Biomedical Engineering Reference
In-Depth Information
tracking single molecules, either directly or as a member of a donor-acceptor pair. There
are several mechanisms available for the acceptor to quench the fluorescence of the donor
species including static quenching, dynamic quenching, and contact mediated quenching
(17). The length and nature of the linker species involved in tethering the donor and accep-
tor moieties to the 3' and 5' ends of the oligonucleotide are also factors that must be con-
sidered when optimizing an MB system. Tan et al. (67) has designed MB species with limits
of detection of 1.7
10
10
M using a simple spectrometer detection scheme, and has used
MB probes inside cellular cytoplasm as miniature biosensors.
Fluorescence resonance energy transfer has been used to investigate conformations of
RNA based on energy transfer between the donor and acceptor (68,69). The advantage of
single-molecule FRET is that it provides for study of time-dependent phenomena. In con-
trast to FRET from ensembles, which rely on signal averaging, single-molecule FRET
measures only one donor-acceptor complex at a time without synchronization (15), and
the observations can be uniquely attributed to a single species. Single-molecule FRET has
been applied to study immobilized molecules at interfaces, such as for investigation of
RNA and protein folding (70-72), and has also found applications in cases where mole-
cules diffuse freely through an excitation laser beam (73-75). Excellent review articles have
been published by Selvin, and by Ha (68,76).
2.4.3
Single-Molecule Electrophoresis
Single-molecule electrophoresis measures the different electrophoretic velocities of each
molecule and this can be used to identify components of mixtures of a sample. The method
has been applied to analyze mixtures of nucleic acids and mixtures of proteins and has the
potential to be applied to the area of organic and inorganic chemical analyses. The detec-
tion instrumentation uses a laser beam that is split. The two beams are focused into a
capillary cell to yield two 5-µm spots, which are separated by 250 µm from each other. An
external electric field is applied between the two ends of the capillary cell and the
individual molecules are detected as they pass through each of the two laser beams. Each
laser beam has a separate time correlated single-photon detector under computer control,
and the electrophoretic velocity can be calculated from the time required for each molecule
to travel the distance between the two beams (77). The computer then produces a histogram
of electrophoretic velocities, which shows a statistical maximum for each molecule present
in the sample. A comparison of measured velocities with the velocity characteristics of a cal-
ibration species allows tentative identification of the molecules in a sample. The elec-
trophoretic velocity of a single molecule is determined by its size, shape, ionic charge, and
the chemical environment of the solution in which it is contained (78). The electrophoretic
velocity, therefore, can provide a unique signature for different molecular species.
The method combines the advantages of bulk solution capillary electrophoresis and sin-
gle molecule detection. It is possible to develop automation, speed, reproducibility, and
high sensitivity by optimizing separation and choosing appropriate detection technology.
Single-molecule electrophoresis has the potential to be used in fluorescence immunoassay,
hybridization, and DNA fingerprinting techniques without the need for extensive DNA
amplification by using PCR or other methods (77).
2.4.4
Single Molecule Detection in the Study of Dynamics
Two different time scales are encountered when studying the dynamics of single mole-
cules. A slow scale of milliseconds to hundreds of seconds provides time for processes
such as spectral fluctuation and diffusion. The scale of picoseconds to nanoseconds is a
