Protein–Inorganic Array Construction: Design and Synthesis of the Building Blocks

Herein we describe the design and synthesis of the first series of di‐functional ligands for the directed construction of inorganic‐protein frameworks. The synthesized ligands are composed of a metal‐ion binding moiety (terpyridine‐based) conjugated to an epoxysuccinyl peptide, known to covalently bind active cysteine proteases through the active‐site cysteine. We explore and optimize two different conjugation chemistries between the di‐functionalized metal‐ion ligand and the epoxysuccinyl‐containing peptide moiety: peptide‐bond formation (with limited success) and Cu^I‐catalysed click chemistry (with good results). Further, the complexation of the synthesized ligands with Fe^II and Ni^II ions is investigated: the di‐functional ligands are confirmed to behave similarly to the parent terpyridine. As designed, the peptidic moiety does not interfere with the complexation reaction, in spite of the presence of two triazole rings that result from the click reaction. ES‐MS together with NMR and UV/Vis studies establish the structure, the stoichiometry of the complexation reactions, as well as the conditions under which chemically sensitive peptide‐containing polypyridine ligands can undergo the self‐assembly process. These results establish the versatility of our approach and open the way to the synthesis of di‐functional ligands containing more elaborated polypyridine ligands as well as affinity labels for different enzyme families. As such, this paper is the first step towards the construction of robust supramolecular species that cover a size‐regime and organization level previously unexplored.     

Macroporous antimony film electrodes for stripping analysis of trace heavy metals

We report on the elaboration of macroporous antimony film electrodes. The strategy to create macroporous electrodes is based on the replication of colloidal crystal templates. These electrodes of controlled porosity show an increased internal electroactive area and a significantly improved electrochemical performance. The application of this novel electrochemical sensor for the sensitive quantification of traces of heavy metals has been demonstrated in combination with differential pulse anodic stripping voltammetry in model solutions.     

Advanced Sensing of Antibiotics with Magnetic Gold Nanocomposite: Electrochemical Detection of Chloramphenicol

The sensing and accurate determination of antibiotics in various environments represents a big challenge, mainly owing to their widespread use in medicine, veterinary practice, and other fields. Therefore, a new, simple electrochemical sensor for the detection of antibiotic chloramphenicol (CAP) has been developed in this work. The amplification strategy of the sensor is based on the application of magnetite nanostructures stabilized with carboxymethyl cellulose (Fe₃O₄‐CMC) and decorated with nanometer‐sized Au nanoparticles (NPs) (Fe₃O₄‐CMC@Au). In this case, CMC serves as a stabilizing agent, preventing the aggregation of Fe₃O₄ NPs, and hence, enabling the kinetic barrier for electron transport to be overcome, and the Au NPs serve as an electron‐conducting tunnel for better electron transport. As a proof of concept, the developed nanosensor is used for the detection of CAP in human urine samples, giving a recovery value of around 97 %, which indicates the high accuracy of the as‐prepared nanosensor.     

Model systems for probing metal cation hydration: the V+(H2O) and ArV+(H2O) complexes

In support of mass-selected infrared photodissociation (IRPD) spectroscopy experiments, coupled-cluster methods including all single and double excitations (CCSD) and a perturbative contribution from connected triple excitations [CCSD(T)] have been used to study the V+(H2O) and ArV+(H2O) complexes. Equilibrium geometries, harmonic vibrational frequencies, and dissociation energies were computed for the four lowest-lying quintet states (5A1, 5A2, 5B1, and 5B2), all of which appear within a 6 kcal mol(-1) energy range. Moreover, anharmonic vibrational analyses with complete quartic force fields were executed for the 5A1 states of V+(H2O) and ArV+(H2O). Two different basis sets were used: a Wachters+f V[8s6p4d1f] basis with triple-zeta plus polarization (TZP) for O, H, and Ar; and an Ahlrichs QZVPP V[11s6p5d3f2g] and Ar[9s6p4d2f1g] basis with aug-cc-pVQZ for O and H. The ground state is predicted to be 5A1 for V+(H2O), but argon tagging changes the lowest-lying state to 5B1 for ArV+(H2O). Our computations show an opening of 2 degrees -3 degrees in the equilibrium bond angle of H2O due to its interaction with the metal ion. Zero-point vibrational averaging increases the effective bond angle further by 2.0 degrees -2.5 degrees, mostly because of off-axis motion of the heavy vanadium atom rather than changes in the water bending potential. The total theoretical shift in the bond angle of about +4 degrees is significantly less than the widening near 9 degrees deduced from IRPD experiments. The binding energies (D0) for the successive addition of H2O and Ar to the vanadium cation are 36.2 and 9.4 kcal mol(-1), respectively.     

Electroactivity of Nonconjugated Proteins and Peptides. Towards Electroanalysis of All Proteins

Present proteomics and biomedicine require sensitive analytical methods for all proteins. Recent progress in electrochemical analysis of peptides and proteins based on their intrinsic electroactivity is reviewed. Tyrosine and/or tryptophan‐containing proteins are oxidizable at carbon electrodes. At mercury electrodes all peptides and proteins (about 13 peptides and >25 proteins were tested) produce chronopotentiometric peak H at nanomolar concentrations. This peak is sensitive to changes in protein structure. Microliter sample volumes are sufficient for the analysis. Electrochemical methods can be used in studies of nucleic acid‐protein interactions and can be applied in biomedicine. Examples of such applications in neurogenerative diseases and cancer are presented.     

Development and optimization of ultra-high performance supercritical fluid chromatography mass spectrometry method for high-throughput determination of tocopherols and tocotrienols in human serum

The goal of this study was to develop an effective supercritical fluid chromatography method using single quadrupole MS for analysis of all isomeric forms of vitamin E. Finally, two fast and effective methods, the high resolution one and the high speed one, for the determination of 8 vitamin E isomers in human serum were developed.     

RP-HPLC analysis of phenolic compounds and flavonoids in beverages and plant extracts using a CoulArray detector

Methods were developed for the analysis of natural antioxidants including phenolic compounds and flavonoids in beverages and plant extracts using gradient HPLC with multi-channel electrochemical coulometric detection. Suitability of various reversed-phase columns for this purpose was compared; pH and mobile phase gradients were optimized with respect to the separation selectivity and sensitivity of detection. Because of different target compounds in various sample types, the overlapping resolution maps and the normalized resolution product approaches described earlier were used to select optimum columns and gradients to suit the analysis of the individual sample types. The methods were applied to the analysis of phenolic compounds and flavonoids in beer, wine, tea, and yacon extracts. 32 phenolic compounds were identified and determined, including derivatives of benzoic and cinnamic acids, flavones, and a few related glycosides. Eight-channel CoulArray detection offers high selectivity and sensitivity with limits of detection in the low microg L(-1) range, at least an order of magnitude lower than single-channel coulometric detection using the Coulochem detector. No special sample pretreatment is necessary and, because of the compatibility of the CoulArray detector with gradient elution, phenolic antioxidants of different polarities can be determined in a single run. In addition to the retention times, the ratios of the areas of the pre-dominant and post-dominant peaks to the area of the dominant peak can be used for improved identification of natural antioxidants.     

Hypericin-mediated photocytotoxic effect on HT-29 adenocarcinoma cells is reduced by light fractionation with longer dark pause between two unequal light doses

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm(2)) to the fractionated light delivery (1 + 11 J/cm(2)) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.