Page 237 - Environmental Nanotechnology Applications and Impacts of Nanomaterials

222 Principles and Methods

as in enzyme systems, rate improvements can occur from proximity
2 3
and geometric effects, with potential enhancements of 10 to 10 at each
junction. Additional advantages arise from the dimensionality at which
detection is conducted. In nanoscale structures, electrons no longer
behave like physical objects that flow in a continuous stream but take
on wave mechanical and quantum properties and have the ability to
tunnel through structures that would ordinarily be insulators. As
single molecule measurements become more feasible with the advent
of methods sensitive enough to study single molecule kinetics, ther-
modynamic, and electronics, significant deviations from ensemble
measurements have been found. With the removal of ensemble aver-
aging, distributions and fluctuations of molecular properties can be
characterized, transient intermediates identified, and catalytic mech-
anisms elucidated. To facilitate single molecule measurements, nano-
electrode platforms have been investigated as nanosensors for enhancing
detection.
We have produced nanobiosensors utilizing various redox enzymes
aligned on nanoelectrode arrays [56]. One of the systems is comprised
of the enzyme, NADH peroxidase, as the specific detector of hydrogen
peroxide, and converts a biological binding event into an electronic
signal[57, 58]. These results demonstrate the use of biosensors to inves-
tigate the ability of nanoparticles to change the redox status of the cell,
as could happen due to the ability of these materials to induce ROS

species such as H O and O . Although this system used an oxidative
2
2
2
metabolism enzyme, other redox proteins, such as glucose oxidase, can
be substituted as the bioelement. The detection event in these redox
enzyme systems is based on generation of electrons as one of the prod-
ucts of an endogenous reaction. In addition to the traditional use of
redox enzymes in biosensors, other nonredox proteins can be used if the
binding of a ligand triggers a conformational change that can be detected
by an induced electronic event or via optical, thermal, or other detectable
physical changes. Alternatively, a virion or particle can theoretically be
the bioelement of a sensor, as structural information is available for
many of these macromolecules. Our strategy integrates desirable prop-
erties of the individual components: the protein machinery for sensi-
tivity and specificity of binding, peptide chemistry for aligning the
various electron transducing units, and the nanoelectrodes for gain
sensitivity in electronic detection (Figure 6.2). Using these NADH per-
oxidase biosensors has allowed us to detect the presence of ROS in ambi-
ent and commercial nanoparticle samples [59]. Comparison to standard
hydrogen peroxide curves permits elucidation of amounts of peroxides
generated. These results highlight the feasibility of utilizing nanobiosen-
sors for detection and, ultimately, quantification of ROS, calcium, and
other fingerprints of activation of specific pathways, thus allowing