Of The University Of California Methods of using synthetic molecules and target sequences
EP1032837A1 (fr) 1997-10-21 2000-09-06 The Regents Of The University Of California Sequences cibles pour molecules synthetiques et procedes d'utilisation de ces dernieres
WO2001053325A2 (fr) * 2000-01-24 2001-07-26 The Regents Of The University Of California Purification par affinite de proteines a l'aide d'une fluoresceine bis-arsenicale modifiee
US20040134565A1 (en) 2003-01-13 2004-07-15 International Business Machines Corporation Process of forming magnetic nanocomposites via nanoparticle self-assembly
WO2005040197A1 (fr) * 2003-10-24 2005-05-06 Invitrogen Corporation Compositions, procedes et necessaires de marquage par fluorophores biarsenicaux
WO2008109464A1 (fr) * 2007-03-02 2008-09-12 University Of Massachusetts Composés de ciblage de spirolactame et composés apparentés

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054271A (en) * 1997-10-21 2000-04-25 The Regents Of The University Of California Methods of using synthetic molecules and target sequences
EP1032837A1 (fr) 1997-10-21 2000-09-06 The Regents Of The University Of California Sequences cibles pour molecules synthetiques et procedes d'utilisation de ces dernieres
WO2001053325A2 (fr) * 2000-01-24 2001-07-26 The Regents Of The University Of California Purification par affinite de proteines a l'aide d'une fluoresceine bis-arsenicale modifiee
US20040134565A1 (en) 2003-01-13 2004-07-15 International Business Machines Corporation Process of forming magnetic nanocomposites via nanoparticle self-assembly
WO2005040197A1 (fr) * 2003-10-24 2005-05-06 Invitrogen Corporation Compositions, procedes et necessaires de marquage par fluorophores biarsenicaux
WO2008109464A1 (fr) * 2007-03-02 2008-09-12 University Of Massachusetts Composés de ciblage de spirolactame et composés apparentés

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
ADAMS ET AL., JACS, 2002
CAO ET AL., JACS, 2007
EMILIE GENIN ET AL: "CrAsH-Quantum Dot Nanohybrids for Smart Targeting of Proteins", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 130, no. 27, 1 July 2008 (2008-07-01), pages 8596 - 8597, XP055025217, ISSN: 0002-7863, DOI: 10.1021/ja802987q *
GAIETTA ET AL., PNAS, 2006
MEDINTZ IGOR L ET AL: "Self-assembled nanoscale biosensors based on quantum dot FRET donors", NATURE MATERIALS, NATURE PUBLISHING GROUP, GB, vol. 2, no. 9, 1 September 2003 (2003-09-01), pages 630 - 638, XP002508986, ISSN: 1476-1122, [retrieved on 20030824], DOI: 10.1038/NMAT961 *
ROBIA ET AL., BIOCHEMISTRY, 2005
SERENA MAZZUCCHELLI ET AL: "Single-Domain Protein A-Engineered Magnetic Nanoparticles: Toward a Universal Strategy to Site-Specific Labeling of Antibodies for Targeted Detection of Tumor Cells", ACS NANO, vol. 4, no. 10, 26 October 2010 (2010-10-26), pages 5693 - 5702, XP055019310, ISSN: 1936-0851, DOI: 10.1021/nn101307r *
SUN; ZENG, J. AM. CHEM. SOC., vol. 124, no. 28, 2002, pages 8204 - 8205
THANH; GREEN, NANO TODAY, vol. 5, 2010, pages 213 - 230
THORN K S ET AL: "A NOVEL METHOD OF AFFINITY-PURIFYING PROTEINS USING A BIS-ARSENICALFLUORESCEIN", PROTEIN SCIENCE, CAMBRIDGE UNIVERSITY PRESS, vol. 9, no. 2, 1 January 2000 (2000-01-01), pages 213 - 217, XP002946402, ISSN: 0961-8368 *
TSIEN ET AL., FEBS LETT., vol. 579, 2005, pages 927 - 932
VOLKER MAILÄNDER ET AL: "Interaction of Nanoparticles with Cells", BIOMACROMOLECULES, vol. 10, no. 9, 14 September 2009 (2009-09-14), pages 2379 - 2400, XP055019306, ISSN: 1525-7797, DOI: 10.1021/bm900266r *
YUN-PEI CHANG ET AL: "Tracking bio-molecules in live cells using quantum dots", JOURNAL OF BIOPHOTONICS, vol. 1, no. 4, 1 September 2008 (2008-09-01), pages 287 - 298, XP055019382, ISSN: 1864-063X, DOI: 10.1002/jbio.200810029 *
ZURN, BIOCONJUGATE CHEM, 2010

Similar Documents

Publication Publication Date Title
Chang et al. Highly fluorescent nanodiamonds protein‐functionalized for cell labeling and targeting
Zong et al. Peptide-functionalized gold nanoparticles: versatile biomaterials for diagnostic and therapeutic applications
Bilan et al. Quantum dot‐based nanotools for bioimaging, diagnostics, and drug delivery
Martynenko et al. Application of semiconductor quantum dots in bioimaging and biosensing
Lee et al. Dual‐mode nanoparticle probes for high‐performance magnetic resonance and fluorescence imaging of neuroblastoma
Jiang et al. Gold–carbon dots for the intracellular imaging of cancer-derived exosomes
Wu et al. Ultrabright and bioorthogonal labeling of cellular targets using semiconducting polymer dots and click chemistry
Wang et al. In vitro and in vivo imaging with quantum dots
Medintz et al. Potential clinical applications of quantum dots
Suh et al. Multifunctional nanosystems at the interface of physical and life sciences
Tyrakowski et al. A primer on the synthesis, water-solubilization, and functionalization of quantum dots, their use as biological sensing agents, and present status
Mao et al. Hydrophobic carbon nanodots with rapid cell penetrability and tunable photoluminescence behavior for in vitro and in vivo imaging
Sosinsky et al. Markers for correlated light and electron microscopy
Durgadas et al. Fluorescent and superparamagnetic hybrid quantum clusters for magnetic separation and imaging of cancer cells from blood
Mi et al. Herceptin functionalized polyhedral oligomeric silsesquioxane–conjugated oligomers–silica/iron oxide nanoparticles for tumor cell sorting and detection
Xue et al. Enhancement of intracellular delivery of CdTe quantum dots (QDs) to living cells by Tat conjugation
Méndez‐Ardoy et al. Where in the cell is our cargo? Methods currently used to study intracellular cytosolic localisation
Terada et al. Nanodiamonds for bioapplications–specific targeting strategies
Chu et al. Preparation of quantum dot-coated magnetic polystyrene nanospheres for cancer cell labelling and separation
Sun et al. A versatile method for generating semiconducting polymer dot nanocomposites
Su et al. Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy
Li et al. Preparation of quantum dot bioconjugates and their applications in bio-imaging
Li et al. Bioconjugated quantum dots as fluorescent probes for biomedical imaging
Morita et al. Controlled synthesis of gold nanoparticles on fluorescent nanodiamond via electron-beam-induced reduction method for dual-modal optical and electron bioimaging
Sotoma et al. Biohybrid fluorescent nanodiamonds as dual‐contrast markers for light and electron microscopies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12791160

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12791160

Country of ref document: EP

Kind code of ref document: A1