Chromosome Research

, Volume 17, Issue 4, pp 519–530 | Cite as

Quantum dots as new-generation fluorochromes for FISH: an appraisal

  • Dimitris Ioannou
  • Helen G. Tempest
  • Benjamin M. Skinner
  • Alan R. Thornhill
  • Michael Ellis
  • Darren K. Griffin
Article

Abstract

In the field of nanotechnology, quantum dots (QDs) are a novel class of inorganic fluorochromes composed of nanometre-scale crystals made of a semiconductor material. Given the remarkable optical properties that they possess, they have been proposed as an ideal material for use in fluorescent in-situ hybridization (FISH). That is, they are resistant to photobleaching and they excite at a wide range of wavelengths but emit light in a very narrow band that can be controlled by particle size and thus have the potential for multiplexing experiments. The principal aim of this study was to compare the potential of QDs against traditional organic fluorochromes in both indirect (i.e. QD-conjugated streptavidin) and direct (i.e. synthesis of QD-labelled FISH probes) detection methods. In general, the indirect experiments met with a degree of success, with FISH applications demonstrated for chromosome painting, BAC mapping and use of oligonucleotide probes on human and avian chromosomes/nuclei. Many of the reported properties of QDs (e.g. brightness, ‘blinking’ and resistance to photobleaching) were observed. On the other hand, signals were more frequently observed where the chromatin was less condensed (e.g. around the periphery of the chromosome or in the interphase nucleus) and significant bleed-through to other filters was apparent (despite the reported narrow emission spectra). Most importantly, experimental success was intermittent (sometimes even in identical, parallel experiments) making attempts to improve reliability difficult. Experimentation with direct labelling showed evidence of the generation of QD-DNA constructs but no successful FISH experiments. We conclude that QDs are not, in their current form, suitable materials for FISH because of the lack of reproducibility of the experiments; we speculate why this might be the case and look forward to the possibility of nanotechnology forming the basis of future molecular cytogenetic applications.

Keywords

quantum dot nanotechnology FISH chromosome painting semiconductor 

Abbreviations

BAC(s)

bacterial artificial chromosome(s)

BSA

bovine serum albumin

DAPI

4′,6-diamidino-2-phenylindole

ddH2O

double-distilled water

DS

dextran sulfate

DOP

degenerate oligo primed

DTT

dithiothreitol

dUTP

2′-deoxyuridine 5′-triphosphate

FA

formamide

FISH

fluorescence in-situ hybridization

FITC

fluorescein isothiocyanate

HFEA

human fertilization and embryology authority

MAA

mercaptoacetic acid

NIR

near infrared

PBS

phosphate-buffered saline

QD

quantum dot

QD-FISH

quantum dot fluorescence in-situ hybridization

RT

room temperature

PCR

polymerase chain reaction

SERT

serotonin transporter protein

SSC

saline sodium citrate

UV

ultraviolet

Supplementary material

10577_2009_9051_Fig8_ESM.jpg (38 kb)
Supplementary Fig. S1

QD585 dissolved in hybridization mix and viewed directly under the microscope using four barrier filters: 525 nm (blue), 565 nm (green), 585 nm (red) and 605 nm (far red but pseudo-coloured purple for the purposes of this figure). The image is a merge of all four filters. The QDs are predominantly red (as would be expected), but a smaller number of green, blue and purple QDs are seen. The discrete appearance of QDs of one or other of the colours indicates there is a mixed population of QDs in each preparation (37.8 KB).

10577_2009_9051_MOESM1_ESM.mpg (118 kb)
Supplementary Movie S2aMovie of QD585 dissolved in hybridization mix and viewed directly under the microscope using 585 nm barrier filter. The phenomenon of ‘blinking’ is clearly seen (MPG 118 KB).
10577_2009_9051_MOESM2_ESM.mpg (128 kb)
Supplementary Movie S2bMovie of QD605 dissolved in hybridization mix and viewed directly under the microscope using 605nm barrier filter. The phenomenon of ‘blinking’ is clearly seen (MPG 128 KB).

References

  1. Akerman ME, Chan WC, Laakkonen P, Bhatia SN, Ruoslahti E (2002) Nanocrystal targeting in vivo. Proc Natl Acad Sci USA 99:12617–12621PubMedCrossRefGoogle Scholar
  2. Alivisatos AP (1996) Nanocrystals:building blocks for modern materials design. Endeavour 21:56–60CrossRefGoogle Scholar
  3. Alivisatos AP, Gu W, Larabell C (2005) Quantum dots as cellular probes. Annu Rev Biomed Eng 7:55–76PubMedCrossRefGoogle Scholar
  4. Arya H, Kaul Z, Wadhwa R, Taira K, Hirano T, Kaul SC (2005) Quantum dots in bio-imaging: revolution by the small. Biochem Biophys Res Commun 329:1173–1177PubMedCrossRefGoogle Scholar
  5. Bailey RE, Smith AM, Nie S (2004) Quantum dots in biology and medicine. Physica E 25:1–12CrossRefGoogle Scholar
  6. Bentolila LA, Weiss S (2006) Single-step multicolor fluorescence in situ hybridization using semiconductor quantum dot-DNA conjugates. Cell Biochem Biophys 45:59–70PubMedCrossRefGoogle Scholar
  7. Bruchez M (2007) Quantum dots for ultra-sensitive multicolor detection of proteins and genes. Paper presented at 16th International Chromosome Conference (16th ICC) AmsterdamGoogle Scholar
  8. Bruchez M Jr, Moronne M, Gin P, Weiss S, Alivisatos AP (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281:2013–2016PubMedCrossRefGoogle Scholar
  9. Chan WC (2006) Bionanotechnology progress and advances. Biol Blood Marrow Transplant 12:87–91PubMedCrossRefGoogle Scholar
  10. Chan WC, Nie S (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281:2016–2018PubMedCrossRefGoogle Scholar
  11. Chan WC, Maxwell DJ, Gao X, Bailey RE, Han M, Nie S (2002) Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol 13:40–46PubMedCrossRefGoogle Scholar
  12. Chan P, Yuen T, Ruf F, Gonzalez-Maeso J, Sealfon SC (2005) Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization. Nucleic Acids Res 33:1–8CrossRefGoogle Scholar
  13. Choi J, Burns AA, Williams RM et al (2007) Core-shell silica nanoparticles as fluorescent labels for nanomedicine. J Biomed Opt 12:064007PubMedCrossRefGoogle Scholar
  14. Dabbousi BO, Rodriguez-Viejo J, Mikulec FV et al (1997) (CdSe)ZnS core-shell quantum dots: synthesis and characterization of a size series of highly luminescent nanocrystallites. J Phys Chem B 101:9463–9475CrossRefGoogle Scholar
  15. Dubertret B, Skourides P, Norris DJ, Noireaux V, Brivanlou AH, Libchaber A (2002) In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298:1759–1762PubMedCrossRefGoogle Scholar
  16. Efros AL, Rosen M (1997) Random telegraph signal in the photolyminescence intensity of a single quantum dot. Physical Rev Lett 78:1110–1113CrossRefGoogle Scholar
  17. Ferrara DE, Weiss D, Carnell PH et al (2006) Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy. Am J Physiol Regul Integr Comp Physiol 290:R114–R123PubMedGoogle Scholar
  18. Fu A, Alivisatos AP, Gu W, Larabell C (2005) Semiconductor nanocrystals for biological imaging. Curr Opin Neurobiol 15:568–575PubMedCrossRefGoogle Scholar
  19. Gao X, Cui Y, Levenson RM, Chung LW, Nie S (2004) In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 22:969–976PubMedCrossRefGoogle Scholar
  20. Gao X, Yang L, Petros JA, Marshall FF, Simons JW, Nie S (2005) In vivo molecular and cellular imaging with quantum dots. Curr Opin Biotechnol 16:63–72PubMedCrossRefGoogle Scholar
  21. Gerion D, Pinaud F, Williams SC et al (2001) Synthesis and properties of biocompatible water-soluble sillica-coated CdSe/ZnS semiconductor quantum dots. J Phys Chem B 105:8861–8871CrossRefGoogle Scholar
  22. Green M (2004) Semiconductor quantum dots as biological imaging agents. Angew Chem Int Ed Engl 43:4129–4131PubMedCrossRefGoogle Scholar
  23. Hohng S, Ha T (2004) Near-complete suppression of quantum dot blinking in ambient conditions. J Am Chem Soc 126:1324–1325PubMedCrossRefGoogle Scholar
  24. Invitrogen (2006) Qdot nanocrystal technology. Vol. 2006. Invitrogen Corporation, Carlsbad CAGoogle Scholar
  25. Jaiswal JK, Mattoussi H, Mauro JM, Simon SM (2003) Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol 21:47–51PubMedCrossRefGoogle Scholar
  26. Jiang Z, Li R, Todd NW, Stass SA, Jiang F (2007) Detecting genomic aberrations by fluorescence in situ hybridization with quantum dots-labeled probes. J Nanosci Nanotechnol 7:4254–4259PubMedCrossRefGoogle Scholar
  27. Kim S, Lim YT, Soltesz EG et al (2004) Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Biotechnol 22:93–97PubMedCrossRefGoogle Scholar
  28. Larson DR, Zipfel WR, Williams RM et al (2003) Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 300:1434–1436PubMedCrossRefGoogle Scholar
  29. Lidke DS, Nagy P, Heintzmann R et al (2004) Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat Biotechnol 22:198–203PubMedCrossRefGoogle Scholar
  30. Lipovskii A, Kolobkova E, Petrikov V et al (1997) Synthesis and characterization of PbSe quantum dots in phosphate glass. Appl Phys Lett 71:3406–3408CrossRefGoogle Scholar
  31. Lounis B, Bechtel HA, Gerion D, Alivisatos PA, Moerner WE (2000) Photon antibunching in single CdSe/ZnS quantum dot fluorescence. Chem Phys Lett 329:399–404CrossRefGoogle Scholar
  32. Ma L, Wu SM, Huang J, Ding Y, Pang DW, Li L (2008) Fluorescence in situ hybridization (FISH) on maize metaphase chromosomes with quantum dot-labeled DNA conjugates. Chromosoma 117:181–187PubMedCrossRefGoogle Scholar
  33. Mansson A, Sundberg M, Balaz M et al (2004) In vitro sliding of actin filaments labelled with single quantum dots. Biochem Biophys Res Commun 314:529–534PubMedCrossRefGoogle Scholar
  34. Mattheakis LC, Dias JM, Choi YJ et al (2004) Optical coding of mammalian cells using semiconductor quantum dots. Anal Biochem 327:200–208PubMedCrossRefGoogle Scholar
  35. Michalet X, Pinaud F, Thilo DL et al (2001) Properties of fluorescent semiconductor nanocrystals and their application to biological labeling. Single Molecules 2:261–276CrossRefGoogle Scholar
  36. Michalet X, Pinaud FF, Bentolila LA et al (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307:538–544PubMedCrossRefGoogle Scholar
  37. Michler P, Imamoglu A, Mason MD, Carson PJ, Strouse GF, Buratto SK (2000) Quantum correlation among photons from a single quantum dot at room temperature. Nature 406:968–970PubMedCrossRefGoogle Scholar
  38. Miller DAB, Chemla DS (1986) Mechanism for enhanced optical nonlinearities and bistability by combined dielectric - electronic confinement in semiconductor microcrystallites. Opt Lett 11:522-524CrossRefGoogle Scholar
  39. Muller F, Houben A, Barker PE, Xiao Y, Kas JA, Melzer M (2006) Quantum dots—a versatile tool in plant science? J Nanobiotechnol 4:5CrossRefGoogle Scholar
  40. Murray CB, Kagan CR, Bawendi M (2000) Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies. Annu Rev Mater Sci 30:545–610CrossRefGoogle Scholar
  41. Parak WJ, Boudreau R, Le Gros MA et al (2002) Cell motility and metastatic potential studies based on quantum dot imaging of phagokinetic tracks. Adv Mater 14:882–885CrossRefGoogle Scholar
  42. Parak WJ, Gerion D, Pellegrino T et al (2003) Biological applications of colloidal nanocrystals. Nanotechnology 14:R15–R27CrossRefGoogle Scholar
  43. Parak WJ, Pellegrino T, Plank C (2005) Labelling of cells with quantum dots. Nanotechnology 16:R9–R25CrossRefGoogle Scholar
  44. Pinaud F, Michalet X, Bentolila LA et al (2006) Advances in fluorescence imaging with quantum dot bio-probes. Biomaterials 27:1679–1687PubMedCrossRefGoogle Scholar
  45. QDCorporation (2006) Qdot nanocrystals anatomy. Vol. 2006. Quantum Dot Corporation (QDC)Google Scholar
  46. Reed MA, Bate RT, Bradshaw WM, Duncan WR, Frensley JWL, Shih HD (1986) Spatial quantization in GaAs-AlGaAs multiple quantum dots. J Vac Sci Technol B 4:358–360CrossRefGoogle Scholar
  47. Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, Nitschke R, Nann T (2008) Quantum dots versus organic dyes as fluorescent labels. Nat Methods 5:763–775PubMedCrossRefGoogle Scholar
  48. Rieger S, Kulkarni RP, Darcy D, Fraser SE, Koster RW (2005) Quantum dots are powerful multipurpose vital labeling agents in zebrafish embryos. Dev Dyn 234:670–681PubMedCrossRefGoogle Scholar
  49. Rosenthal SJ, Tomlinson A, Adkins EM et al (2002) Targeting cell surface receptors with ligand-conjugated nanocrystals. J Am Chem Soc 124:4586–4594PubMedCrossRefGoogle Scholar
  50. Wu X, Liu H, Liu J et al (2003) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol 21:41–46PubMedCrossRefGoogle Scholar
  51. Xiao Y, Barker PE (2004a) Semiconductor nanocrystal probes for human chromosomes and DNA. Minerva Biotec 16:281–288Google Scholar
  52. Xiao Y, Barker PE (2004b) Semiconductor nanocrystal probes for human metaphase chromosomes. Nucleic Acids Res 32:e28PubMedCrossRefGoogle Scholar
  53. Xiao Y, Telford WG, Ball JC, Locascio LE, Barker PE (2005) Semiconductor nanocrystal conjugates, FISH and pH. Nat Methods 2:723PubMedCrossRefGoogle Scholar
  54. Yao G, Wang L, Wu Y et al (2006) FloDots: luminescent nanoparticles. Anal Bioanal Chem 385:518–524PubMedCrossRefGoogle Scholar
  55. Zheng J, Ghazani AA, Song Q, Mardyani S, Chan WC, Wang C (2006) Cellular imaging and surface marker labeling of hematopoietic cells using quantum dot bioconjugates. Lab Hematol 12:94–98PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Dimitris Ioannou
    • 1
  • Helen G. Tempest
    • 1
    • 2
  • Benjamin M. Skinner
    • 1
  • Alan R. Thornhill
    • 1
    • 2
  • Michael Ellis
    • 3
  • Darren K. Griffin
    • 1
  1. 1.Department of BiosciencesUniversity of KentCanterburyUK
  2. 2.The London Bridge Fertility, Gynaecology and Genetics Centre and Bridge GenomaLondonUK
  3. 3.Digital Scientific UK LtdCambridgeUK

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