Amperometric microcells for alkaline phosphatase assay

To develop simple electrochemical immunoassays, a screen printed amperometric microcell with graphite working and Ag/AgCl reference electrodes was tested for the determination of alkaline phosphatase enzyme (ALP) and anti-humanIgG conjugated ALP (alpha-hIgG-ALP) activity in 5-10 microl samples. To ensure reproducible, steady state conditions, the working electrode surface was coated with mass-transport controlling hydrogel layer. The kinetic response curves of the hydrogel coated electrodes were linear. In addition, the hydrogel layer reduced the nonspecific adsorption of the alpha-hIgG-ALP conjugate on the working electrode surface. The measurements were made in the range of 2 divided by 4000 mU ml(-1) enzyme activities using ascorbic acid 2-phosphate (AAP) as the enzyme substrate. AAP is commercially available, non-toxic and has excellent stability. The sensitivity of the determinations was about 71% of the sensitivity which could be achieved using p-aminophenylphosphate (PAPP), a not easily accessible and unstable enzyme substrate. The experimentally determined kinetic parameters of the ALP enzyme catalyzed reactions were the same with the bare and hydrogel layer coated electrodes.     

Multiple charging of poly(propylene glycol) by binary mixtures of cations in electrospray

Single, double and triple charging of poly(propylene glycol) (PPG) (Mn = 1900 g/mol) in the presence of binary mixtures of cations (Li+, Na+, K+, Cs+, and NH4+) under electrospray ionization (ESI) conditions were investigated. For these studies, sodium ion was selected as the reference cation, and the resulting ion-intensities were evaluated as a function of the [Na+]/[C+] ratio (where C+ is the other cation, i.e., Li+, K+, Cs+ and NH4+). A linear relationship was found between INa+/IC+)and [Na+]/[C+] (INa+ and IC+ stand for the intensity of the singly charged PPG molecules cationized with Na+ and C+ ions, respectively). The slope of the INa+/IC+--[Na+]/[C+] plot (alpha) indicates the binding selectivity of Na+ ions to PPG chains with respect to cation C+. In the case of the doubly charged PPG chains, the INaNa2+/INaC2+ and INaC2+/ICC2+ versus [Na+]/[C+] ratio also yield straight lines with slopes of approximately alpha/2 and 2alpha, respectively (INaNa2+, INaC2+ and ICC2+ are the intensity of the doubly charged PPG chains cationized with two Na+ ions, Na+ and C+ ions, and two C+ ions, respectively). Similarly, linear dependences with the [Na+]/[C+] ratio for the corresponding intensity ratios of the triply charged PPG were found. Based on the value of alpha, the selectivity of the cations was found to increase in the order of Li+ < Cs+ approximately Na+ < K+ approximately NH4+. The observed relative ion intensities are interpreted on the basis of the solution state equilibrium between PPG and the cations. In addition, the investigations showed that the abundances of the doubly and triply charged PPG-containing mixed cations can be optimized in a simple way using the value of alpha.     

Orientation and structure-building role of the water molecules bound at the contact surface of the dihydrofolate reductase-methotrexate complex

Summary Orientation of ten water molecules bound strongly at the contact surface of the dihydrofolate reductase-methotrexate enzyme-inhibitor complex was determined theoretically. To optimize the orientation of the water molecules, a recent method based on a simple electrostatic model was applied. The electrostatic complementarity in the binary complex was investigated using the lock-and-key model, considering the effect of the water molecules as well. The strongly bound water molecules improve the electrostatic fit in the pteridine region of methotrexate. Their role in the benzoic amide and-glutamate region is to decrease the internal energy by creating water bridges among remote polar sites making it possible to form H-bonds. Some modifications in the inhibitor structure were proposed for achieving greater inhibitor potency. The presumably enhanced effect is ascribed to the free energy gain in repelling the water molecules from the contact surface to the bulk of the solvent, and, in other cases, to internal energy decreases due to better electrostatic fit in the enzyme-inhibitor complex.     

Amperometric measuring cell for the determination of putrescine oxidase activity

A microfabricated, flat form, amperometric microcell (microchip) is used in a simple, two-electrode arrangement for putrescine oxidase enzyme activity determinations. The cell contains a platinum microdisk working electrode and an Ag/AgCl reference electrode covered by a porous, hydrophilic membrane. An electrochemically-prepared size-exclusion layer is applied on the working electrode surface, to avoid the effect of electroactive interferences in the sample. The hydrophilic membrane, resting on the bottom of the cell, is soaked with a small volume of buffered substrate solution and a few mul enzyme containing sample solution is dispensed over the electrodes. During the enzyme activity measurement a catalytic reaction takes place in the membrane-supported liquid film over the working electrode surface. The hydrogen peroxide produced in the reaction is detected amperometrically. The amperometric current-time curves are used for evaluation. In our work putrescine was used as a substrate to determine the unknown putrescine oxidase enzyme activity of the sample. Elevated diamine oxidase enzyme activity in the vaginal milieu can indicate premature rupture of the amniotic membrane. Results with membrane discs, containing all the necessary chemicals in solid or lyophilized form, are very encouraging with respect of a single use, 'reagentless' biosensor for home care.     

Ferrocenyl-palladium complexes in cross-coupling reactions: a comparative study

Ferrocenecarboxaldehyde hydrazones were converted into palladium complexes on treatment with sodium tetrachloropalladate. The substitution pattern of the ferrocenylhydrazones was found to have a marked influence on the mode the palladium was attached to the organic moiety. The catalytic activity of the new palladium complexes in cross-coupling reactions was examined in detail, and it was compared with conventional catalyst systems.     

The synthesis of amino acids by 1,3-dipolar cycloadditions of azomethine ylides

Thiazolium ylides react with a variety of dipolarophiles to afford adducts. After filtration chromatography, a tricyclic adduct is obtained. The tricyclic adducts react with potassium t-butoxide/t-butanol to provide dihydropyrroles. The adducts also react with tributyltin hydride to form compounds in which the thiazolidine ring has been cleaved. These adducts can be hydrolyzed under acidic conditions to form pyrrolidines. The desulfurization procedure is significant in that none of the relative asymmetry derived from the dipolar cycloaddition is lost. The synthesis of α-allokainic acid has been achieved from adduct 16s.     

Preparation of uracil by cycloreversion. Structure of cycloalkane/ene- and norbornane/ene-fused dihydrouracils

The reactions of the 2-amino-1-cycloalkane-, cycloalkene-, norbornane- and norbornenecarboxylates 1–9 with potassium cyanate gave urea esters, which were cyclized to cycloalkane-, cycloalkene-, norbornane- and norbornene-fused 5,6-dihydrouracils 10–17 . On cyclization, the urea ester formed from trans-4-cyclohexene-1-carboxylate, furnished the cis-fused 5,6-dihydropyrimidine-2,4(1H,3H)-dione. On heating, the norbornene-diexo-fused dihydrouracil 16 yielded 2,4-pyrimidinedione through the splitting-off of cyclopentadiene. The structures of the compounds were proved by ¹H and ¹³C nmr spectroscopy.     

Terent'ev low-energy theorem in the Extended Nambu-Jona-Lasinio model

The low-energy theorem, derived by Terent'ev within the framework of current algebra, is proved at the quark level within the framework of the Extended Nambu-Jona-Lasinio model.One of the authors (A.N.I.) is pleased to thank Dr. A. Nogová and Dr. J. Boháik for a very kind hospitality during the stay at the Institute of Physics in Bratislava, where this work was finished.     

Intracavity photoacoustic gas detection with an external cavity diode laser

The first use of an external cavity diode-laser light source in combination with a photoacoustic detector for high-sensitivity gas detection is described. This combined system is applicable for detecting gases with absorption coefficients as low as 5 x 10^–8 cm^–1 by operating the photoacoustic cell in an intracavity mode. Measurements were made on the 1.13 m absorption lines of water vapour. For quantitative measurements, it was found to be necessary to introduce a reference cell into the system.     

New insights into the metal ion–peptide hydroxamate interactions: Metal complexes of primary hydroxamic acid derivatives of common dipeptides in aqueous solution

Complex formation of primary dipeptide hydroxamic acids, L-Ala-L-AlaNHOH and L-Ala-L-SerNHOH, as well as the corresponding Z-protected ones, Z-L-Ala-L-AlaNHOH and Z-L-Ala-L-SerNHOH (Z = benzyloxycarbonyl), with iron(III), aluminium(III), nickel(II), copper(II) and zinc(II) was studied in aqueous solution by pH-potentiometric and spectroscopic (UV–Vis, EPR, CD, ¹H NMR) methods. The exclusive formation of [O,O] chelated hydroxamate complexes was found with iron(III) and aluminium(III) with all the ligands. Formation of linkage isomers with the involvement of either [O,O] hydroxamate or [NH₂,CO] chelates was detected both in the zinc(II)-L-Ala-L-AlaNHOH and -L-Ala-L-SerNHOH systems. Upon increasing the pH, none of these chelating sets are capable of preventing the hydrolysis of the metal ion. The formation of stable complexes was found in the nickel(II) and copper(II) systems above pH ∼ 6 with a [NH₂, N_amide, N_hydrox.] binding mode after deprotonation and coordination of the peptide amide and the hydroxamate group. With an excess of copper(II), the formation of trinuclear [Cu₃H_xL₂]^x+4 type (x = −4 to −6) complexes as the major species was also detected. Blocking the terminal amino group in the Z-protected ligands results in a dramatic decrease of the nickel(II) and zinc(II) binding strengths, and insoluble complexes with copper(II). No indication was found for the role of the hydroxyl group of the serine moiety in metal ion binding.