Improved Detection Limit and Stability of Amperometric Carbon Nanotube-Based Immunosensors by Crosslinking Antibodies with Polylysine

Amperometric immunosensor configurations featuring covalently bound anti-biotin antibodies (Ab) embedded into a polylysine (PLL)-single walled carbon nanotube (SWCNT) composite layer were evaluated. Assemblies were made by first oxidizing pyrolytic graphite (PG) electrodes to form surface carboxylic acid groups, to which PLL, SWCNTs and anti-biotin were covalently linked. Incorporating SWCNT into PLL-antibody assemblies improved the amperometric detection limit for biotin (Ag) labeled with horseradish peroxidase to 10 fmol mL(-1). Anti-biotin embedded into the PLL matrix had improved thermal stability and retained its binding ability for biotin after exposure to temperatures of 42 degrees C for up to 3 hours, while the noncrosslinked antibody was inactivated at this temperature in several minutes.     

Secondary nucleation and accessible surface in insulin amyloid fibril formation

At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical study of the fibrillation kinetics suggests that the early stages of the process are affected by stochastic nucleation events.     

Conjugating luminescent CdTe quantum dots with biomolecules

Newly prepared CdTe quantum dots ( QD) bearing shells of water solubility providing capping agents (i.e., thioglycolic acid ( TGA) and 2-(dimethylamino)ethanethiol hydrochloride (DMAET) were subjected to electrostatic assays with several proteins (i.e., cytochrome c (cyt c) and human serum albumin (HSA). In particular, we employed absorption, emission, transient absorption and time-resolved emission spectroscopic means to test their response to light. Only for negatively capped QDs spectroscopic and kinetic evidence were gathered that corroborate the successful bioconjugation of QDs with cyt c to yield QD- cyt c bioconjugates. In fact, photoexcitation of QD-cyt c leads to a fast deactivation of the QD band gap emission and of the QD excited state. Notably, these interactions depend on the size of the QDs. Repulsive forces, on the other hand, are operative between the positively capped QDs and cyt c, hampering any bioconjugation.     

Langmuir-Blodgett films of a cationic zinc porphyrin-imidazole-functionalized fullerene dyad: formation and photoelectrochemical studies

Electron donor-acceptor dyad ensembles of a water-soluble cationic zinc porphyrin (viz., zinc tetrakis(N-methylpyridinium)porphyrin tetrachloride, Zn(TMPyP)) and a C60 derivative that bears an imidazole ligand (viz., 2-(phenylimidazolyl)fulleropyrrolidine, C60im) were assembled during the formation of Langmuir and then Langmuir-Blodgett (LB) films. Surface pressure versus surface area isotherms and surface pressure time profiles, as well as Brewster angle microscopic images documented that the Langmuir films formed were remarkably stable. Subsequently, these Langmuir films were transferred onto different solid substrates, by using the LB technique, for spectroscopic and photoelectrochemical characterization. The UV-vis spectroscopic investigations confirmed that the water-soluble Zn(TMPyP) was, indeed, transferred together with C60im in the LB films. Upon visible light illumination of these LB films, deposited on the ITO transparent conductive supports, a photocurrent generated in the C60im-Zn(TMPyP) system is ascribed to an efficient photoinduced electron transfer from the electron donor, porphyrin singlet excited-state to the electron acceptor, C60. Overall, internal photon-to-current efficiency, IPCE, of the photoanodic current generation (with ascorbate as a sacrificial electron donor) in the ITO/C60im-Zn(TMPyP)/ascorbate/Pt construct is over 5x larger than that of the photocathodic system (with methyl viologen, MV2+, as a sacrificial electron acceptor) in the ITO/Zn(TMPyP)-C60im/MV2+/Pt construct. Highly ordered film stacking favors vectorial electron transfer within the dyad, giving rise to the highest IPCE values of 2.5% determined for a photoanode that was composed of around 20 monolayer films.     

Exploring the subtleties of drug-receptor interactions: the case of matrix metalloproteinases

By solving high-resolution crystal structures of a large number (14 in this case) of adducts of matrix metalloproteinase 12 (MMP12) with strong, nanomolar, inhibitors all derived from a single ligand scaffold, it is shown that the energetics of the ligand-protein interactions can be accounted for directly from the structures to a level of detail that allows us to rationalize for the differential binding affinity between pairs of closely related ligands. In each case, variations in binding affinities can be traced back to slight improvements or worsening of specific interactions with the protein of one or more ligand atoms. Isothermal calorimetry measurements show that the binding of this class of MMP inhibitors is largely enthalpy driven, but a favorable entropic contribution is always present. The binding enthalpy of acetohydroxamic acid (AHA), the prototype zinc-binding group in MMP drug discovery, has been also accurately measured. In principle, this research permits the planning of either improved inhibitors, or inhibitors with improved selectivity for one or another MMP. The present analysis is applicable to any drug target for which structural information on adducts with a series of homologous ligands can be obtained, while structural information obtained from in silico docking is probably not accurate enough for this type of study.     

Growth and characterization of films containing fullerenes and water soluble porphyrins for solar energy conversion applications

Thin films consisting of two fulleropyrrolidine derivatives 1 or 2 and a water-soluble porphyrin, TPPS4, were prepared by the Langmuir-Sch?fer (LS, horizontal lifting) method. In particular, a solution of the fulleropyrrolidine in chloroform and dimethyl sulfoxide was spread on the water surface, while the porphyrin (bearing peripheral anionic sulfonic groups) was dissolved into the aqueous subphase. To the best of our knowledge, such a versatile method for film fabrication of fullerene/porphyrin mixed composite films has never been used by other researchers. Evidence of the effective interactions between the two components at the air-water interface was obtained from the analysis of the floating layers by means of surface pressure vs area per molecule Langmuir curves, Brewster angle microscopy, and UV-visible reflection spectroscopy. The characterization of the LS films by UV-visible spectroscopy reveals that in each case the two constituents behave as strongly interacting pi systems. The use of polarized light suggests the existence of a preferential direction of the TPPS4 macrocyclic rings with an edge-on arrangement with respect to the substrate surface, regardless which fulleropyrrolidine derivative is in the composite film. Atomic force microscopy investigations give evidence of morphologically flat layers even for LS transfer at low surface pressures. Photoaction spectra were recorded from films deposited by only one horizontal lifting onto indium-tin-oxide (ITO) electrodes, and the observed photocurrent increased notably with increasing transfer surface pressure for both 1/TPPS4 and 2/TPPS4 composite films. IPCE values are larger for 2/TPPS4 systems in comparison with 1/TPPS4 composite layers. Finally, a nonconventional approach to photoinduced phenomena is proposed by differential spectroscopy in the FT-IR attenuated total reflectance (ATR) mode.     

Switching-On Fluorescence by Copper (II) and Basic Anions: A Case Study with a Pyrene-Functionalized Squaramide

The new symmetric acyclic N,N’-bis(1-pyrenyl) squaramide (H₂L) functionalized with the pyrene moiety as a fluorogenic fragment has been designed and its ability to selectively detect specific anions and metals investigated. H₂L selectively binds Cl⁻ both in solution (DMSO 0.5% H₂O and MeCN) and in the solid state, and allows to selectively detect Cu²⁺ in MeCN with the formation of a 2:1 metal-receptor complex, with a green intense emission appreciable by naked eye under the UV lamp. The H₂L copper complex preserves its emission properties in the presence of Cl⁻. The addition of basic anions (OH⁻, CN⁻, and F⁻) up to 10 equivalents caused the deprotonation of the squaramide NHs and a dramatic change of the emission properties of the H₂L copper complex.     

Regio- and stereoselective lithiation of terminal oxazolinylaziridines: the aziridine N-substituent and the oxazolinyl group effect

The regioselective lithiation of terminal oxazolinylaziridines has been investigated. The steric hindrance of the nitrogen substituent in 1-trityl-2-oxazolinylaziridine 3a, combined with the coordinating ability of the oxazolinyl group, causes beta-lithiation, whereas a completely regioselective alpha-lithiation is observed with the much less sterically demanding 1-benzyl-2-oxazolinylaziridine 3c and a competition between alpha- and beta-lithiation occurs with 1-cumyl-2-oxazolinylaziridine 3b in which the N-substituent has a steric hindrance in between the trityl and the benzyl groups. The application of the lithiation-trapping sequence for the preparation of enantioenriched 2,3-cis-disubstituted oxazolinylaziridines and aziridino-gamma-lactones is also reported.     

A double inequality for a generalized-Euler-constant function

The rate of convergence of the sequence , a>0, towards the generalized Euler?s constant , where γ(1) is the Euler-Mascheroni constant, is accurately estimated using the Euler-Maclaurin summation formula. The expression

     

Study of the Ternary Complex Formation Between Vanadium(III), Dipicolinic Acid and Small Blood Serum Bioligands

Ternary complex formation reactions were studied between vanadium(III), dipicolinic acid and small molecular weight blood serum components: lactic, oxalic, citric and ortophosphoric acids. The electromotive force measurement permitted us to determine the chemical speciation of the complexes formed. In the vanadium(III)–dipicolinic acid–lactic acid system the complexes detected were: V(dipic)(lac), V(dipic)(lac)(OH)⁻ and V(dipic)(lac)(OH)₂^2-(\mathrm{OH})_{2}^{2-}. In the vanadium(III)–dipicolinic acid–oxalic acid system the observed complexes were: V(dipic)(ox)⁻, V(dipic)(ox)(Hox)²⁻ and V(dipic)(ox)₂^3-(\mathrm{ox})_{2}^{3-}. In the vanadium(III)–dipicolinic acid–citric acid system the complexes V(dipic)(Hcit)⁻, V(dipic)(cit)²⁻, V(dipic)(cit)(OH)³⁻, V(dipic)(cit)(OH)₂^4-(\mathrm{OH})_{2}^{4-} and V(dipic)(cit)(OH)₃^5-(\mathrm{OH})_{3}^{5-} were detected. Finally in the vanadium(III)–dipicolinic acid–phosphoric acid system the complexes V(dipic)(H₂PO₄) and V(dipic)(HPO₄)⁻ were observed. The UV-vis spectra allowed us to perform a qualitative characterization of the complexes formed in aqueous solution.