Electrochemical Determination of Fentanyl Using Carbon Nanofiber-Modified Electrodes.

In this work, we report the direct electrochemical oxidation of fentanyl using commercial screen-printed carbon electrodes (SPCEs) modified with carboxyl-functionalized carbon nanofibers (fCNFs). CNFs have surface chemistry and reactivity similar to carbon nanotubes (CNTs), yet they are easier to produce and are of a lower cost than CNTs. By monitoring the current produced during the electrochemical oxidation of fentanyl, variables such as fCNF loading, fentanyl accumulation time, electrolyte pH, and differential pulse voltammetry parameters were optimized. Under an optimized set of conditions, the fCNF/SPCEs responded linearly to fentanyl in the concentration range of 0.125-10 µM, with a limit of detection of 75 nM. The fCNF/SPCEs also demonstrated excellent selectivity against common cutting agents found in illicit drugs (e.g., glucose, sucrose, caffeine, acetaminophen, and theophylline) and interferents found in biological samples (e.g., ascorbic acid, NaCl, urea, creatinine, and uric acid). The performance of the sensor was also successfully tested using fentanyl spiked into an artificial urine sample. The straightforward electrode assembly process, low cost, ease of use, and rapid response make the fCNF/SPCEs prime candidates for the detection of fentanyl in both physiological samples and street drugs.

A Molecularly Imprinted Sol-Gel Electrochemical Sensor for Naloxone Determination.

A molecularly imprinted sol-gel is reported for selective and sensitive electrochemical determination of the drug naloxone (NLX). The sensor was developed by combining molecular imprinting and sol-gel techniques and electrochemically grafting the sol solution onto a functionalized multiwall carbon nanotube modified indium-tin oxide (ITO) electrode. The sol-gel layer was obtained from acid catalyzed hydrolysis and condensation of a solution composed of triethoxyphenylsilane (TEPS) and tetraethoxysilane (TES). The fabrication, structure and properties of the sensing material were characterized via scanning electron microscopy, spectroscopy and electrochemical techniques. Parameters affecting the sensor's performance were evaluated and optimized. A sensor fabricated under the optimized conditions responded linearly between 0.0 µM and 12 µM NLX, with a detection limit of 0.02 µM. The sensor also showed good run-to-run repeatability and batch-to-batch performance reproducibility with relative standard deviations (RSD) of 2.5-7.8% (n = 3) and 9.2% (n = 4), respectively. The developed sensor displayed excellent selectivity towards NLX compared to structurally similar compounds (codeine, fentanyl, naltrexone and noroxymorphone), and was successfully used to measure NLX in synthetic urine samples yielding recoveries greater than 88%.

Type effect of inhibitory KT tape on measured vs. perceived maximal grip strength.

This study examined the effects of KT tape (KT) applied in an inhibitory manner on muscle activity, measured maximal grip strength, and perceived maximal grip strength in regular KT-users and non-users. This study was a single-blinded crossover study with sixty participants including 27 kT-users and 33 non-users. Participants underwent maximal grip strength tests with and without inhibitory KT applied across the wrist extensors. Muscle activity and maximal grip strength were measured, while perceived maximal grip strength was rated using a visual analogue scale. No significant interaction effect was found between taping conditions and participant KT-experience for muscle activity (F = 0.825, p = 0.367), measured grip strength (F = 1.018, p = 0.317) or perceived grip strength (F = 0.122, p = 0.728). No significant differences were observed in the EMG activity between taping conditions for either KT-users (p = 0.367) or non-users (p = 0.215). A similar trend was found in the measured grip strength (KT-users: p = 0.317; non-users: p = 0.294) and perceived grip strength (KT-users: p = 0.728; non-users: p = 0.063). KT applied in an inhibitory manner does not impede EMG activity, measured maximal grip strength, or perceived maximal grip strength in adults, regardless of their preconceived notions of KT.

Orbital angular momentum eigenfunctions for fast and numerically stable evaluations of closed-form pseudopotential matrix elements.

The computation of s-type Gaussian pseudopotential matrix elements involving low powers of the distance from the pseudopotential center using Gaussian orbitals can be reduced to familiar integrals. They may be directly expressed as either simple three-center overlap integrals for even powers of the radial distance from the pseudopotential center or related to the three-center nuclear integrals of a Gaussian charge distribution for odd powers. Orbital angular momentum about each atom is added to these integrals by solid-harmonic differentiation with respect to its center. The solid-harmonic addition theorem allows all the integrals to be factored into products of invariant one-dimensional integrals involving the Gaussian exponents and angular factors that contain the azimuthal quantum numbers but are independent of all Gaussian exponents. Precomputing the angular factors allow looping over all Gaussian exponents about the three centers. The fact that solid harmonics are eigenstates of angular momentum removes the singularities seen in previous treatments of pseudopotential matrix elements.

Investigation of the utility of complementary electrochemical detection techniques to examine the in vitro affinity of bacterial flagellins for a toll-like receptor 5 biosensor.

An initial investigation of the fabrication of a novel biosensor utilizing toll-like receptor 5 (TLR5) has been conducted. The detection assay using this sensor platform has been carried out using two complementary electrochemical techniques. The electrochemical properties of the modified bare gold surface following TLR5 immobilization were characterized. The electrochemical response to changes in the sensor film resistance and electron charge-transfer permittivity triggered by independent exposures to flagellins from Salmonella typhimurium (S. typhimurium) and Bacillus subtilis (B. subtilis) were examined and observed. The quantified film resistance data gathered using electrochemical impedance spectroscopy (EIS) over a macroscopic scale are in significant agreement with the corresponding electron charge-transfer permittivity measured locally by scanning electrochemical microscopy (SECM). Unlike other sensors that exploit pathogen recognition elements, TLR5 biosensors have the potential to carry out broad-spectrum detection of flagellated bacterial pathogens in near real time. This broad-spectrum detection platform is a significant step toward the development of fast, inexpensive clinical tools for early warning diagnoses and immediate on-site treatment.

Frontal affinity chromatography-mass spectrometry.

Frontal affinity chromatography (FAC) is a biophysical method for the discovery and characterization of molecular interactions in a flow-based system. Several different modes of analysis are possible by interfacing to the mass spectrometer, including robust single-compound characterizations as well as high-throughput screening of over 1,000 compounds per run. The method supports thermodynamic and kinetic characterization of interactions for a wide range of molecular species and possesses similarities to flow-based biosensors such as surface plasmon resonance. It offers sensitive detection of ligands present well below their respective dissociation constants, and can be assembled from readily available laboratory components. Direct coupling of the FAC cartridge to the mass spectrometer is useful for the interrogation of single compounds or mixtures of limited complexity. An offline fractionation schema is more appropriate for discovery-mode applications. A high-performance FAC system enabling both modes can be assembled in 2-3 h. Measurements of dissociation constants can be made with such a system in 0.5-3 h, and the system supports higher-throughput screening modes at a rate of 10,000 compounds d(-1).

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: deconvolution strategy.

Abstract Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.

Frontal affinity chromatography-mass spectrometry assay technology for multiple stages of drug discovery: applications of a chromatographic biosensor.

This article presents new concepts in affinity chromatography/mass spectrometry for the study of molecular interactions. Chromatographic assays involving estrogen receptor-beta, sorbitol dehydrogenase, human alpha-thrombin, cholera toxin B subunit, beta-galactosidase, and Griffonia simplicifolia isolectin B(4) were established in microaffinity columns and operated in frontal analysis mode. Methods and formalism are presented for the measurement of dissociation constants, using direct methods in which the mass spectrometric signature of the ligand is used to measure breakthrough time and, hence, binding strength. The direct approach is capable of measuring sub-micromolar K(d) and higher, on sub-pmol amounts of immobilized protein, as shown in the cholera toxin assay. Indirect assays that demonstrate the advantage of routine, rugged performance were developed. By tracking the effect of a test ligand on a selected probe, or indicator ligand, dissociation constants in the low nanomolar range could be reliably determined for ligands to estrogen receptor-beta. Mass spectrometry supports the resolution of complex ligand mixtures, and it is demonstrated in the sorbitol dehydrogenase assay that ligands can be rank ordered across approximately three orders of magnitude in K(d), in a single run. A new concept for rapid mixture prescreening is presented, in which an indicator ligand can be used to discriminate between mixtures that contain high levels of weak ligands and those that contain single strong ligands.

Novel Helicobacter pylori alpha1,2-fucosyltransferase, a key enzyme in the synthesis of Lewis antigens.

Helicobacter pylori lipopolysaccharides (LPS) contain complex carbohydrates known as Lewis antigens which may contribute to the pathogenesis and adaptation of the bacterium. Involved in the biosynthesis of Lewis antigens is an alpha1,2-fucosyltransferase (FucT) that adds fucose to the terminal betaGal unit of the O-chain of LPS. Recently, the H. pylori (Hp) alpha1,2-FucT-encoding gene (fucT2) was cloned and analysed in detail. However, due to the low level of expression and instability of the protein, its enzymic activity was not demonstrated. In this study, the Hp fucT2 gene was successfully overexpressed in Escherichia coli. Sufficient amounts of the protein were obtained which revealed alpha1,2-fucosyltransferase activity to be associated with the protein. A series of substrates were chosen to examine the acceptor specificity of Hp alpha1,2-FucT, and the enzyme reaction products were identified by capillary electrophoresis. In contrast to the normal mammalian alpha,2-FucT (H or Se enzyme), Hp alpha1,2-FucT prefers to use Lewis X [betaGal1-4(alphaFuc1-3)betaGlcNAc] rather than LacNAc [betaGal1-4betaGIcNAc] as a substrate, suggesting that H. pylori uses a novel pathway (via Lewis X) to synthesize Lewis Y. Hp alpha1,2-FucT also acts on type 1 acceptor [betaGal1-3betaGlcNAc] and Lewis a [betaGal1-3(alphaFuc1-4)betaGIcNAc], which provides H. pylori with the potential to synthesize H type 1 and Lewis b epitopes. The ability to transfer fucose to a monofucosylated substrate (Lewis X or Lewis a) makes Hp alpha1,2-FucT distinct from normal mammalian alpha1,2-FucT.