Electrochemical Detection of MS2 Phage Using a Bead‐based Immunoassay and a NanoIDA

A microbead based sandwich immunoassay for MS2 bacteriophage was developed using an interdigitated array (IDA) electrode with nanoscale dimensions (220 nm electrode width, 620 nm gap). The IDA was fabricated using an electron beam lithographic lift‐off technique. After an antibody‐assisted capture of MS2 using paramagnetic microbeads, a β‐galactosidase labeled secondary antibody was used to convert p‐aminophenyl‐β‐D ‐galactopyranoside (PAPG) into the redox active p‐aminophenol (PAP). Amperometric detection of PAP with IDA electrodes at +300 and ?200 mV vs. a Ag/AgCl reference electrode was used to measure the result, detecting MS2 concentrations as low as 10 ng/mL.     

Estimation of the absolute internal-rotation entropy of molecules with two torsional degrees of freedom from stochastic simulations

A method of statistical estimation is applied to the problem of evaluating the absolute entropy of internal rotation in a molecule with two torsional degrees of freedom. The configurational part of the entropy is obtained as that of the joint probability density of an arbitrary form represented by a two-dimensional Fourier series, the coefficients of which are statistically estimated using a sample of the torsional angles of the molecule obtained by a stochastic simulation. The internal rotors in the molecule are assumed to be attached to a common frame, and their reduced moments of inertia are initially calculated as functions of the two torsional angles, but averaged over all the remaining internal degrees of freedom using the stochastic-simulation sample of the atomic configurations of the molecule. The torsional-angle dependence of the reduced moments of inertia can be also averaged out, and the absolute internal-rotation entropy of the molecule is obtained in a good approximation as the sum of the configurational entropy and a kinetic contribution fully determined by the averaged reduced moments of inertia. The method is illustrated using Monte Carlo simulations of isomers of stilbene and halogenated derivatives of propane. The two torsional angles in cis-stilbene are found to be much more strongly correlated than those in trans-stilbene, while the degree of the angular correlation in propane increases strongly on substitution of hydrogen atoms with chlorine.     

Eliminating positively charged lysine epsilon-NH3+ groups on the surface of carbonic anhydrase has no significant influence on its folding from sodium dodecyl sulfate

This study compares the folding of two polypeptides--bovine carbonic anhydrase (BCA) and peracetylated BCA (BCA-Ac(18))--having the same sequence of amino acids but differing by 18 formal units of charge, from a solution containing denaturing concentrations of sodium dodecyl sulfate (SDS). Acetylation of BCA with acetic anhydride converts all 18 lysine-epsilon-NH(3)(+) groups to lysine-epsilon-NHCOCH(3) groups and generates BCA-Ac(18). Both BCA and BCA-Ac(18) are catalytically active, and circular dichroism spectroscopy (CD) suggests that they have similar secondary and tertiary structures. SDS at concentrations above approximately 10 mM denatured both proteins. When the SDS was removed by dialysis, both proteins were regenerated in native form. This study suggests that large differences in the net charge of the polypeptide have no significant influence on the structure, the ability to refold, or the rate of refolding of this protein from solutions containing SDS. This study reinforces the idea that charged residues on the surface of BCA do not guide protein folding and raises the broader question of why proteins have charged residues on their surface, outside of the region of the active site.     

Kinetics of the manganese(III)-oxalate reaction in acidic sulphate media

The kinetics of the reaction of manganese(III) with oxalic acid (OA) has been studied in H₂SO₄ solutions. Under the experimental conditions of 6 × 10^–3 <>₀ < 0.4=" mol=">^–3 and [H₂SO₄]₀ 0.2 mol dm^–3 the observed pseudo-first order rate constant k _obs follows the expression

Chemical Biological Sensors Based on Advances in Conducting Electroactive Polymers

Advances in conducting electroactive polymers (CEPs) have driven the design of novel chemical and biochemical sensors. The redox properties of CEPs have been intensely studied for more than two decades with emphasis on their synthesis and characterization. Little attention has been paid to the importance of mechanism in sensor designs. However, in order to design robust and stable sensors, it is important to understand how the polymer structure, morphology, adhesion properties and microenvironment affect sensor performance. This work describes how chemical and biological sensors have been designed, fabricated, characterized and tested based on the fundamental understanding in CEPs. The use of photopolymerized conducting polymers in sensor designs is described. Four focus areas are presented in which the electronic properties of CEPs have enabled the design of novel sensors for organics, nucleic acids, biological molecules, vapors and metal ions.     

Ion pair generation rates near planar3H and63Ni electron capture detector foils

The ion-pair generation rate (ionization topography) in plasmas from⁶³Ni and particularly Ti³H₄ foils, as used in electron capture detectors, was measured at room temperature using large, parallel plates of low backscattering ability in nitrogen gas of varying density. For one atmosphere pressure, the fall-off of ion pair formation as calculated from the exponential region equalsN ₀·e ^–0.19d for⁶³Ni andN ₀·e ^–1.4d for³H (whereN ₀ is the initial ionization rate immediately adjacent to the foil andd is the distance from the foil in mm). The experimentally measured half ranges (distances from the foil within which 50% of all possible ion pairs are created) are 2.7 mm for⁶³Ni and 0.27 mm for³H. The half ranges calculated from the exponential region where there is less interference from electron backscattering, are 3.7 and 0.5 mm, respectively. The latter values are considered closer to the true, unimpeded ionization topography near planar⁶³Ni and³H foils.Material taken from doctoral thesis     

(2+2)-cycloaddition of triazolinediones to strained bicycloalkenes

Substituted norbornenes and dibenzobarrelene react under forced conditions with $\text{N}$-methyl-1,2,4-triazolin-3,5-diones to afford urazoles via (2+2)-cycloaddition.     

Homolysis of weak Ti-O bonds: experimental and theoretical studies of titanium oxygen bonds derived from stable nitroxyl radicals

Titanium-oxygen bonds derived from stable nitroxyl radicals are remarkably weak and can be homolyzed at 60 degrees C. The strength of these bonds depends sensitively on the ancillary ligation at titanium. Direct measurements of the rate of Ti-O bond homolysis in Ti-TEMPO complexes Cp2TiCl(TEMPO) (3) and Cp2TiCl(4-MeO-TEMPO) (4) (TEMPO = 2,2,6,6-tetramethylpiperidine-N-oxyl, 4-MeO-TEMPO = 2,2,6,6-tetramethyl-4-methoxypiperidine-N-oxyl) were conducted by nitroxyl radical exchange experiments. Eyring plots gave the activation parameters, deltaH++ = 27(+/- 1) kcal/mol, deltaS++ = 6.9(+/- 2.3) eu for 3 and deltaH++ = 28(+/- 1) kcal/mol, deltaS++ = 9.0(+/- 3.0) eu for 4, consistent with a process involving the homolysis of a weak Ti-O bond to generate the transient Cp2Ti(III)Cl and the nitroxyl radical. Thermolysis of the titanocene TEMPO complexes in the presence of epoxides leads to the Cp2Ti(III)Cl-mediated ring-opening of the epoxide followed by trapping by the nitroxyl radical. The X-ray crystal structure of the Ti-TEMPO derivative, Cp2TiCl(4-MeO-TEMPO) (4), is reported. DFT (B3LYP/6-31G*) calculations and experimental studies reveal that the strength of the Ti-O bond decreases dramatically with the number of cyclopentadienyl groups on titanium. The calculated Ti-O bond strength of the monocyclopentadienyl complex 2 is 43 kcal/mol, whereas that of the biscyclopentadienyl complex 3 is 17 kcal/mol, a difference of 26 kcal/mol. These studies reveal that the strength of these Ti-O bonds can be tuned over an interesting and experimentally accessible temperature range by appropriate ligation on titanium.     

ASSESSMENT OF GENOTOXICITY AND MUTAGENICITY OF 1,2-DIOXETANES IN HUMAN CELLS USING A PLASMID SHUTTLE VECTOR

Abstract— 1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds on thermal decomposition. They cause photochemical and photobiological transformations in the dark. In order to study the genotoxicity and mutagenicity of 1,2-dioxetanes, the replicating shuttle vector pZ189 was damaged with 3,3,4-trimethyl-l,2-dioxetane(TrMD) or 3-hydroxymethyl-3,4,4-trimethyl-l,2-dioxetane (HTMD) in vitro and subsequently transfected into normal human lymphoblasts. We found a dose-dependent increase of genotoxicity (decrease of plasmid survival) and increase of mutation frequency with both dioxetanes. However, TrMD was less mutagenic than HTMD at similar genotoxicity. Sequence analysis of the supF gene revealed more point mutations than deletions. Single base substitutions occurred exclusively at G:C sites: 94.6% of point mutations with TrMD and 100% with HTMD were G:C to T:A and G:C to C:G transversions. These are the typical mutations following 7,8-dihydro-8-oxoguanine (8-oxo-G) formation, the main DNA lesion induced by TrMD and HTMD. Only with TrMD we found 5.4% G:C to A:T transitions, probably reflecting the more pronounced ability of TrMD to form some pyrimidine dimers. Our results indicate that 8-oxo-G is also the most relevant modification in in vivo mutagenesis.