A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K _D ) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC₅₀ of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 μg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).     

Structural modifications of aqueous ionic micelles in the presence of denaturants as studied by DLS and viscometry

Hydrophobic interactions control the morphologies of both surfactant aggregates and proteins. Globular proteins "denature" upon addition of excess amounts of denaturants such as urea. Understanding the microscopic basis of the urea effect on proteins or supramolecular aggregates such as micelles has always been a debated issue. Inspired by this need, the effect of urea (U), thiourea (TU), monomethylurea (MMU), dimethylurea(DMU), tetramethylurea (TMU), dimethylthiourea (DMTU), and tetramethylthiourea (TMTU) on the structural transition (spherical micelles to rod-shaped micelles, s --> r) in the sodium dodecylbenzenesulfonate (SDBS)-1-pentanol system has been investigated through dynamic light scattering(DLS) and viscosity measurements at 25 degrees C. 1-Pentanol, at 0.14 M, is found to promote s --> r in this system (0.2 M SDBS). The presence of the additives causes, in almost all cases, a decrease and increase in this 1-pentanol concentration depending upon the concentration and nature of the additive. These effects are explained in terms of an increased dielectric constant of the solvent medium due to the presence of additives and increased micellar hydration due to the repulsion of charged monomers caused by adsorption of the additives. Taken together, the data signal the exposure of biological assemblies to water at higher [additive], which causes a decrease in hydrophobic interactions responsible for compact structure formation (i.e., native protein).     

PHOTOREACTIVATION OF THYMINE-STARVED ESCHERICHIA COLIBs-1

Abstract —Thymine starvation prior to 254 nm ultraviolet light (UV) exposures has been found to decrease the level of maximum photoreactivation in Escherichia coli B _s-1. The dark equilibrium level of photoreactivating enzyme-substrate complexes was determined from the levels of photoreactivation obtained with exposures to single flashes of high-intensity light. The kinetics indicate that photoreactivating enzyme concentration does not decrease as a result of thymine starvation. The UV sensitivities of normal and thymine-starved cells are found to be the same. Photoreactivation by sequential flashes shows a lesser number of total photorepairable lesions in starved cells. It is concluded that thymine starvation renders a portion of the dimers inaccessible to the photoreactivating enzyme, thus lowering the level of maximum photoreactivation.     

Interaction of acridine orange monohydrochloride dye with sodiumdodecylsulfate, (SDS) cetyltrimethylammoniumbromide (CTAB) and p-tert-octylphenoxypolyoxy ethanol (Triton X 100) surfactants

Summary Results of spectrophotometric, conductometric and dialysis studies on the interaction of acridine orange monohydrochloride dye with sodiumdodecylsulfate (anionic), cetyltrimethylammoniumbromide (cationic) and Triton X 100 (nonionic) surfactants have been reported. The anionic surfactant, SDS has been observed to undergo both electrostatic and hydrophobic interactions with the dye cation. Aggregation of the dye molecules can be destroyed when the surfactant is in large excess, whereas, excess dye can check micellization of SD S. At a ratio of AO:SDS=1:7 and above, dye embedded mixed micelles are formed. These remain in a separate phase, probably as coacervates. At lower ratios than 1:7, aggregation of dye molecules is induced, which being complexed with SDS become stabilized as colloids. The colloid and the coacervate have been observed to be thermally stable, negatively charged materials that can be broken by salts, and cations of higher valency are more effective in this regard. An 1:3 = AO:SDS colloid has beeen found to be sufficiently large like the coacervates to pass through a membrane having cut off permeability for molecular weights 12,000 and above. All the above features of AO-SDS interaction have been observed to be absent for AO-CTAB and AO-TX 100 systems, Even hydrophobic interaction has played an insignificant role in these cases. Thus, the dye cation, the cationic and the nonionic surfactants have almost retained their self physicochemical identities in solution in the presence of each other. Electrostatic interaction is thus the primary requirement for acridine orange-surfactant (anionic) system; the hydrophobic effect is secondary and may become co-operative.With 9 figures and 2 tables     

After-effects of Auger ionization following electron capture in organic 125iodine compounds

The emission Mössbauer spectra of ¹²⁵I labeled iodobenzene, methyl iodide, and of their dilute solutions in benzene and hexane, were computer analyzed. Two species were observed, in one of which tellurium is presumably attached to two organic moieties, while in the other, tellurium is attached to a single organic moiety.     

Use of cluster expansion techniques in quantum chemistry. A linear response model for calculating energy differences

In this paper we have reviewed the theoretical framework of the coupled-cluster (cc) based linear response model as a tool for directly calculating energy differences of spectroscopic interest like excitation energy (ee), ionisation potential (ip) or electron affinity (ea). In this model, the ground state of a many-electron system is described as in a coupled cluster theory for closed shells. The electronic ground state is supposed to interact with an external photon field of frequencyw, and the poles of the linear response function as a function ofw furnish with the elementary excitations of the system. Depending on the general form of the coupling term chosen, appropriate difference energies like ee, ip or EA may be generated. Pertinent derivations of the general working equations are reviewed, and specific details as well as approximations for ee, ip or ea are indicated. It is shown that the theory bears a close resemblance to the equation of motion (eom) method but is superior to the latter in that the ground state correlation is taken to all orders and may be looked upon as essentially a variant of renormalisedtda. A perturbative analysis elucidating the underlying perturbative structure of the formulation is also given which reveals that the theory has a hybrid structure: the correlation terms are treated akin to an open shellmbpt, while the relaxation terms are treated akin to a Green function theory. A critique of the methodvis-a-vis other cc-based approaches for difference energies forms the concluding part of our review.     

Counterions of BINAP-Pt(II) and -Pd(II) complexes: novel catalysts for highly enantioselective Diels-Alder reaction

[formula: see text] Platinum and palladium chiral bisphosphine complexes and their counterion effects in asymmetric Diels-Alder reactions have been investigated. The reaction of cyclopentadiene and various bidentate dienophiles in the presence of a catalytic amount of Pt(II)- or Pd(II)-BINAP complex proceeds with excellent endo/exo selectivity as well as endo enantioselectivity (up to 99% ee).     

Interfacial phase transition of an environmentally responsive elastin biopolymer adsorbed on functionalized gold nanoparticles studied by colloidal surface plasmon resonance

The change in optical properties of colloidal gold upon aggregation has been used to develop an experimentally convenient colorimetric method to study the interfacial phase transition of an elastin-like polypeptide (ELP), a thermally responsive biopolymer. Gold nanoparticles, functionalized with a self-assembled monolayer (SAM) of mercaptoundecanoic acid onto which an ELP was adsorbed, exhibit a characteristic red color due to the surface plasmon resonance (SPR) of individual colloids. Raising the solution temperature from 10 degrees C to 40 degrees C thermally triggered the hydrophilic-to-hydrophobic phase transition of the adsorbed ELP resulting in formation of large aggregates due to interparticle hydrophobic interaction. Formation of large aggregates caused a change in color of the colloidal suspension from red to violet due to coupling of surface plasmons in aggregated colloids. The surface phase transition of the ELP was reversible, as seen from the reversible change in color upon cooling the suspension to 10 degrees C. The formation of colloidal aggregates due to the interfacial phase transition of adsorbed ELP was independently verified by dynamic light scattering of ELP-modified gold colloids as a function of temperature. Colloidal SPR provides a simple and convenient colorimetric method to study the influence of the solution environment, interfacial properties, and grafting method on the transition properties of ELPs and other environmentally responsive polymers at the solid-water interface.     

Magnetic nanoparticle assemblies on denatured DNA show unusual magnetic relaxivity and potential applications for MRI

Denatured (substantially single-stranded) herring sperm DNA acts as a template for the preparation of magnetic nanowires, forming stable aqueous suspensions, which exhibit unprecedentedly high relaxivity at low field, suggesting that the material may be a potentially useful reagent for MRI.     

Electrocatalytic Proton Reduction by a Cobalt Complex Containing a Proton-Responsive Bis(alkylimdazole)methane Ligand: Involvement of a C−H Bond in H2 Formation

Homogeneous electrocatalytic proton reduction is reported using cobalt complex [ 1 ](BF₄)₂. This complex comprises two bis(1-methyl-4,5-diphenyl-1H-imidazol-2-yl)methane (HBMIM ) ligands that contain an acidic methylene moiety in their backbone. Upon reduction of [ 1 ](BF₄)₂ by either electrochemical or chemical means, one of its HBMIM ligands undergoes deprotonation under the formation of dihydrogen. Addition of a mild proton source (acetic acid) to deprotonated complex [ 2 ](BF₄) regenerates protonated complex [ 1 ](BF₄)₂. In presence of acetic acid in acetonitrile solvent [ 1 ](BF₄)₂ shows electrocatalytic proton reduction with a k_obs of ≈200 s⁻¹ at an overpotential of 590 mV. Mechanistic investigations supported by DFT (BP86) suggest that dihydrogen formation takes place in an intramolecular fashion through the participation of a methylene C−H bond of the HBMIM ligand and a Co^II−H bond through formal heterolytic splitting of the latter. These findings are of interest to the development of responsive ligands for molecular (base)metal (electro)catalysis.