Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads

Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (~75%), the best compromise between plasmid yield (~96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.     

Chemiluminescent bead-based hybridization assay for the detection of genomic DNA from <Emphasis Type="Italic">E. coli</Emphasis> in purified plasmid samples

A bead-based hybridization assay was developed for detection of traces of E. coli genomic DNA (gDNA) present in purified plasmid DNA (pDNA) samples. Standards of gDNA and pDNA samples were sheared by sonication and adsorbed onto aminopropyl controlled pore glass (CPG) particles (130 μm). A preliminary study was conducted to optimize the amount of DNA adsorbed on the particles. Results indicated that maximum attachment efficiency was obtained by adsorbing DNA for 2 h in 0.2 × SSC, pH 5.7. The DNA-bound particles were hybridized overnight with a 181-bp digoxigenin-labeled probe, specific for gDNA. Following a chemiluminescent detection protocol, signal intensities of the standards were plotted as a function of initial gDNA concentration. The calculated detection limit (LOD) was 1.4 pM of gDNA. The assay was able to detect gDNA in pure plasmid preparations at the 1% level even in the presence of 1,000-fold excess of noncomplementary target. Hybridization results were compared with a quantitative real-time PCR assay. Both methods afforded similar accurate results at the 95% confidence level.     

The first total synthesis of the (±)-17-methyl-trans-4,5-methyleneoctadecanoic acid and related analogs with antileishmanial activity

The first total synthesis of the marine cyclopropane fatty acid (±)-17-methyl-trans-4,5-methyleneoctadecanoic acid was accomplished in eight steps and in 9.1% overall yield starting from 1-bromo-12-methyltridecane. The cis analog (±)-17-methyl-cis-4,5-methyleneoctadecanoic acid was also synthesized but in seven steps and in 16.4% overall yield. With the two isomeric cyclopropane fatty acids at hand it was possible to unequivocally corroborate the trans relative configuration of the naturally occurring fatty acid by gas chromatographic co-elution of the corresponding methyl esters. The cis isomer was cytotoxic to Leishmania donovani promastigotes with an IC₅₀ of 300.2 ± 4.2 μM.     

The conformational stability of gaseous 1-butene studied by femtosecond nonlinear spectroscopy and ab initio calculations

1-Butene was investigated by rotational femtosecond degenerate four-wave mixing spectroscopy (fs DFWM) under supersonic expansion conditions as well as by quantum chemical calculations. Fs DFWM is a time-resolved rotational Raman spectroscopy, which allows for precise determination of rotational constants. The experimental fs DFWM spectrum was successfully reproduced by a fitted simulation using a single structure ascribed to the gauche conformer of 1-butene. The obtained rotational constants A = 22.6 ± 1.7 GHz, B = 4.1554 ± 0.0004 GHz and C = 4.0550 ± 0.0004 GHz are in excellent agreement with the ones from microwave spectroscopy. The cis conformer was not observed in the fs DFWM spectrum recorded under the low-temperature conditions of a supersonic expansion. The absence of the cis form along with simulations of a fs DFWM spectrum indicate that the cis conformer is at most equally but most likely less stable than the gauche rotamer. The experimental findings were supplemented by high level quantum chemical calculations, which predict the gauche form to have lower total energy than the cis structure.     

Modelling of Pb-(TAPS)x-(OH)y system and refinement of stability constants in the region of lead hydrolysis and lead hydroxide precipitation

The influence of [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS) on solutions containing lead(II) was studied by direct current polarography (DCP) and glass electrode potentiometry (GEP). The readings were taken at fixed total TAPS to total lead(II) concentration ratios and various pH values, at 25.0 ± 0.1 °C and ionic strength 0.1 M KNO₃.Due to the basic pK_a of the ligand, which occurs in the pH range where large amount of lead polynuclear species are formed, and the occurrence of ligand adsorption, that disabled the use of high concentrations of TAPS on DCP experiments, GEP and DCP experimental conditions were put to the limit in order to provide the correct Pb-TAPS-OH model and reliable stability constants.The proposed final model is: PbL, PbL₂, PbL₂(OH) and PbL₂(OH)₂ with overall stability constants values, as log β, 3.27 ± 0.06, 6.5 ± 0.1, 12.7 ± 0.1 and 17.27 ± 0.06, respectively.A comparative analysis of the strength of complexation of TAPS and a structural related buffer, 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO), with lead is also discussed.     

Two-dimensional liquid chromatographic methods to examine phenylboronate interactions with recombinant antibodies

A two-dimensional liquid chromatography (2D-LC) system incorporating Agilent 1100 components was set up and 2D-LC methods were developed to determine how recombinant antibodies (rAbs) interact with a commercial boronate column. Analyses of ten rAbs demonstrated that a simple boronate affinity chromatography method cannot be generalized to separate or detect the glycation level for all of rAbs as the aggregates were also retained on the boronate column. A quantitative boronate affinity chromatography method was developed to examine the extent of glycation of a recombinant humanized monoclonal antibody (rhuMAb). With the method, only the glycated components of intact or papain-treated rhuMAb were bound to the boronate column. When denatured, rhuMAb heavy chain was also bound. However, enzymatic removal of the N-oligosaccharides did not delete the binding, indicating that the interaction between the heavy chain and the phenylboronate functionality is not mediated through the N-oligosaccharides. Two-stage 2D-LC analysis with boronate affinity chromatography in the first dimension and reversed-phase chromatography in the second demonstrated that the interaction between the antibody heavy chain and boronate column is not specific, as urea is able to selectively elute retained heavy chain without disrupting the retention of glycated light chain. Quantum mechanic calculations show that the vicinal cis-diols in β-pyranose conformations in N-glycosylated antibodies need to overcome at least 7.172 kcal/mol to become co-planar (zero-dihedral angle). However, the cis-diols on β-furanose rings of glycated species only have to overcome a 0.885 kcal/mol energy barrier to achieve the co-planar configuration that facilitates its complex formation with the phenylboronate groups. This minimal energy barrier, coupled with the stabilizing effect from the electrostatic interaction between the oppositely charged phenylboronate group on the column and the fructosamine group of glycated species, explains why the boronate column exhibits high binding selectivity towards the glycated proteins.     

Microfabricated bioreactor chips for immobilised enzyme assays

A microfabricated device is reported that has been designed to permit the in situ packing of a section of channel with enzyme immobilised onto controlled pore glass (CPG). It is fabricated from glass and polydimethylsiloxane and to prevent dead volumes, has dedicated channels for packing the reactor. The device has the advantage of being simple in design, the flow through enzyme reactor channel being simply a widened section of the analyte channel. The system is suitable for both hydrodynamic and electro-osmotic pumping, and is designed such that when the packing is exhausted it can be repacked. Controlled pore glass provides a reproducible none swelling, high porosity medium onto which the enzyme could be immobilised. The large particle size meant that it was vital to optimise the immobilisation procedure in order to achieve acceptable enzyme activity. The microfabricated device was developed with two enzymes of different molecular masses; alkaline phosphatase and xanthine oxidase. The pore size of the CPG was found to be very important for xanthine oxidase, where the 697 Å pore size (120-200 mesh) CPG was found to give the highest activity (18-20% activity retained after immobilisation). The microfabricated device was used for the assay of p-nitrophenyl phosphate and hypoxanthine with spectrophotometric detection at 405 and 470 nm, respectively. The limits of detection were 5 and 8 μM, respectively.     

Novel polymeric ionic liquid microspheres with high exchange capacity for fast extraction of plasmid DNA

A novel polymeric ionic liquid (PIL) microsphere, poly(1-vinyl-3-(2-methoxy-2-oxyl ethyl)imidazolium) hexafluorophosphate, is prepared via W/O emulsion polymerization. Rapid ion-exchange between the anionic moieties of PIL and DNA fragments is demonstrated facilitating the exchange equilibrium to be reached within 1 min. The PIL microspheres exhibit a high capacity of 190.7 μg mg⁻¹ for DNA adsorption. A fast DNA isolation protocol is thus developed with the PIL microspheres as solid phase adsorbent. It is feasible to facilitate DNA adsorption or stripping from the microspheres by simply regulating the concentration of salt. DNA adsorption is facilitated at low salt concentration, while higher concentration of salt entails DNA recovery from the microspheres. In practice, the retained DNA could be readily recovered with 1.0 mol L⁻¹ NaCl as stripping reagent, giving rise to a recovery of ca. 80.7%. The PIL microspheres are used for the adsorption/isolation of plasmid DNA from E. coli cell culture, demonstrating a superior adsorption performance with respect to that achieved by a commercial Plasmid Miniprep Kit.     

Polystyrene with different topologies: Study of the glass transition temperature in confined geometry of thin films

The glass transition behaviour of polystyrene (PS) with systematically varied topologies (linear, star-like and hyperbranched) confined in nanoscalic films was studied by means of spectroscopic vis-ellipsometry. All applied PS samples showed no or only a marginal depression in glass transition temperature T_g in the order hyperbranched PS (5 K) > star-like PS (3 K) > linear PS (0 K) for the thinnest films analyzed. The T_g behaviour was accompanied by the observation of the film density in dependence of film thickness. A maximum decreased density of about 7% for hyperbranched PS and 5% for star-like PS and again no deviation in density of bulk was found for linear PS. Accordingly, we deduce from these results considering an experimental accuracy of about ± 2 K for T_g and up to ±3% for film density, that the polymer topology only barely influences T_g in the confinement of thin films.     

Binding of mucin to water-soluble and surface-grafted boronate-containing polymers

The binding of mucin to water-soluble copolymers of N,N-dimethylacrylamide and N-acryloyl-m-aminophenylboronic acid grafted on the surface of glass is studied. Atomic force microscopy studies show that many graft copolymer islands 20–200 nm in diameter and 50 nm in height occur on the modified surface of flat glass. Owing to the presence of phenyl boronate groups, the copolymer behaves as a weak polyelectrolyte (pK _a = 9.0) and, in the grafted state in an aqueous solution, experiences reversible transitions between states with higher and lower degrees of ionization. As evidenced by spectral correlation interferometry, this phenomenon brings about a change in the thickness of the grafted layer by approximately 0.5 nm. The ability of phenyl boronate groups to form cyclic esters with diol and polyol groups results in complexation of the soluble copolymer with mucin oligosaccharides and entails the appearance of slowly growing submicron particles formed by similarly charged polymers. The specificity of complexation is confirmed by dissolution of particles in the presence of fructose: a saccharide with a strong affinity for phenyl boronate groups. The binding of mucin to glass, which is chemically modified with the above copolymer, leads to formation of an adsorption layer with a thickness of 1.2–1.8 nm. Thus, boronate-containing copolymers are suitable for preparing carriers with controllable adsorption properties with respect to polyols, including mucinlike proteins of cellular glycocalyxes.     

Evaluation of electroless gold deposited on porous silica for ligand attachment for metal ion-exchange

A novel ion-exchange system was developed by tethering a small, monofunctional metal ligand, 3-mercaptopropionic acid (MPA), through thiol linkages onto a glass substrate overlaid with gold, using electroless deposition of Au onto 100 mesh controlled pore glass (CPG) particles. Two different CPG glass pore sizes of 22.6 and 104.9 nm were used in an attempt to optimize Au deposition and ligand coverage. It was found that the Au was present on both of the substrates as a nonuniform deposit, possibly existing as small multilayer islands, throughout the CPG particles. The deposit also did not fill the intricate CPG pore network.Au-CPG is a reasonable substrate for the support of small metal binding ligands such as the monofunctional MPA. Typical metal binding capacities were in the range of 7-24 μmol metal/g column, with the smaller pore system binding approximately twice as much metal as the larger pore system due to increased surface area and fractional ligand coverage. The immobilized binding systems were also found to remain chemically and structurally stable in pH environments ranging from pH∼9 to <1 (viz. 1 M HNO₃).     

Magnetic N-doped carbon nanotubes: A versatile and efficient material for the determination of polycyclic aromatic hydrocarbons in environmental water samples

This paper describes a new, efficient and versatile method for the sampling and preconcentration of PAH in environmental water matrices using special hybrid magnetic carbon nanotubes. These N-doped amphiphilic CNT can be easily dispersed in any aqueous matrix due to the N containing hydrophilic part and at the same time show high efficiency for the adsorption of different PAH contaminants due to the very hydrophobic surface. After adsorption, the CNT can be easily removed from the medium by a simple magnetic separation. GC/MS analyses showed that the CNT method is more efficient than the use of polydimethylsiloxane (PDMS) with much lower solvent consumption, technical simplicity and time, showing good linearity (range 0.18–80.00 μg L⁻¹) and determination coefficient (R² > 0.9810). The limit of detection ranged from 0.05 to 0.42 μg L⁻¹ with limit of quantification from 0.18 to 1.40 μg L⁻¹. Recovery (n = 9) ranged from 80.50 ± 10 to 105.40 ± 12%. Intraday precision (RSD, n = 9) ranged from 1.91 to 9.01%, whereas inter day precision (RSD, n = 9) ranged from 7.02 to 17.94%. The method was applied to the analyses of PAH in four lake water samples collected in Belo Horizonte City, Brazil.     

Raman and infrared spectra, conformational stability, barriers to internal rotation, vibrational assignment, and ab initio calculations of 3-methyl-3-butenenitrile

The Raman (3500-30 cm⁻¹) spectra of liquid and solid and the infrared (3500-40 cm⁻¹) spectra of gaseous and solid 3-methyl-3-butenenitrile, CH₂C(CH₃)CH₂CN, have been recorded. Both cis and gauche conformers have been identified in the fluid phases but only the cis form remains in the solid. Variable temperature (−55 to −100 °C) studies of the infrared spectra of the sample dissolved in liquid xenon have been carried out. From these data, the enthalpy difference has been determined to be 163±16 cm⁻¹ (1.20±0.19 kJ mol⁻¹), with the cis conformer the more stable rotamer. It is estimated that there is 48±2% of the gauche conformer present at  25°C. A complete vibrational assignment is proposed for the cis conformer based on infrared band contours, relative intensities, depolarization ratios and group frequencies. Several of the fundamentals for the gauche conformer have also been identified. The vibrational assignments are supported by normal coordinate calculations utilizing ab initio force constants. Complete equilibrium geometries have been obtained for both rotamers by ab initio calculations employing the 6-31G(d), 6-311G(d,p), 6-311+G(d,p) and 6-311+G(2d,2p) basis sets at the levels of restricted Hartree-Fock (HF) and/or Møller-Plesset perturbation theory to the second order (MP2). Only with the 6-311G(2d,2p) and 6-311G(2df,2pd) basis sets with or without diffuse functions is the cis conformer predicted to be more stable than the gauche form. The potential energy terms for the conformational interchange have been obtained at the MP2(full)/6-311+G(2d,2p) level, and compared to those obtained from the experimental data. The results are discussed and compared to the corresponding quantities obtained for some similar molecules.     

Affinity-based microdialysis sampling using heparin for in vitro collection of human cytokines

Microdialysis sampling is a widely used method to sample from complex biological matrices. Cytokines are important signaling proteins that are typically recovered with low relative recovery values during microdialysis sampling. Heparin was included in the microdialysis perfusion fluid as an affinity agent to increase in vitro recovery of different cytokines through polyethersulfone (PES) microdialysis membranes with 100 kDa molecular weight cutoff. No change in fluid volumes collected from the microdialysis probes occurred when heparin was included in the perfusion fluid up to concentrations of 10 μM. The loss of heparin (10 μM) across the dialysis membrane was minimal (2.7 ± 0.9%, n = 3). Additionally, heparin at these concentrations did not interfere with the cytokine immunoassays. The control and heparin-enhanced relative recoveries for five human cytokines using 0.1 μM heparin in the microdialysis perfusion fluid flowing at 0.5 μL min⁻¹ were (n = 3): interleukin-4 (IL-4), 4.2 ± 0.5% and 7.2 ± 3.1%; interleukin-6 (IL-6), 1.4 ± 0.8% and 3.6 ± 1.3%; interleukin-7 (IL-7), 1.3 ± 0.8% and 4.8 ± 1.8%; monocyte chemoattractant protein-1 (MCP-1), 9.0 ± 1.6% and 19.5 ± 2.7%; and tumor necrosis factor-α (TNF-α), 7.4 ± 1.3% and 16.9 ± 1.6%, respectively. Heparin increased the microdialysis sampling relative recovery of several human cytokines in vitro.     

cis- and trans-Bis(pyridine)chromium(III) complexes containing mixed-valence chrysenesemiquinonate–chrysenecatecholate ligands: Synthesis,characterization and interpretation of the electronic absorption spectra using ZINDO/S method

Two new geometrical isomers of [Cr(py)₂(chrySQ)(chryCat)] (chrySQ = chrysenesemiquinonate; chryCat = chrysenecatecholate; py = pyridine) were synthesized by two different synthetic procedures. In the first, an acetonitrile solution containing a stoichiometric mixture of Cr(CO)₆, chrysenequinone and pyridine was photolyzed with a Hg-lamp. The second procedure was based on substituting one of the chrysenesemiquinonate ligand in the tris-[Cr(chrySQ)₃] complex with two pyridine ligands. In both procedures two isomeric forms of [Cr(py)(chrySQ)(chryCat)] were isolated with the trans-isomer obtained in higher yield. The structures of the two isomers have been modeled using parameterized PM3 semiempirical method. Theoretical harmonic vibrational frequencies of the cis- and trans-isomers have been computed and compared with the experimental vibrational frequencies. Variable-temperature magnetic susceptibility has been studied for the two isomers in the 10–300 K temperature range. Theoretical modeling of the magnetic data indicated strong antiferromagnetic exchange interaction between Cr^III (S = 3/2) and chrySQ (S = 1/2) with J = −365 ± 6 and −395 ± 4 cm⁻¹ for the cis- and trans-isomers, respectively. The electrochemical behavior of cis- and trans-[Cr(py)₂(chrySQ)(chryCat)] complexes were studied by cyclic voltammetry in acetonitrile solvent. Both complexes showed two one-electron redox processes attributable to reversible reduction and oxidation of the chrySQ and chryCat ligands. Reduction of the Cr(III) to Cr(II) was observed for both complexes near−1300 mV. The electronic spectra of the two isomers were dominated by charge-transfer (LMCT, MLCT and ILCT) transitions. In addition, a low-energy intervalence charge-transfer (IVCT) transition was observed for the cis-isomer at 1085 nm. Theoretical studies of the electronic spectra by ZINDO/S-CI method were useful in interpreting the observed electronic transitions.     

DNA interaction probed by evanescent wave cavity ring-down absorption spectroscopy via functionalized gold nanoparticles

Evanescent wave cavity ring-down absorption spectroscopy (EW-CRDS) is employed to study interaction and binding kinetics of DNA strands by using gold nanoparticles (Au NPs) as sensitive reporters. These Au NPs are connected to target DNA of study that hybridizes with the complementary DNA fixed on the silica surface. By the absorbance of Au NPs, the interaction between two DNA strands may be examined to yield an adsorption equilibrium constant of 2.2 × 10¹⁰ M⁻¹ using Langmuir fit. The binding efficiency that is affected by ion concentration, buffer pH and temperature is also examined. This approach is then applied to the label-free detection of the DNA mutation diseases using the sandwich hybridization assay. For monitoring a gene associated with sickle-cell anemia, the detection limit and the adsorption equilibrium constant is determined to be 1.2 pM and (3.7 ± 0.8) × 10¹⁰ M⁻¹, distinct difference from the perfectly matched DNA sequence that yields the corresponding 0.5 pM and (1.1 ± 0.2) × 10¹¹ M⁻¹. The EW-CRDS method appears to have great potential for the investigation of the kinetics of a wide range of biological reactions.     

Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N-dG adducts by capillary electrophoresis immunoassay

The stereospecific binding of monoclonal antibody (mAb) 8E11 to anti-benzo(a)pyrene diol epoxide (BPDE)-dG adducts in single nucleoside, long oligonucleotide, and genomic DNA were quantitatively evaluated using noncompetitive and competitive capillary electrophoresis (CE) immunoassays. Two single-stranded TMR-BPDE-90mers containing a single anti-BPDE-dG adduct with defined stereochemistry and a fluorescent label at 5′-end were used as fluorescent probes for competitive CE immunoassay. To quantitatively evaluate the binding affinity through competitive CE immunoassays, a series of equations were derived according to the binding stoichiometry. The binding of mAb 8E11 to trans-(+)-anti-BPDE-dG displays strongest affinity (K_b: 3.57 × 10⁸ M⁻¹) among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(−)-anti-BPDE-dG displays lowest affinity (K_b: 1.14 ×10⁷ M⁻¹). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE-90mers (K_b: 6.36 ± 0.54 × 10⁸ M⁻¹) than trans-(−)-anti-BPDE-90mers (K_b: 4.52 ± 0.52 × 10⁸ M⁻¹). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (K_b: 3.74 × 10⁸ M⁻¹), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA.     

Application of surface plasmon resonance spectroscopy for adsorption studies of different types of components on poly(dimethylsiloxane)

Surface plasmon resonance (SPR) spectroscopy is utilized to study in real-time and, by label-free means, the reversible and quasi-irreversible adsorption of small ionic or neutral molecules, pharmaceuticals, and proteins on poly(dimethylsiloxane) (PDMS) surfaces. The SPR sensor is covered with 0.2% (w/v) PDMS in octane. During the timescale of a typical lab-on-a-chip analysis or an electrophoretic separation, it was found that small neutral components containing a hydrophobic part do not adsorb or absorb onto PDMS, while larger, water-soluble polymer-like materials like proteins generally irreversibly adsorb to PDMS. The technique can be used to monitor the kinetics of adsorption and desorption of the molecules. For the non-specific adsorption of teicoplanin to PDMS, a Langmuir-like adsorption isotherm was obtained (K_d = 32 ± 2 μmol L⁻¹).     

Nanostructured progesterone immunosensor using a tyrosinase-colloidal gold-graphite-Teflon biosensor as amperometric transducer

A novel progesterone immunosensor using a colloidal gold-graphite-Teflon-tyrosinase composite biosensor as amperometric transducer is reported. A sequential competitive configuration between the analyte and progesterone labelled with alkaline phosphatase (AP) was used. Phenyl phosphate was employed as the AP-substrate and the enzyme reaction product, phenol, was oxidized by tyrosinase to o-quinone, which is subsequently reduced at −0.1 V at the biocomposite electrode. Variables such as the concentration of phenyl phosphate, the amount of antibody attached to the electrode surface, immersion time in a 2% BSA solution, working pH and incubation times in progesterone and AP conjugate were optimized. A linear calibration graph for progesterone was obtained between 0 and 40 ng mL⁻¹ with a slope value of −82.3 nA ng⁻¹ mL, and a detection limit of 0.43 ng mL⁻¹. The time needed to reach the steady-state current from the addition of phenyl phosphate was 30-40 s. These analytical characteristics improve substantially those reported for other progesterone immunosensors. A lifetime of 14 days with no need to apply any regeneration procedure was also achieved. The usefulness of the immunosensor was evaluated by determining progesterone in milk samples spiked with the analyte at 5.0 and 1.5 ng mL⁻¹ concentration levels. Following a very simple procedure, involving only sample dilution, mean recoveries (n = 7) of 98 ± 3% and 99 ± 3%, respectively, were obtained.     

Extremely facile ring inversion and rearrangement in fluorobicyclo[2.1.0]pentanes

cis-1,2,3,4,5,5-Hexafluorobicyclo[2.1.0]pentane and 1,2,4,5-tetrafluorobicyclo[2.1.0]pentane have been synthesized from hexafluorobenzene. The former hydrofluorocarbon, which exists entirely in the endo configuration, rearranges to cis-1,2,3,3,4,5-hexafluorocyclopentene below room temperature (E_a = 21.9 kcal/mol, log A = 13.4). The latter undergoes degenerate ring inversion with extraordinary ease (ΔG^‡ = 6.8 ± 0.2 kcal/mol at −55 °C). Density functional calculations indicate that significant bonding between the bridgehead carbons is retained in the ring inversion transition state. Analogous calculations predict for hexafluorobicyclo[1.1.0]butane a considerably lower barrier for ring inversion and more 1,3-bonding in the transition state.