The origin of high electrocatalytic activity of hydrogen peroxide reduction reaction by a g-C3N4/HOPG sensor.

Graphitic carbon nitride (g-C3N4) was synthesized from a low-cost precursor by means of a thermal process. The product was characterized by several spectroscopic techniques and the crystallinity was analyzed by X-ray diffraction. In the manufacture of the sensor, g-C3N4 was chemically exfoliated and a film was placed on the surface of a Highly Oriented Pyrolytic Graphite (HOPG). We compared the electrocatalytic activities of g-C3N4/HOPG and pristine HOPG surfaces as sensors for H2O2 quantification in buffer solution at pH 7. The results indicate that the surface of g-C3N4/HOPG exhibits striking analytical stability as well as reproducibility, enabling a reliable and sensitive determination within the 0.12-120 µM interval with a detection limit of 0.12 µM. These results suggest that this g-C3N4 film is a really particularly good nano-structured material to be applied as a biosensor. Chemical and physical factors are responsible for the outstanding electrocatalytic activity observed. The N in the g-C3N4 allows huge uptake of H2O2 through the hydrogen-bonding interaction and the change in the electronic structure since the HOPG/g-C3N4 heterojunction favors the charge transfer process through the interface.

Compositional studies of human RPE lipofuscin.

Age-related macular degeneration (AMD) is an ocular disease that causes visual loss and legal blindness in the elderly population. The etiology of AMD is complex and may include genetic predispositions, accumulation of lipofuscin and drusen, local inflammation and neovascularization. The accumulation of lipofuscin has been shown to precede the death of photoreceptor cells and the deterioration of the RPE. As a result, the determination of the photosensitive components of lipofuscin has been of major interest. One of these components, previously identified as a bis-retinoid pyridinium compound, is referred to as A2E. A2E has been characterized by mass spectrometry and is known to have a mass of 592 Da. Most remaining chromophores in RPE lipofuscin are structurally related to A2E as determined by their fragmentation pattern with losses of M ± 190, 174 and/or 150 Da. Analysis of lipofuscin from various donors indicated that the extracts consist of as many as 15 of these hydrophobic components, which are also observed to form spontaneously in vitro over extended periods of time. These consist of ca 90% of the A2E-like components in RPE lipofuscin and correspond to derivatized A2E with discrete molecular weights of 800-900 m/z, 970-1080 m/z and above 1200 m/z regions. It was determined that these species are formed from self-reaction of A2E oxidation products or their reaction with A2E itself to form higher molecular weight products. The majority of modifications are much more hydrophobic than A2E and exhibit increasingly higher values of log P. This acts as a driving force for the sequestering of A2E into granules resulting in a concomitant diminution of its reactivity in vivo.

A new approach to measuring the action spectrum for singlet oxygen production by human retinal lipofuscin.

The human retinal pigment epithelial (RPE) layer contains a complex mixture of components called lipofuscin; this mixture forms with age and with various genetic disorders such as Stargardt's disease. Its presence may contribute to retinal deterioration via several mechanisms including photochemical processes. In the lipofuscin mixture, both type I and II mechanisms have been identified, with the latter consisting of the generation of singlet oxygen. Several components of that mixture have been identified, most notably a bis-retinoid pyridinium compound called A2E and its derivatives. Photooxidative studies on the compound A2E have revealed that its dominant photochemical mechanism is via free radical or type I processes. Because singlet oxygen is an important photooxidative intermediate in tissue, its generation in the RPE may contribute to retinal maculopathies. It is therefore necessary to determine which specific component(s) in the lipofuscin mixture produce singlet oxygen upon excitation with light. This was ascertained by evaluating the action spectrum for singlet oxygen production for the whole lipofuscin mixture using time-resolved spectroscopy. Singlet oxygen was generated by excitation of the sample at different wavelengths while maintaining a constant beam energy, and was directly detected by its phosphorescence decay at 1270 nm using a Ge photodiode. The action spectrum for singlet oxygen sensitization by the organic soluble portion of lipofuscin had an absorption maximum at ca 380 nm, which is to the blue of A2E (maximum at 430 nm). Compounds with a similar absorption maximum eluted in the HPLC earlier than A2E and were detected in human lipofuscin. The concentration of this component apparently increased in concentration in human RPE lipofuscin mixture as a function of age up to 90 years old.

The photochemical oxidation of A2E results in the formation of a 5,8,5',8'-bis-furanoid oxide.

With age and certain diseases, the retinal pigment epithelium (RPE) accumulates a complex mixture of fluorophores generally called lipofuscin. This mixture has been implicated in those diseases via several possible mechanisms including photochemically mediated damage and detergent effects. Recently, a component of that mixture has been identified, a bis-retinoid pyridinium compound called A2E. Furthermore, the photolysis of this compound has been shown to lead to a series of photooxidation products each differing by 16 amu; with the addition of up to a total of nine oxygen atoms to A2E. It has been suggested that these products consist of a series of epoxides forming along the acyclic side chains. The present study reinvestigates this hypothesis and demonstrates that the bis-oxygenated product is in fact a relatively stable 5-8 5'-8'-bis-furanoid oxide. This is in agreement with the oxidation products observed for a homologue of A2E, beta-carotene. In addition, it was found that the bis-oxygenated photoproduct of A2E contained a mixture of symmetrical and unsymmetrical oxidized products where the second oxygen was present in the cyclohexenyl ring structure in the unsymmetrical product. Most importantly, both of these oxidation products of A2E were detected in lipofuscin extracts from human RPE cells, suggesting that these processes occur in vivo.

A mechanistic study of the photooxidation of A2E, a component of human retinal lipofuscin.

A major constituent of human retinal lipofuscin is A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-hexatrienyl]-pyridinium). Light transmitted by the lens is absorbed by A2E and the processes initiated by this absorption has been implicated in several maculopothies. The purpose of this study was to evaluate the dominant photochemical mechanisms involved in these reactions, whether through free radical or singlet oxygen intermediacy. The photodestruction of A2E occurs faster in water vs. chloroform and hydrogenated vs. perdeuterated methanol. Both results suggest a free radical mechanism. Product distributions indicate sequential oxygen addition rather than the addition of two oxygen atoms which would be expected if singlet oxygen was an intermediate. Finally, EPR trapping studies lead to the detection of superoxide as the primary intermediate in the photochemical reactions. It is concluded that if singlet oxygen is involved in these photochemical processes it is of minor importance.

Observation of A2E oxidation products in human retinal lipofuscin.

Several retinal dystrophies are associated with the accumulation of lipofuscin in the retinal pigment epithelium (RPE). The only structurally characterized component of human retinal lipofuscin is the bis-retinoid pyridinium compound A2E. We report here on the observation of a monooxygenated product of A2E in the organic soluble portion of human retinal lipofuscin and in the organic extract of bovine RPE cells that have been fed A2E and irradiated. Liquid chromatography mass spectrometry confirms that the products are identical. This is the first observation of a photoproduct of A2E in human retinal lipofuscin.

Study of human serum albumin-TiO(2) nanocrystalline electrodes interaction by impedance electrochemical spectroscopy.

The adsorption of human serum albumin (HSA) onto nanocrystalline TiO(2) electrodes was studied by electrochemical impedance spectroscopy (EIS) in function of pH and electrode potential. The characterization and physico-chemical properties of the TiO(2) electrode were investigated by scanning electron microscopy (SEM), UV-photoelectron spectroscopy (UPS), cyclic voltammetry and capacitance measurements. The impedance response of the particulate TiO(2) electrode/protein interface was fitted using an equivalent circuit model to describe the adsorption process. The adsorbed protein layer, which is formed as soon as the protein is injected into the solution and becomes in contact with the electrode, was investigated as a function of electrode potential and solution pH. The measurements were performed under pseudo-steady-state and steady-state conditions, which gave information about the different states of the system. With the pseudo-steady state measurements, it was possible to determine two rate constants of the protein adsorption process, which correspond to two different states of the protein. The shortest one was associated with the first contact between the protein and the substrate and the second relaxation time, with the protein suffering an structural rearrangement due to the interaction with the TiO(2) electrode. It was detected that at sufficiently long times (approx. 1 h, where the system was under steady state conditions), a quasi-reversible protein adsorption mechanism was established. The measurements performed as a function of frequency under steady-state conditions, an equivalent circuit with a Warburg element gave the better fitting to data taken at -0.585 V closer to the oxide flat band potential and it was associated with protein diffusion. Experimental results obtained at only one frequency as a function of potential could be fitted to a model that takes into account non-specific and probable specific protein adsorption, which renders to be potential- and pH-dependent. Low capacity values were obtained in the whole potential range, which were measured in the presence and in the absence of the protein layer. The capacity dependence on potential and pH were associated with the generation of surface states on TiO(2). A surface state concentration of 4.1x10(18) cm(-2) was obtained by relating the parallel capacitance with oxide surface states arising from the protein-oxide interaction.