Review
Luminescent quantum dots for multiplexed biological detection and imaging

https://doi.org/10.1016/S0958-1669(02)00282-3Get rights and content

Abstract

Recent advances in nanomaterials have produced a new class of fluorescent labels by conjugating semiconductor quantum dots with biorecognition molecules. These nanometer-sized conjugates are water-soluble and biocompatible, and provide important advantages over organic dyes and lanthanide probes. In particular, the emission wavelength of quantum-dot nanocrystals can be continuously tuned by changing the particle size, and a single light source can be used for simultaneous excitation of all different-sized dots. High-quality dots are also highly stable against photobleaching and have narrow, symmetric emission spectra. These novel optical properties render quantum dots ideal fluorophores for ultrasensitive, multicolor, and multiplexing applications in molecular biotechnology and bioengineering.

Introduction

Metal and semiconductor nanoparticles in the 2–6 nm size range are of considerable current interest, not only because of their unique size-dependent properties but also because of their dimensional similarities with biological macromolecules (e.g. nucleic acids and proteins) 1., 2., 3., 4.. These similarities could allow an integration of nanotechnology and biology, leading to major advances in medical diagnostics, targeted therapeutics, molecular biology and cell biology [5•]. Recent research by several groups has linked colloidal nanoparticles to bio-molecules such as peptides [6], proteins 7••., 8••., 9., and DNA 10., 11.. These nanoparticle bioconjugates are being used for assembling new materials 12., 13., for developing homo-geneous bioassays 14., 15., 16., and as multicolor fluorescent labels for ultrasensitive detection and imaging 7••., 8••., 9., 10., 11..

This review focuses on the synthesis, optical properties, surface chemistry, and biological applications of semi-conductor nanocrystals, also known as quantum dots (QDs). These nanocrystals are often composed of atoms from groups II-VI or III-V elements in the periodic table, and are defined as particles with physical dimensions smaller than the exciton Bohr radius 1., 2., 3., 4.. For spherical CdSe particles, this occurs when the particle diameter is less than ∼10 nm. The effect of quantum confinement gives rise to unique optical and electronic properties that are not available in either discrete atoms or in bulk solids. Extensive research in the past 20 years has focused on the photophysics of nanostructures and their applications in microelectronics and optoelectronics 1., 2., 3., 4.; however, recent developments indicate that the first practical applications of QDs are occurring in biology and medicine 17•., 18•.. Key advances that have enabled these applications include the synthesis of highly luminescent QDs in large quantities [19••], a reasonable understanding of the surface chemistry, the preparation of water-soluble and biocompatible nanocrystals 7••., 8••., and the incorporation of multicolor QDs into microbeads and nanobeads for multiplexed optical encoding of biomolecules [20••].

Section snippets

Synthesis

Both group II-VI (e.g. CdSe, CdTe, CdS, and ZnSe) and group III-V (e.g. InP and InAs) nanocrystals have been synthesized and studied extensively in the past 1., 2., 3., 4.. Before 1993, QDs were mainly prepared in aqueous solution with added stabilizing agents (e.g. thioglycerol or polyphosphate). This procedure yielded low-quality QDs with poor fluorescence efficiencies and large size variations (relative standard deviation [RSD]>15%). In 1993, Bawendi and coworkers [21] synthesized highly

Optical properties

The optical properties of semiconductor nanoclusters arise from interactions between electrons, holes, and their local environments. Semiconductor QDs absorb photons when the excitation energy exceeds the bandgap. During this process, electrons are promoted from the valence band to the conduction band. Measurements of UV–visible spectra reveal a large number of energy states in QDs. The lowest excited energy state is shown by the first observable peak, known as the quantum-confinement peak.

Surface chemistry

The complex surface chemistry of nanocrystals has been studied by NMR spectroscopy and X-ray photoemission spectroscopy (XPS) 36., 37.. Morphologically, QDs are not smooth spherical particles, but are faceted with many planes and edges. They are generally considered to be negatively charged owing to molecules adsorbed on the surface [38]. TOPO strongly coordinates to the surface metal atoms, whereas tri-n-octylphosphine (TOP) or tributylphosphine (TBP) are only weakly bound. The surface

Bioconjugation and applications

Reactive functional groups include primary amines, carboxylic acids, alcohols, and thiols. Primary amines react with carboxylic acids, catalyzed with a carbodiimide or sulfo N-hydroxysuccinimide ester, to form a stable amide bond. A thioether bond is formed when sulfhydryl groups react with maleimides. Another approach for linking biomolecules to nanocrystals is to use a thiol-exchange reaction. Here, mercapto-coated QDs (prepared by direct adsorption) are mixed with thiolated biomolecules

Conclusions

A pervasive trend in biotechnology is the development of ultrasensitive and high-throughput technologies for the rapid detection and quantification of genes, proteins, and cells. The ability to quickly screen a large number of biomolecules is important in several research areas, such as drug discovery and medical diagnostics. In the next 10 years, we envision that novel platforms based on multicolor QDs will be developed for massive parallel biosensing and analytical detection. These

Acknowledgements

We thank Michael Kennedy of Purdue University for help in folate conjugation and cell culturing. This work was supported by grants from the National Institutes of Health (R01 GM 58173 and R01 GM60562) and the Department of Energy (DOE FG02-98ER14873). SN acknowledges the Whitaker Foundation for a Biomedical Engineering Award and the Beckman Foundation for a Beckman Young Investigator Award.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

References (45)

  • A. Henglein

    Small-particle research: physiochemical properties of extremely small colloidal metal and semiconductor particles

    Chem Rev

    (1989)
  • G. Schmid

    Large clusters and colloids: metals in the embryonic state

    Chem Rev

    (1992)
  • A.P. Alivisatos

    Semiconductor clusters, nanocrystals, and quantum dots

    Science

    (1996)
  • M. Nirmal et al.

    Luminescence photophysics in semiconductor nanocrystals

    Acc Chem Res

    (1999)
  • C.M. Niemeyer

    Nanoparticles, proteins, and nucleic acids: biotechnology meets materials science

    Angew Chem Int Ed Engl

    (2001)
  • S.R. Whaley et al.

    Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly

    Nature

    (2000)
  • M. Bruchez et al.

    Semiconductor nanocrystals as fluorescent biological labels

    Science

    (1998)
  • W.C.W. Chan et al.

    Quantum dot bioconjugates for ultrasensitive nonisotopic detection

    Science

    (1998)
  • H. Mattoussi et al.

    Self-assembly of CdSe–ZnS quantum dots bioconjugates using an engineered recombinant protein

    J Am Chem Soc

    (2000)
  • G.P. Mitchell et al.

    Programmed assembly of DNA functionalized quantum dots

    J Am Chem Soc

    (1999)
  • S. Pathak et al.

    Hydroxylated quantum dots as luminescent probes for in situ hybridization

    J Am Chem Soc

    (2001)
  • C.A. Mirkin et al.

    A DNA-based method for rationally assembling nanoparticles into macroscopic materials

    Nature

    (1996)
  • A.P. Alivisatos et al.

    Organization of nanocrystal molecules using DNA

    Nature

    (1996)
  • R. Elghanian et al.

    Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles

    Science

    (1997)
  • B. Dubertret et al.

    Single-mismatch detection using gold-quenched fluorescent oligonucleotides

    Nat Biotechnol

    (2001)
  • R.A. Reynolds et al.

    Homogeneous, nanoparticle-based quantitative colormetric detection of oligonucleotides

    J Am Chem Soc

    (2000)
  • E. Klarreich

    Biologists join the dots

    Nature

    (2001)
  • P. Mitchell

    Turning the spotlight on cellular imaging

    Nat Biotechnol

    (2001)
  • Z.A. Peng et al.

    Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor

    J Am Chem Soc

    (2001)
  • M.Y. Han et al.

    Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules

    Nat Biotechnol

    (2001)
  • C.B. Murray et al.

    Synthesis and characterization of nearly monodisperse CdE (ES, Se, Te) semiconductor nanocrystallites

    J Am Chem Soc

    (1993)
  • B.O. Dabbousi et al.

    (CdSe)ZnS core-shell quantum dots: synthesis and characterization of a size series of highly luminescent nanocrystallites

    J Phys Chem B

    (1997)
  • Cited by (2039)

    View all citing articles on Scopus
    View full text