Development of an electrochemical immunosensor for direct detection of interferon-gamma at the attomolar level.

An electrochemical immunosensor for direct detection of the 15.5-kDa protein interferon-gamma (IFN-gamma) at attomolar level has been developed. Self-assembled monolayers (SAMs) of cysteine or acetylcysteine are formed on electropolished polycrystalline Au electrodes. IFN-gamma adsorbs physically to each of these SAMs. With injections of 100 mM KCl, IFN-gamma can be removed in the flow without damaging the acetylcysteine SAM. However, the cysteine SAM is affected by these KCl injections. In an on-line procedure in the flow, a specific antibody (MD-2) against IFN-gamma is covalently attached following carbodiimide/succinimide activation of the SAM. The activation of the carboxylic groups, attachment of MD-2, and deactivation of the remaining succinimide groups with ethanolamine are monitored impedimetrically at a frequency of 113 Hz, a potential of +0.2 V versus SCE, and an ac modulation amplitude of 10 mV. Plots of the real (Z') and imaginary (Z") component of the impedance versus time provide the information to control these processes. In the thermostated setup (23.0 degrees C), samples of unlabeled IFN-gamma (in phosphate buffer pH 7.4) are injected and the binding with immobilized MD-2 is monitored with ac impedance or potential-step methods. While the chronoamperometric results are rather poor, the ac impedance approach provides unsurpassed detection limits, as low as 0.02 fg mL-1 (approximately 1 aM) IFN-gamma. From a calibration curve (i.e. Z" versus the amount injected), recorded by multiple 50-microL injections of 2 pg mL(-1) of IFN-gamma, a dynamic range of 0-12 pg mL(-1) could be derived. However, when nonspecific adsorption is taken into account, which has been found to be largely reduced through injections of 100 mM KCl, a much smaller dynamic range of 0-0.14 fg mL(-1) remains. The immunosensor can be regenerated by using a sequence of potential pulses in the flow by which the SAM with attached MD-2 and bound IFN-gamma is completely removed. When the developed procedures described above are repeated, the response of the immunosensor is reproducible within 10%.

Reductive activation of potential antitumor mitosene compounds.

The reductive activation of mitosene compounds was studied with cyclic voltammetry and HPLC analysis. Reduction of mitosenes, possessing good leaving groups at C-1 and C-10, was shown to result in loss of these groups at pH 7.0 and pH 6.0. The loss of leaving groups from mitosenes occurred faster at lower pH. Mitosenes without good leaving groups were found to be stable upon reduction. In the presence of acetoxy groups at C-1 and C-10, the C-10 site is the most reactive site upon reductive activation. This is opposite to the case of mitomycin C, where the C-1 site is the first to react upon reduction. At pH 6.0 without reduction, acid degradation also caused the loss of leaving groups of mitosenes, although at a very slow rate. In contrast to reductive activation, upon acid degradation of a diacetoxymitosene the C-1 group appeared to be lost faster. Electrochemical as well as dithionite reduction of a bifunctional (diacetoxy) mitosene compound in the presence of calf thymus DNA at pH 5.5 resulted in the formation of DNA interstrand cross-links. Depending on activation method, this diacetoxymitosene was at least as efficient in DNA cross-linking as mitomycin C under comparable conditions.

Reaction products of 1-naphthol with reactive oxygen species prevent NADPH oxidase activation in activated human neutrophils, but leave phagocytosis intact.

Activation of human neutrophils with opsonized particles in the presence of a nontoxic dose of 1-naphthol resulted in inhibition of superoxide anion production but not of the phagocytotic activity of the cells. In this study we have investigated the mechanism of action of 1-naphthol. The inhibition is not at the level of cellular activation since the FMLP-induced rise of intracellular free calcium was unaffected. Our results show that the (metabolic) activation of 1-naphthol to 1,4-naphthoquinone by reaction with H2O2 from the oxidative burst is a necessary event for the inhibition to occur. The study provides evidence that by its reactivity with essential thiol groups 1,4-naphthoquinone (1,4-NQ) prevents the assembly of a functional NADPH-oxidase in the neutrophil membrane.

Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens. 2. Investigations by chronoamperometry.

The electron transfer kinetics between the hydrogenase from Desulfovibrio vulgaris (strain Hildenborough) and the mediators methyl viologen, di-(n-aminopropyl) viologen and propyl viologen sulfonate have been investigated by chronoamperometry. Second-order rate constants were calculated on basis of the theory for a simple catalytic mechanism and are compared with the results obtained before by cyclic voltammetry (preceding paper in this journal). From the ionic-strength dependence and the observed differences in the rate constants for the differently charged viologens, the existence of an electrostatic interaction between mediator and a negatively charged part of the protein is confirmed. Chronoamperometry (computer-controlled) was found to possess advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate, and better reproducibility).

Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens. 1. Investigations by cyclic voltammetry.

The electron transfer kinetics between the hydrogenase from Desulvovibrio vulgaris (strain Hildenborough) and three different viologen mediators has been investigated by cyclic voltammetry. The mediators methyl viologen, di(n-aminopropyl) viologen and propyl viologen sulfonate differ in redox potential and in net charge. Dependent on the pH both the one- and two-electron-reduced forms or only the two-electron-reduced form of the viologens are effective in electron exchange with hydrogenase. Calculations of the second-order rate constant k for the reaction between reduced viologen and hydrogenase are based on the theory of the simplest electrocatalytic mechanism. Values for k are in the range of 10(6)-10(7) M-1 s-1 and increase in the direction propyl viologen sulfonate----methyl viologen----di(n-aminopropyl) viologen. An explanation is based on electrostatic interactions. It is proposed that the electron transfer reaction is the rate-determining step in the catalytic mechanism.