Biomedical Engineering Reference
In-Depth Information
phenomena observed from these experiments may be due to slow conformational changes
of proteins (89). Single pair FRET has also been used for such studies (92,93). Taking
advantage of the distance dependence of FRET, the intermediate states during protein-
folding processes have been monitored at different denaturant concentrations. The fluo-
rescence polarization method has also been used to determine the motion of proteins
(94-98).
Besides enzyme reactivity and protein dynamics, various types of biomolecule meas-
urements have been made at single-molecule level. These nanometer-sized biomolecules
use low energy to work efficiently and accurately in our everyday life. The single mole-
cule detection measurements have provided a clue to understand the mechanism of these
processes (89). Though using recent advanced ensemble measurements, the structure of
the biomolecules and the roles they play have already been determined, the dynamic
properties of these molecules are averaged and could not be observed. Therefore, single
molecule detection technique made it possible for us to observe the structure change and
the interaction between these biomolecules dynamically. Furthermore, this technique has
allowed us to record the behavior of individual molecules in real time.
2.5
Conclusions
Optical single molecule detection techniques have been widely developed and applied in
physical chemistry, material science, and the biosciences (102). Compared with the ensem-
ble average experiment, observations of single molecule structure and function provide
advantages in understanding reactivity and dynamics. The optical approach permits such
interrogation without contacting the molecule, although there must be consideration of
the influence and perturbations caused by spectroscopic labels in cases where these are
added. Other single molecule methods that do not make use of optical probing have been
described, and include surface force microscopies, electrochemical wiring to single mole-
cules, and lipid membranes that can detect the transit of single molecules such as DNA
oligonucleotides through artificial pores.
Optical methods that have been adopted for single molecule detection include FCS,
NSOM, CM, TIR, and wide-field epi-illumination. These methods have already been used
to provide much insight into particular chemical and biochemical systems of interest. A
number of other methods are suitable for single molecule analysis, including frequency
modulation spectroscopy and two-photon excitation. It is clear that substantial advantage
can be gained by integration of methods, such as NSOM and AFM. This combination can
provide molecular information by fluorescence detection and nanometer-resolved topo-
graphic information by AFM (11,99-101).
References
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Ambrose, W.P., Goodwin, P.M., Keller, R.A. (1994). Alterations of single molecule fluorescence
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Shera, E.B., Seitzinger, N.K., Davis, L.M., Keller, R.A., Soper, S.A. (1990). Detection of single
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174: 553-557.
3.
Schmidt, T., Schutz, G.J., Baumgartner, W., Gruber, H.J., Schindler, J. (1996). Imaging of single
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