Properties of mixed alkanethiol-dendrimer layers and their applications in biosensing.

We studied the properties of mixed alkanethiol-dendrimer layers on a gold support and their application in biosensing. We showed that properties of glucose sensor can be modified using a different ratio of 1-hexadecanethiol (HDT) and poly(amidoamine) dendrimer of first generation (G1). The cyclic voltammetry in the presence of the redox couple, Fe(CN)(6)(3-)/Fe(CN)(6)(4-), was used for estimating how effectively the layer blocks the redox probe's access to the electrode surface. A scanning electrochemical microscope (SECM) was used to image the resulting distribution of the organic compounds. We found that with increasing content of dendrimers, the integrity of the layers was improved.

A chronoamperometric method to estimate changes in the membrane composition of ion-selective membranes.

A new chronoamperometric method is used to estimate changes in the membrane composition of mobile-site, ionophore-based membranes. The characteristic features of the chronoamperometric curves (initial current, slope, break time) of valinomycin-based, potassium-selective membranes loaded with potassium tetrakis(4-chlorophenyl)borate are correlated with the mobile-site and free ionophore concentration in the membrane. limiting cases for strong and negligible ion pair formation are distinguished. Replicate measurements indicate a relative standard deviation in the calculated values less than 10%. The practical applicability of the method was tested with membranes incorporated into conventional ion-selective electrode bodies or cast onto microfabricated planar sensor structures.

Picomolar detection limits with current-polarized Pb2+ ion-selective membranes.

Minor ion fluxes across ion-selective membranes bias submicromolar activity measurements with conventional ion-selective electrodes. When ion fluxes are balanced, the lower limit of detection is expected to be dramatically improved. As proof of principle, the flux of lead ions across an ETH 5435 ionophore-based lead-selective membrane was gradually compensated by applying a few nanoamperes of galvanostatic current. When the opposite ion fluxes were matched, and the undesirable leaching of primary ions was eliminated, Nernstian response down to 3 x 10(-12) M was achieved.

Direct evidence of ionic fluxes across ion-selective membranes: a scanning electrochemical microscopic and potentiometric study.

Scanning electrochemical microscopy (SECM) supplemented with potentiometric measurements was used to follow the time-dependent buildup of a steady-state diffusion layer at the aqueous-phase boundary of lead ion-selective electrodes (ISEs). Differential pulse voltammetry is adapted to SECM for probing the local concentration profiles at the sample side of solvent polymeric membranes. Major factors affecting the membrane transport-related surface concentrations were identified from SECM data and the potentiometric transients obtained under different experimental conditions (inner filling solution composition, membrane thickness, surface pretreatment). The amperometrically determined surface concentrations correlated well with the lower detection limits of the lead ion-selective electrodes.

Analytical performance characteristics of thin and thick film amperometric microcells.

The analytical performance of amperometric microcells with different electrode geometries is compared for enzyme activity measurements. The microcells were fabricated with thin film photolithography or thick film screen-printing in four different designs. The cells made with the thin film process used flexible substrate with microelectrode array or a circular, disk-shaped working electrode. The screen-printed working electrodes had semicircle or disk shape on ceramic chips. Putrescine oxidase (PUO) activity measurement was used as a model. The determination of PUO activity is important in the clinical diagnosis of premature rupture of the amniotic membrane. An electropolymerized m-phenylenediamine size-exclusion layer was used to eliminate common interferences. The size exclusion layer revealed also to be advantageous in protecting the electrodes from fouling by putrescine (enzyme substrate). The electrode fouling of bare electrodes was insignificant for screen-printed electrodes, but very severe for electroplated platinum working electrodes. The microelectrode array electrodes demonstrated smaller RSD and higher normalized sensitivities for hydrogen peroxide and PUO activity. All the other electrodes were demonstrating comparable analytical performances.

Screen-printed amperometric microcell for proline iminopeptidase enzyme activity assay.

A microfabricated amperometric microcell was designed and used for the determination of proline iminopeptidase (PIP) enzyme activity in 2-10-microl samples. The measurements were made in the range of 10.3-841.5 mU/ml enzyme activities. The sensitivity of the determinations was between - 0.0195 and - 0.0203 microA ml/mU per min. The coefficient of variation of the determined values ranged between 2.8 (at 561.2 mU/ml) and 24.1% (at 10.3 mU/ml). The microcell was manufactured on an alumina substrate using screen-printed graphite working and Ag/AgCl reference electrodes. Elevated PIP activity in the vaginal fluid is a biochemical indicator of bacterial vaginosis. The method is appropriate to differentiate between normal (66+/-145 mU/ml) and elevated, diseased (704+/-145 mU/ml), values.

Study of the determination of acetylcholine after enzymatic hydrolysis by triangle programmed coulometric flow titration.

A new method for the determination of acetylcholine is introduced and studied. The method uses enzymatic hydrolysis of acetylcholine carried out in a flow-through analytical reactor column, while for the detection triangle programmed coulometric flow titration is employed. The flow-through coulometric titration system and the preparation of the enzyme reactor are described. The operation conditions for the hydrolysis and for the titration were optimized. The flow-through coulometric titration technique offers a better precision for the determination of acetylcholine in small concentration ranges than methods based on the detection of pH change. Working conditions of the system can be easily adjusted for different sample concentration ranges. The limit of detection for the determination of acetylcholine was found to be 8x10(-5) M. The method described could be advantageously used for indirect determination of acetylcholinesterase inhibitors.