Biomedical Engineering Reference
In-Depth Information
The groups of Aartsma and Davis
139
demonstrated that instead
of the intrinsic fluorescence of a reactant, a fluorescent reporter
label can be attached to a non-fluorescent redox molecule. They
attached Cy5 to the blue copper protein azurin, which has a strong
absorption band around 600 nm when oxidized, but is colorless
when reduced. The Cu(II) state absorption overlaps with the dye
emission spectrum, resulting in quenching of the fluorescence due
to FRET. The labeled protein was immobilized on an alkanethiol
SAM on transparent gold, and TIRF microscopy was used to fol-
low the redox-state dependent switching of the fluorescent intensi-
ty during cyclic voltammetry. Although not yet accomplished at
single-molecule level, this promises to be a powerful and versatile
method that does not require an intrinsically fluorogenic reactant.
Surface enhancement results in a spectacular amplification of
Raman scattering, provided that the molecules are in direct contact
with the electrode, the excitation wavelength matches the plasmon
frequency of the metal, and the surface is nanoscopically struc-
tured.
140
This has opened the way to single-molecule Surface-
enhanced Raman scattering (SERS) studies.
141
Electrochemical
SERS developments are now also approaching the single-molecule
level. Islam et al.
142,143
combined SERS spectroscopy with
nanostructured interdigitated array electrodes. The potential-
dependent orientation of the molecules resulted in notably poten-
tial-dependent SERS response. By varying the electrode spacing,
the redox cycling by ferricyanide or crystal violet
molecules, the
collection efficiency, and surface amplification could be optimized.
A real step towards single-molecule SERS spectro-electro-
chemistry was reported by DosSantos and co-workers.
144
Time-
dependent fluctuations were observed from a roughened silver
electrode in dilute solutions of rhodamine and congo red, attribut-
ed to the very small number of SERS-active molecules probing
nanoscopic
hot spots
. The tailed statistics as a function of applied
potential were linked to single-molecule dynamics. An interesting
development is to use an AFM or STM tip as an external Raman-
enhancing unit for single-molecule studies.
145,146
This technically
challenging method has recently been applied
in-situ
.
147
When a single molecule is trapped between two electrodes, a
continuous current can be passed through. This amplification al-
lows for the purely electronic detection of individual molecules.
Such two-electrode junctions can either be pre-designed and nano-
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