Biomedical Engineering Reference
In-Depth Information
This effect can however be used to one's advantage to study the dynamics (see
Section 8.3.1.1 for a description of the FRAP technique).
Exogenous Fluorophores: Micro- and Nanoparticles
Fluorescent latex beads are plastic beads (typically a few hundred nm in diameter)
loaded with organic fluorophores. These relatively large particles are quite use-
ful to reveal the structures of larger structures or to probe flows in microchannels
geometries (see Chapter 2). Their surface can be tailored to match one's particular
application by grafting particular molecules to them. For instance, immunoassays
that use antibodies coupled to latex beads allow detecting specific proteins (see Sec-
tion 8.2.2.1).
Quantum dots (QDs) are fluorescent nanoparticles (typically 10 nm) whose
use in biology oriented applications is relatively recent. These inorganic particles
are made of semiconductors (very often ZnSe crystals surrounded by a thin ZnS
shell), and besides their small size, have a few remarkable properties that explain
their popularity. They all share a broad excitation spectrum in the blue and their
emission wavelength depends only on their size. They are extremely bright and
show practically no photobleaching. Moreover, they are small enough to be in-
corporated in many systems, even at the surface of live cells. For instance, by
using two sizes, two different proteins can be labeled and excited with the same
wavelength making colocalization experiments particularly easy. The applications
of QDs in biology-related applications have long been delayed mainly because of
the difficulties in dispersing these hydrophobic particles in water, not to mention
their difficult coupling to biomolecules due to a particularly inert surface. How-
ever these difficulties have been solved in particular by their encapsulation with
amphiphilic molecules such as block copolymers [12]. QDs are now commercially
available with different surface groups and couplings. Because they are so bright
and quite small, they can be used to track particular proteins at the surface of cells
[13] or even within cells [14].
Endogenous Fluorophores
Green fluorescent protein (GFP) is a naturally fluorescent protein present in the jel-
lyfish
Aequorea Victoria
. The GFP reporter gene can be fused by genetic engineer-
ing to the one of the protein of interest so that the resulting protein is a fusion of
both, coupling the desired function with fluorescence. This way, proteins in living
cells can be directly observed by fluorescence. Better efficiency and other colors
have been developed by mutating the original GFP. Using these proteins, dynamic
fluorescent imaging of proteins can be performed on live cells (for which the cou-
pling with organic fluorophore would have been prohibited for toxicity reasons).
Strategies exist to get transfected cell lines that have a transient response but it is
also possible to get stable clones.
8.2.2 Other Micro- and Nanoparticles
We review here some of the particles used in tests or in biotechnology-related appli-
cations. Because of their potential, magnetic beads deserves a chapter by themselves
and are described in Chapter 9.
