Claudio G. Rodrigues

Single-molecule mass spectrometry in solution using a solitary nanopore.

We introduce a two-dimensional method for mass spectrometry in solution that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) molecules that enter a single α-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of molecules in solution.

Mechanism of KCl Enhancement in Detection of Nonionic Polymers by Nanopore Sensors

The mechanisms of KCl-induced enhancement in identification of individual molecules of poly(ethylene glycol) using solitary alpha-hemolysin nanoscale pores are described. The interaction of single molecules with the nanopore causes changes in the ionic current flowing through the pore. We show that the on-rate constant of the process is several hundred times larger and that the off-rate is several hundred times smaller in 4 M KCl than in 1 M KCl. These shifts dramatically improve detection and make single molecule identification feasible. KCl also changes the solubility of poly(ethylene glycol) by the same order of magnitude as it changes the rate constants. In addition, the polymer-nanopore interaction is determined to be a strong non-monotonic function of voltage, indicating that the flexible, nonionic poly(ethylene glycol) acts as a charged molecule. Therefore, salting-out and Coulombic interactions are responsible for the KCl-induced enhancement. These results will advance the development of devices with sensor elements based on single nanopores.

Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha‐toxin in planar lipid bilayers

Staphylococcal alpha‐toxin forms homo‐oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single‐channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha‐toxin in planar lipid membranes is a heptamer.

Lumen geometry of ion channels formed by Vibrio cholerae EL Tor cytolysin elucidated by nonelectrolyte exclusion

Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes. In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores. It was established that all cytolysin channels were inserted into membranes with the same orientation. Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm). The channel lumen appeared constricted, with a diameter of < or = 1.2 nm. Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.

Identificação de resíduos de disparos de armas de fogo por meio da técnica de espectrometria de massas de alta resolução com fonte de plasma indutivo

The violence derived from crimes involving firearms represents one of the main concerns of society. For this reason modern techniques have emerged in forensic science to identify suspects at crime scenes. This work describes a methodology to identify residues present in the hands of suspect by using a high resolution inductively coupled plasma mass spectrometry and collection procedure based on ethylenediaminetetraacetic acid (EDTA) solution as a complexing agent in moistened swabs. In order to distinguish real gunshot residues from others types of residues present in the hand of suspect, ternary ratio per cent diagrams were developed for antimony (Sb), barium (Ba) and lead (Pb) detected on the hands of volunteers, before and immediately after shooting tests, revealing a remarkable difference in both situations.

Hofmeister Effect in Confined Spaces: Halogen Ions and Single Molecule Detection

Despite extensive research in the nanopore-sensing field, there is a paucity of experimental studies that investigate specific ion effects in confined spaces, such as in nanopores. Here, the effect of halogen anions on a simple bimolecular complexation reaction between monodisperse poly(ethylene glycol) (PEG) and α-hemolysin nanoscale pores have been investigated at the single-molecule level. The anions track the Hofmeister ranking according to their influence upon the on-rate constant. An inverse relationship was demonstrated for the off-rate and the solubility of PEG. The difference among anions spans several hundredfold. Halogen anions play a very significant role in the interaction of PEG with nanopores although, unlike K(+), they do not bind to PEG. The specific effect appears dominated by a hydration-dehydration process where ions and PEG compete for water. Our findings provide what we believe to be novel insights into physicochemical mechanisms involved in single-molecule interactions with nanopores and are clearly relevant to more complicated chemical and biological processes involving a transient association of two or more molecules (e.g., reception, signal transduction, enzyme catalysis). It is anticipated that these findings will advance the development of devices with nanopore-based sensors for chemical and biological applications.

Identificação da influência do descarte de lodo de estações de tratamento de água

The growing concern of environmental surveillance of the quality of hydric resources guides the development of research on management of residues generated in water treatment plants (WTP). Approximately 8.000 WTPs in Brazil operate without a treatment program of the residues, disposing these effluents in the environment. This work evaluated WTP discharges into watercourses by collecting superficial waters, sediments and benthic samples at the town of Registro, Sao Paulo State, Brazil. Even though superficial waters and benthic samples showed no further contamination, sediment analysis pointed out that aluminum deposits detected near sludge discharges may represent a potential risk to the environment.

Inhibition of the hemolytic activity caused by Staphylococcus aureus alpha-hemolysin through isatin-Schiff copper(II) complexes.

A great number of pathogens secrete pore-forming proteins during infection. Such molecules, from either bacterial or viral origin, are considered important virulence factors, which makes them attractive targets in the study of new therapeutic agents. Thus, the inhibitory activity of isatin-Schiff base copper(II) complexes was evaluated against membrane damage activity of Staphylococcus aureus α-hemolysin (α-HL). For this purpose, a standard hemolysis assay with rabbit erythrocytes and micromolar concentrations of the compounds was employed. Additionally, planar artificial lipid membranes with a single α-HL ion channel and molecular docking studies were used to elucidate the molecular mechanism of the complexes. Accordingly, the compounds were observed to possess a significant anti-hemolytic activity, capable of interacting with the constriction region of α-HL channel and blocking it in a potential dependent manner. Based on these results, it is expected that such isatin-Schiff base Copper(II) complexes may be employed as cotherapeutic agents for the treatment of staphylococcal infections.

Non stochastic distribution of single channels in planar lipid bilayers

The selectivity of the planar lipid bilayers modified by two channel-forming proteins (alpha-toxin S. aureus and colicin Ia) was examined. It was established that in all cases the value of zero current potential depended on the amount of open ion channels and increased with the number of channels (from one to about 5-7). These facts point out both the interactions among ion channels and their non stochastic distribution on the membrane surface.

BIOSSENSORIAMENTO ESTOCÁSTICO VIA NANOPORO PROTEICO INDIVIDUAL NO DESENVOLVIMENTO DE FERRAMENTAS ANALÍTICAS

Studies employing a single protein nanopore as a molecular recognition element in the development of analytical devices - biosensors, spectrometers, DNA sequencing - have increased considerably in the last decade. Several studies show the potential of these bionanostructures for future stochastic biosensing technology. Stochastic biosensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. This approach is inherent to the organisms that use a single protein nanopore as a key element to start, manage and maintain the chemical and biophysical processes of living cells. Here, we discuss alpha-toxin as a bacterial exotoxin and prototype of a protein nanopore in real-time detection and characterization of molecules in aqueous systems.