Surface‐Tuned Electron Transfer and Electrocatalysis of Hexameric Tyrosine‐Coordinated Heme Protein

Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine‐coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self‐assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl‐terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this “disc” orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi‐reversible redox behavior with rate constant k_s values between 0.93 and 2.86 s^?1 and apparent formal potentials ${E{{0{^{\prime }}\hfill \atop {\rm app}\hfill}}}$ between ‐131.1 and ‐249.1 mV. On the MUA/MU‐modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.     

Direct Electron Transfer and Bioelectrocatalysis by a Hexameric,Heme Protein at Nanostructured Electrodes

A nanohybrid consisting of poly(3‐aminobenzenesulfonic acid‐co‐aniline) and multiwalled carbon nanotubes [MWCNT‐P(ABS‐A)]) on a gold electrode was used to immobilize the hexameric tyrosine‐coordinated heme protein (HTHP). The enzyme showed direct electron transfer between the heme group of the protein and the nanostructured surface. Desorption of the noncovalently bound heme from the protein could be excluded by control measurements with adsorbed hemin on aminohexanthiol‐modified electrodes. The nanostructuring and the optimised charge characteristics resulted in a higher protein coverage as compared with MUA/MU modified electrodes. The adsorbed enzyme shows catalytic activity for the cathodic H₂O₂ reduction and oxidation of NADH.     

Spectroscopic Observation of Calcium‐Induced Reorientation of Cellobiose Dehydrogenase Immobilized on Electrodes and its Effect on Electrocatalytic Activity

Cellobiose dehydrogenase catalyzes the oxidation of various carbohydrates and is considered as a possible anode catalyst in biofuel cells. It has been shown that the catalytic performance of this enzyme immobilized on electrodes can be increased by presence of calcium ions. To get insight into the Ca²⁺‐induced changes in the immobilized enzyme we employ surface‐enhanced vibrational (SERR and SEIRA) spectroscopy together with electrochemistry. Upon addition of Ca²⁺ ions electrochemical measurements show a shift of the catalytic turnover signal to more negative potentials while SERR measurements reveal an offset between the potential of heme reduction and catalytic current. Comparing SERR and SEIRA data we propose that binding of Ca²⁺ to the heme induces protein reorientation in a way that the electron transfer pathway of the catalytic FAD center to the electrode can bypass the heme cofactor, resulting in catalytic activity at more negative potentials.     

Enhanced monitoring of biopharmaceutical product purity using liquid chromatography-mass spectrometry

LC-MS is a widely used technique for impurity detection and identification. It is very informative and generates huge amounts of data. However, the relevant chemical information may not be directly accessible from the raw data map, particularly in reference to applications where unknown impurities are to be detected. This study demonstrates that multivariate statistical process control (MSPC) based on principal component analysis (PCA) in conjunction with multiple testing is very powerful for comprehensive monitoring and detection of an unknown and co-eluting impurity measured with liquid chromatography-mass spectrometry (LC-MS). It is demonstrated how a spiked impurity present at low concentrations (0.05% (w/w)) is detected and further how the contribution plot provides clear diagnostics of the unknown impurity. This tool makes a fully automatic monitoring of LC-MS data possible, where only relevant areas in the LC-MS data are highlighted for further interpretation.     

SAXS investigation of a cubic to a sponge (L3) phase transition in self-assembled lipid nanocarriers

The encapsulation and release of peptides, proteins, nucleic acids, and drugs in nanostructured lipid carriers depend on the type of the self-assembled liquid-crystalline organization and the structural dimensions of the aqueous and membraneous compartments, which can be tuned by the multicomponent composition of the systems. In this work, small-angle X-ray scattering (SAXS) investigation is performed on the 'melting' transition of the bicontinuous double diamond cubic phase, formed by pure glycerol monooleate (MO), upon progressive inclusion of varying fractions of pharmaceutical-grade glycerol monooleate (GO) in the hydrated system. The self-assembled MO/GO mixtures are found to form diamond (Pn3m) inverted cubic, inverted hexagonal (H(II)), and sponge (L(3)) phases at ambient temperature in excess of aqueous medium without heat treatment. Mixing of the inverted-cubic-phase-forming MO and the sponge-phase-forming GO components, in equivalent proportions (50/50 w/w), yields an inverted hexagonal (H(II)) phase nanostructured carrier. Scattering models are applied for fitting of the experimental SAXS patterns and identification of the structural changes in the aqueous and lipid bilayer subcompartments. The possibility of transforming, at ambient temperature (20 °C), the bicontinuous cubic nanostructures into inverted hexagonal (H(II)) or sponge (L(3)) mesophases may facilitate novel biomedical applications of the investigated liquid crystalline self-assemblies.     

Inhibition of amyloid aggregation by formation of helical assemblies

The formation of amyloid aggregates is responsible for a wide range of diseases, including Alzheimer's and Parkinson's disease. Although the amyloid-forming proteins have different structures and sequences, all undergo a conformational change to form amyloid aggregates that have a characteristic cross-β-structure. The mechanistic details of this process are poorly understood, but different strategies for the development of inhibitors of amyloid formation have been proposed. In most cases, chemically diverse compounds bind to an elongated form of the protein in a β-strand conformation and thereby exert their therapeutic effect. However, this approach could favor the formation of prefibrillar oligomeric species, which are thought to be toxic. Herein, we report an alternative approach in which a helical coiled-coil-based inhibitor peptide has been designed to engage a coiled-coil-based amyloid-forming model peptide in a stable coiled-coil arrangement, thereby preventing rearrangement into a β-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, we show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils.     

Multilayered artifacts in the pre-Columbian metallurgy from the North of Peru

Three types of alloys were recognized when analyzing pre-Columbian artifacts from the North of Peru: gold, silver, and copper alloys; gilded copper and silver; silvered copper; tumbaga, i.e., copper or silver enriched on gold at the surface by depletion gilding. In this paper, a method is described to differentiate gold alloys from gilded copper and from copper–gold tumbaga, and silver alloys from silvered copper and copper–silver tumbaga. This method is based on the use of energy-dispersive X-ray fluorescence, i.e., on a sophisticated analysis of XRF-spectra carrying out an accurate determination of Cu(K _α /K _β ), Ag(K _α /K _β ), Au(L _α /L _β ), and Au-L _α /Cu-K _α or Ag-K _α /Cu-K _α ratios. That implies a dedicated software for the quantitative determination of the area of X-ray peaks. This method was first checked by a relevant number of standard samples and then it was applied to pre-Columbian alloys from the North of Peru.     

Biodistribution of gold nanoparticles in mouse lung following intratracheal instillation

Background  

The fate of gold nanoparticles, 2, 40 and 100 nm, administered intratracheally to adult female mice was examined. The nanoparticles were traced by autometallography (AMG) at both ultrastructural and light microscopic levels. Also, the gold content was quantified by inductively coupled plasma mass spectrometry (ICP-MS) and neutron activation analysis (NAA). The liver is the major site of deposition of circulating gold nanoparticles. Therefore the degree of translocation was determined by the hepatic deposition of gold. Mice were instilled with 5 intratracheal doses of gold nanoparticles distributed over a period of 3 weeks and were killed 24 h after the last dose. One group of mice were given a single intratracheal dose and were killed after 1 h.     

Direct Determination of Bacterial Cell Viability Using Carbon Nanotubes Modified Screen‐printed Electrodes

For the early detection of bacterial infection, there is a need for rapid, sensitive, and label‐free assays. Thus, in this study, nanostrucured microbial electrochemical platform is designed to monitor the viability and cell growth of S. aureus. Using multi‐walled carbon nanotube modified screen‐printed electrodes (MWCNTs/SPE), the cyclic voltammetric measurements showed only one irreversible oxidation peak at 600 mV vs Ag/AgCl that accounts for the viable and metabolically active bacterial cells. The assay was optimized and the secreted metabolites, in the extracellular matrix, were directly detected. The peak current showed a positive correlation with viable cell numbers ranging from OD_{600 nm} of 0.1 to 1.1, indicating that the activity of live cells can be quantified. Consequently, responses of viable and non‐viable cells of S. aureus to the effects of antibiotic and respiratory chain inhibitors were determined. Thus, the proposed nanostructure‐based bacterial sensor provides a reasonable and reliable way for real‐time monitoring of live‐dead cell functions, and antibacterial profiling.