Nanosensors Sensitive Probes for the Biodetection of ROS
In the context of testing, a nanobiosensor can involve a biological molecule that serves as a detector, linker, or mediator, and nanoelectrodes. Various components can be equated with the electronic elements of a sensor, as every component has to transduce the signal generated at the source (biomolecule) to the detector (electrode) (Figure 6.2). Consequently,
NADH peroxidase Multi-histidine peptide AuNP
Figure 6.2 Nanobiosensor assembly based on the atomic coordinates of the NADH peroxidase and MHP (multi-histidine peptide). The peptide coordinates cobalts (small sphere) through histidine residues at every i, i + 4 positions. AuNP was modeled in as a sphere, to scale with the biological molecules, with a diameter of 14 A.
NADH peroxidase Multi-histidine peptide AuNP
Figure 6.2 Nanobiosensor assembly based on the atomic coordinates of the NADH peroxidase and MHP (multi-histidine peptide). The peptide coordinates cobalts (small sphere) through histidine residues at every i, i + 4 positions. AuNP was modeled in as a sphere, to scale with the biological molecules, with a diameter of 14 A.
as in enzyme systems, rate improvements can occur from proximity and geometric effects, with potential enhancements of 102 to 103 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 averaging, distributions and fluctuations of molecular properties can be characterized, transient intermediates identified, and catalytic mechanisms 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 investigate 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 H2O2 and O2 . Although this system used an oxidative 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 products 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 properties of the individual components: the protein machinery for sensitivity 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 peroxidase biosensors has allowed us to detect the presence of ROS in ambient 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 confirmation of induction of specific cellular response elements due to exposure to various NM.
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