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Hydrogen peroxide (H2O2) sensing has been widely investigated using various electrochemical methods, yet the challenge of finding an imaging technique capable of real-time, spatially resolved detection remains. Addressing this, we introduce a Prussian blue (PB) nanofilm-sensitized plasmonic electrochemical microscopy (PEM) technique that successfully visualizes the localized delivery of H2O2. The PB nanofilm was carefully characterized, and its sensing capability towards H2O2 was demonstrated in amperometric mode. Employing a precise micromanipulator system, we controlled a micropipette to create a localized concentration gradient on the sensor surface and monitored the gradient through the PB nanofilm-sensitized PEM. The accuracy of the obtained concentration values was further validated by numerical simulations based on finite-element methods. Our technique ensures dependable localized detection, and we anticipate that advancements in film uniformity will further improve the resolution. The potential applications of this technique are broad and significant, including the opportunity to investigate single-cell exocytosis with neurotransmitters like dopamine, thus offering a promising avenue for future biomedical research.

t‐Darpp (truncated isoform of dopamine‐ and cAMP‐regulated phosphoprotein) is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t‐Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells, t‐Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t‐Darpp function, we studied its secondary structure, oligomerization status, metal‐binding properties, and phosphorylation by cyclin‐dependent kinases 1 and 5. t‐Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn, and 35% random coil structures. It binds calcium, but not other metals commonly found in biological systems. The T39 site, critical for t‐Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin‐dependent kinase 1 and cyclin‐dependent kinase 5. Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde‐mediated cross‐linking experiments demonstrate that the majority of t‐Darpp exists as a monomer, but forms low levels (

The effect of potential bias, light intensity, and the concentration of the photohole scavenger (glucose) on the photocurrent responses of the nanoporous TiO2 film electrodes is studied focusing on the overall photoelectrocatalytic oxidation process. It was observed that at high glucose concentrations the photocurrent increases linearly with potential positive of the zero current potential.

The complexation of Cu(II) by amyloid-[beta] (A[beta]) is examined through the tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. With the help of tyrosine fluorescence spectroscopy has provided mechanistic insights into the oxidation state of copper ions bound to A[beta], the binding heterogeneity and the impact of solution conditions on complex formation.

The redox reactions of copper complexes and the binding stoichiometry between Cu(II) and the full-length [beta]-amyloid A[beta](1-42) was determined. A stoichiometric ratio of 1:1 was observed which remains the same in the presence of more than 10--fold excess of Cu(II) and the A[beta]-Cu(II) complex could be involved in the consumption of antioxidants causing their depletion in neurons and damaging the neuronal defense system.

Copper coexists with amyloid-β (Aβ) peptides at a high concentration in the senile plaques of Alzheimer’s disease (AD) patients and has been linked to oxidative damage associated with AD pathology. However, the origin of copper and the driving force behind its accumulation are unknown. We designed a sensitive fluorescent probe, Aβ(1–16)(Y10W), by substituting the tyrosine residue at position 10 in the hydrophilic domain of Aβ(1–42) with tryptophan. Upon mixing Cu (II), Aβ(1–16)(Y10W), and aliquots of Aβ(1–42) taken from samples incubated for different lengths of time, we found that the Cu(II) binding strength of aggregated Aβ(1–42) has been elevated by more than 2 orders of magnitude with respect to that of monomeric Aβ(1–42). Electron paramagnetic spectroscopic measurements revealed that the Aβ(1–42) aggregates, unlike their monomeric form, can seize copper from human serum albumin, an abundant copper-containing protein in brain and cerebrospinal fluid. The significantly elevated binding strength of the Aβ(1–42) aggregates can be rationalized by a Cu(II) coordination sphere constituted by three histidines from two adjacent Aβ(1–42) molecules. Our work demonstrates that the copper binding affinity of Aβ(1–42) is dependent on its aggregation state and provides new insight into how and why senile plaques accumulate copper in vivo. [ABSTRACT FROM AUTHOR]

The prion protein (PrP) takes up 4-6 equiv of copper in its extended N-terminal domain, composed of the octarepeat (OR) segment (human sequence residues 60-91) and two mononuclear binding sites (at His96 and His111; also referred to as the non-OR region). The OR segment responds to specific copper concentrations by transitioning from a multi-His mode at low copper levels to a single-His, amide nitrogen mode at high levels (Chattopadhyay et al. J. Am. Chem. Soc.2005, 127, 12647-12656). The specific function of PrP in healthy tissue is unclear, but numerous reports link copper uptake to a neuroprotective role that regulates cellular stress (Stevens, et al. PLoS Pathog.2009, 5 (4), e1000390). A current working hypothesis is that the high occupancy binding mode quenches copper's inherent redox cycling, thus, protecting against the production of reactive oxygen species from unregulated Fenton type reactions. Here, we directly test this hypothesis by performing detailed pH-dependent electrochemical measurements on both low and high occupancy copper binding modes. In contrast to the current belief, we find that the low occupancy mode completely quenches redox cycling, but high occupancy leads to the gentle production of hydrogen peroxide through a catalytic reduction of oxygen facilitated by the complex. These electrochemical findings are supported by independent kinetic measurements that probe for ascorbate usage and also peroxide production. Hydrogen peroxide production is also observed from a segment corresponding to the non-OR region. Collectively, these results overturn the current working hypothesis and suggest, instead, that the redox cycling of copper bound to PrP in the high occupancy mode is not quenched, but is regulated. The observed production of hydrogen peroxide suggests a mechanism that could explain PrP's putative role in cellular signaling. [ABSTRACT FROM AUTHOR]

This paper describes the construction of a mixed monolayer of ferrocenylalkanethiol and encapsulated horseradish peroxidase (HRP) at a gold electrode for amperometric detection of H2O2 at trace levels. By tuning the alkanethiol chain lengths that tether the HRP enzyme and the ferrocenylalkanethiol (FcC11SH) mediator, facile electron transfer between FcC11SH and HRP can be achieved. Unlike most HRP-based electrochemical sensors, which rely on HRP-facilitated H2O2 reduction (to H2O), the electrocatalytic current is resulted from an HRP-catalyzed oxidation reaction of H2O2 (to O2). Upon optimizing other experimental conditions (surface coverage ratio, pH, and flow rate), the electrocatalytic reaction proceeding at the electrode was used to attain a low amperometric detection level (0.64 nM) and a dynamic range spanning over 3 orders of magnitude. Not only does the thin hydrophilic porous HRP capsule allow facile electron transfer, it also enables H2O2 to permeate. More significantly, the enzymatic activity of the encapsulated HRP is retained for a considerably longer period (>3 weeks) than naked HRP molecules attached to an electrode or those wired to a redox polymer thin film. By comparing to electrodes modified with denatured HRP that are subsequently encapsulated or embedded in a poly-L-lysine matrix, it is concluded that the encapsulation has significantly preserved the native structure of HRP and therefore its enzymatic activity. The electrode covered with FcC11SH and encapsulated HRP is shown to be capable of rapidly and reproducibly detecting H2O2 present in complex sample media. [ABSTRACT FROM AUTHOR]

The interaction of amyloid-β (Aβ) and redox-active metals, two important biomarkers present in the senile plaques of Alzheimer's disease (AD) brain, has been suggested to enhance the Aβ aggregation or facilitate the generation of reactive oxygen species (ROS). This study investigates the nature of the interaction between the metal-binding domain of Aβ, viz., Aβ(1-16), and the Fe(III) or Fe(II) complex with nitrilotriacetic acid (NTA). Using electrospray ionization mass spectrometry (ESI-MS), the formation of a ternary complex of Aβ(1-16), Fe(III), and NTA with a stoichiometry of 1:1:1 was identified. MS also revealed that the NTA moiety can be detached via collision-induced dissociation. The cumulative dissociation constants of both Aβ-Fe(III)-NTA and Aβ-Fe(II)-NTA complexes were deduced to be 6.3 × 10-21 and 5.0 × 10-12 M2, respectively, via measurement of the fluorescence quenching of the sole tyrosine residue on upon formation of the complex. The redox properties of these two complexes were investigated by cyclic voltammetry. The redox potential of the Aβ-Fe(III)-NTA complex was found to be 0.03 V versus Ag/AgCl, which is negatively shifted by 0.54 V when compared to the redox potential of free Fe(III)/Fe(II). Despite such a large potential modulation, the redox potential of the Aβ-Fe(III)-NTA complex is still sufficiently high for a range of redox reactions with cellular species to occur. The Aβ-Fe(II)-NTA complex electrogenerated from the Aβ-Fe(III)-NTA complex was also found to catalyze the reduction of oxygen to produce H2O2. These findings provide significant insight into the role of iron and Aβ in the development of AD. The binding of iron by Aβ modulates the redox potential to a level at which its redox cycling occurs. In the presence of a biological reductant (antioxidant), redox cycling of iron could disrupt the redox balance within the cellular milieu. As a consequence, not only is ROS continuously produced, but oxygen and biological reductants can also be depleted. A cascade of biological processes can therefore be affected. In addition, the strong binding affinity of Aβ toward Fe(III) and Fe(II) indicates Aβ could compete for iron against other iron-containing proteins. In particular, its strong affinity for Fe(II), which is 8 orders of magnitude stronger than that of transferrin, would greatly interfere with iron homeostasis. [ABSTRACT FROM AUTHOR]

A novel fluorescent CdS-encapsulated DNA nanocomposite was synthesized via alternate adsorption of Cd2+ and S2- onto the DNA template affixed inside an agarose gel. Confining DNA molecules in the gel matrix reduces the flexibility of the DNA strand, which facilitates the formation of a uniform coating of CdS onto the DNA template. The resultant rod-shaped nanocomposite (40-90 nm in width and 200-300 nm in length) is well dispersed in solution and fluoresces at 330 nm upon excitation at either 228 or 280 nm. The fluorescence is attributed to tiny particles present in the CdS coating. It was found that the fluorescence can be significantly quenched by trace amount of Hg2+. The high selectivity toward Hg2+ and the apparent change in the CdS coating upon exposure to Hg2+ indicate that Hg2+ has reacted with the CdS coating through formation of the much more insoluble HgS and the bridging S-Hg-S bonds at the surface. The extent of quenching is dependent on the concentration of Hg2+ in the range of 0.04-13 μM, and a remarkable detection limit (8.6 nM at 30 °C and 4.3 nM at 50 °C) can be achieved. The feasibility of the method for the analysis of Hg2+ in a wastewater sample was demonstrated with an excellent relative standard deviation (RSD, 3.4%). The method described herein is simple, selective, and sensitive and obviates the need of extensive sample pretreatment or special instrumentation. [ABSTRACT FROM AUTHOR]

At the air/buffer solution interface the kinetics of adsorption of amyloid beta peptide, Aβ(1−42), whose bulk concentration (submicromolar) is more than 2 orders of magnitude lower than that typically used in other in vitro aggregation studies, has been studied using a Langmuir−Blodgett trough. The pressure−time curves exhibit a lag phase, wherein the surface pressure essentially remains at zero, and a rising phase, corresponding to the Aβ adsorption at the interface. The duration of the lag phase was found to be highly dependent on both the Aβ bulk concentration and the solution temperature. A large activation energy (62.2 ± 4.1 KJ/mol) was determined and the apparent adsorption rate constant was found to be linearly dependent on the Aβ bulk concentration. Attenuated total reflection-IR spectra of the adsorbed Aβ transferred to a solid substrate and circular dichroism measurements of Aβ in the solution layer near the interface reveal that the natively unstructured Aβ in the bulk undergo a conformation change (folding) to mainly the α-helical structure. The results suggest that, prior to the adsorption step, an equilibrium between Aβ conformations is established within the subsurface. The kinetic equation derived from this model confirms that the overall Aβ adsorption is kinetically controlled and the apparent rate constant is proportional to the Aβ bulk concentration. This model also indicates that interfaces such as cell membranes and lipid bilayers may facilitate Aβ aggregation/fibrillation by providing a thin hydrophobic layer adjacent to the interface for the initial Aβ conformation change (misfolding) and accumulation. Such a preconcentration effect offers a plausible explanation of the fact that Aβ fibrillation occurs in vivo at nanomolar concentrations. Another important biological implication from our work is that Aβ misfolding may occur before its adsorption onto a cell membrane. This general kinetic model should also find applications in adsorption studies of other types of biomolecules whose overall kinetics exhibits a lag phase that is dependent on the bulk concentration of the adsorbate. [ABSTRACT FROM AUTHOR]

Due in large part to the lack of crystal structures of the amyloid-β (Aβ) peptide and its complexes with Cu(II), Fe(II), and Zn(II), characterization of the metal−Aβ complex has been difficult. In this work, we investigated the complexation of Cu(II) by Aβ through tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. EPR experiments indicate that Cu(II) bound to Aβ can be reduced to Cu(I) using sodium borohydride and that both Aβ−Cu(II) and Aβ−Cu(I) are chemically stable. Upon reduction of Cu(II) to Cu(I), the Aβ fluorescence, commonly reported to be quenched upon Aβ−Cu(II) complex formation, can be regenerated. The absence of the characteristic tyrosinate peak in the absorption spectra of Aβ−Cu(II) complexes provides evidence that the sole tyrosine residue in Aβ is not one of the four equatorial ligands bound to Cu(II), but remains close to the metal center, and its fluorescence is sensitive to the copper oxidation state and perturbations in the coordination sphere. Further analysis of the quenching and Cu(II) binding behaviors at different Cu(II) concentrations and in the presence of the competing ligand glycine offers evidence supporting the operation of two binding regimes which demonstrate different levels of fluorescence recovery upon addition of the reducing agent. We provide results that suggest the fluorescence quenching is likely caused by charge transfer processes. Thus, by using tyrosine to probe the coordination site, fluorescence spectroscopy provides valuable mechanistic insights into the oxidation state of copper ions bound to Aβ, the binding heterogeneity, and the influence of solution conditions on complex formation. [ABSTRACT FROM AUTHOR]

The binding stoichiometry between Cu(II) and the full-length β-amyloid Aβ(1-42) and the oxidation state of copper in the resultant complex were determined by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) and cyclic voltammetry. The same approach was extended to the copper complexes of Aβ(1-16) and Aβ(1-28). A stoichiometric ratio of 1:1 was directly observed, and the oxidation state of copper was deduced to be 2+ for all of the complexes, and residues tyrosine-10 and methionine-35 are not oxidized in the Aβ(1-42)-Cu(II) complex. The stoichiometric ratio remains the same in the presence of more than a 10-fold excess of Cu(II). Redox potentials of the sole tyrosine residue and the Cu(II) center were determined to be ca. 0.75 and 0.08 V vs Ag/AgCl [or 0.95 and 0.28 V vs normal hydrogen electrode (NHE)], respectively. More importantly, for the first time, the Aβ-Cu(I) complex has been generated electrochemically and was found to catalyze the reduction of oxygen to produce hydrogen peroxide. The voltammetric behaviors of the three Aβ segments suggest that diffusion of oxygen to the metal center can be affected by the length and hydrophobicity of the Aβ peptide. The determination and assignment of the redox potentials clarify some misconceptions in the redox reactions involving Aβ and provide new insight into the possible roles of redox metal ions in the Alzheimer's disease (AD) pathogenesis. In cellular environments, the reduction potential of the Aβ-Cu(II) complex is sufficiently high to react with antioxidants (e.g., ascorbic acid) and cellular redox buffers (e.g., glutathione), and the Aβ-Cu(I) complex produced could subsequently reduce oxygen to form hydrogen peroxide via a catalytic cycle. Using voltammetry, the Aβ-Cu(II) complex formed in solution was found to be readily reduced by ascorbic acid. Hydrogen peroxide produced, in addition to its role in damaging DNA, protein, and lipid molecules, can also be involved in the further consumption of antioxidants, causing their depletion in neurons and eventually damaging the neuronal defense system. Another possibility is that Aβ-Cu(II) could react with species involved in the cascade of electron transfer events of mitochondria and might potentially sidetrack the electron transfer processes in the respiratory chain, leading to mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Nanoporous TiO2 film electrodes with a mixed anatase/rutile phase were prepared by dip-coating TiO2 nanoparticle colloid onto Indium Tin Oxide (ITO) conducting glass substrates and a subsequent calcination process at 700 °C for 16 h. The photocatalytic oxidation of a wide range of organic compounds has been studied using the photo-electrochemical method under the conditions that the photohole capturing step controls the overall photocatalytic processes. The characteristics of the mixed anatase/rutile phase TiO2 film electrodes were compared with pure anatase phase TiO2 film electrodes to identify the key differences between them. The results revealed that different organic compounds, despite their difference in chemical entities, can be stoichiometrically mineralized at the mixed-phase TiO2 electrode under diffusion-controlled conditions, which is in great contrast to the situation at the pure anatase phase TiO2 electrode. The exceptional ability of the mixed-phase TiO2 electrodes for mineralization of organic compounds and their remarkable resistance to the inhibition by aromatic compounds at higher concentration has been explained by the synergetic effect of the rutile and anatase phases. For this type of mixed phase electrodes, upon absorption of UV light, the electron-transfer pathway from anatase phase to rutile phase facilitates the separation of photoelectron and photohole, extending the lifetime of the photoelectron and photohole. [ABSTRACT FROM AUTHOR]

A novel rapid methodology for the determination of chemical oxygen demand (COD) based on photoelectrochemical oxidative degradation principle (PECOD) was proposed and experimentally validated. With this new method, the extent of degradation of dissolved organic matter in a water sample is measured simply by directly quantifying the extent of electron transfer at a TiO[SUB2] nanoporous film electrode during an exhaustive photoelectrocatalytic degradation of organic matter in a thin layer photoelectrochemical cell. The PECOD method demonstrated in this work is a direct and absolute method. It does not require the use of standard for calibration. The method, in principle, measures the theoretical COD value due to the extraordinary high oxidation efficiency and accuracy of charge measurement. This new approach overcomes many of the current problems associated with existing oxygen demand techniques (e.g., the matrix effect, one of the serious practical problems that most rapid COD methods suffered because of the insufficient oxidation efficiency). The PECOD method overcomes the matrix effect by employing a highly effective photoelectrochemical system that is capable of fully oxidizing a wide spectrum of organics in the water sample. The method was successfully applied to determine the COD of a range of synthetic and real samples. Excellent agreement with a standard dichromate method was achieved. The practical detection limit of 0.2 mg L[SUP-1] COD with the linear range of 0-200 mg L[SUP-1] was also achieved. The PECOD method is a method that is environmentally friendly, robust, rapid, and easily automated. It requires only 1-5 min to complete an assay and consumes very limited reagent (electrolyte only). [ABSTRACT FROM AUTHOR]