Nanocrystals and Nanoparticles Versus Molecular Fluorescent Labels as Reporters for Bioanalysis and the Life Sciences: A Critical Comparison - Advanced Fluorescence Reporters in Chemistry and Biology - page 4

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2 Properties of Molecular and Nanoparticular Labels and Reporters . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1 Spectroscopic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2 Solubility and Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.3 Thermal and Photochemical Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4 Cyto- and Nanotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3 Application of Molecular and Nanoparticulate Fluorophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1 Coupling Chromophores to Biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.2 Extra- and Intracellular Targeting of Biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.3 Interactions Between Chromophores and their Microenvironment . . . . . . . . . . . . . . . . . . . 24
3.4 Exploitation of F¨rster Resonance Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.5 Multiplexing Detection Schemes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.6 Strategies for Signal Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.7 Reproducibility, Quality Assurance and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4 Applications of Nanoparticles: State-of-the-Art
and Future Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

1 Introduction

The investigation of many fundamental processes in the life sciences requires
straightforward tools for the fast, sensitive, reliable, and reproducible detection of
the interplay of biomolecules with one another and with various molecular or ionic
species. One of the best suited and most popular methods to meet these challenges
presents the use of photoluminescence or fluorescence techniques in conjunction
with functional dyes and labels [ 1 - 3 ]. Advantages of fluorescence methods, which
range from fluorescence spectroscopy over fluorescence microscopy and flow
cytometry to in vivo fluorescence imaging, include the comparatively simple
measurement of a number of unique experimental parameters (excitation wave-
length, emission wavelength, intensity/quantum yield, fluorescence lifetime, and
emission anisotropy) with nanometer scale resolution and possible sensitivity down
to the single molecule level [ 4 ]. The potential of these methods, e.g., the achievable
sensitivity (detection limit), the dynamic range, and the number of emissive species
to be distinguished or detected simultaneously (multiplexing capability), is con-
trolled by the physico-chemical properties of the fluorescent reporter(s) employed.
Generally, a suitable label or reporter must be (1) conveniently excitable, without
excitation of the (biological) matrix, and detectable with conventional instrumen-
tation; (2) bright, i.e., possess a high molar absorption coefficient at the excitation
wavelength and a high fluorescence quantum yield; (3) soluble in application-
relevant media such as buffers, cell culture media, or body fluids; and (4) thermally
and photochemically stable under relevant conditions. (5) For site-specific labeling,
functional groups, often in conjunction with spacers, are beneficial. Depending on