Amos Bardea

Chronopotentiometry and Faradaic impedance spectroscopy as signal transduction methods for the biocatalytic precipitation of an insoluble product on electrode supports : routes for enzyme sensors, immunosensors and DNA sensors

The biocatalyzed precipitation of an insoluble product produced on electrode supports is used as an amplification path for biosensing. Enzyme-based electrodes, immunosensors and DNA sensors are developed using this biocatalytic precipitation route. Faradaic impedance spectroscopy and chronopotentiometry are used as transduction methods to follow the precipitation processes. While Faradaic impedance spectroscopy leads to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provides the total resistance at the interfaces of the modified electrodes. A horseradish peroxidase, HRP, monolayer-functionalized electrode is used to sense H(2)O(2) by the biocatalyzed oxidation of 4-chloro-1-naphthol (1), to the insoluble product benzo-4-chlorohexadienone (2). An antigen monolayer electrode is used to sense the dinitrophenyl antibody, DNP-Ab, applying an anti-antibody-HRP conjugate as a biocatalyst for the oxidative precipitation of 1 by H(2)O(2) to yield the insoluble product 2. An oligonucleotide (3) functionalized monolayer electrode is used to sense the DNA-analyte (4), that is one of the Tay-Sachs genetic disorder mutants. Association of a biotin-labeled oligonucleotide to the sensing interface, followed by the association of the avidin-HRP conjugate and the biocatalyzed precipitation of 2 leads to the amplified sensing of 4. The amount of the precipitate accumulated on the conductive support is controlled by the concentration of the respective analytes and the time intervals employed for the biocatalytic precipitation of 2. The electron-transfer resistances of the electrodes covered by the insoluble product (2) are derived from Faradaic impedance measurements, whereas the total electrode resistances are extracted from chronopotentiometric experiments. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports are found. Chronopotentiometry is suggested as a rapid transduction means (a few seconds). The precautions needed to apply chronopotentiometry in biosensors are discussed.

Sensing and amplification of oligonucleotide-DNA interactions by means of impedance spectroscopy: a route to a Tay–Sachs sensor

A three-component oligonucleotide/DNA layered assembly on a Au-electrode acts as a specific biosensor for the analysis of the Tay–Sachs mutant by means of Faradaic impedance spectroscopy.

Protection against developmental retardation in apolipoprotein E-deficient mice by a fatty neuropeptide: Implications for early treatment of Alzheimer's disease

Stearyl-Nle17-VIP (SNV) is a novel agonist of vasoactive intestinal peptide (VIP) exhibiting a 100-fold greater potency than the parent molecule and specificity for a receptor associated with neuronal survival. Here, mice deficient in apolipoprotein E (ApoE), a molecule associated with the etiology of Alzheimers disease, served as a model to investigate the developmental and protective effects of SNV. In comparison to control animals, the deficient mice exhibited (a) reduced amounts of VIP messenger RNA; (b) decreased cholinergic activity (c) significant retardation in the acquisition of developmental milestones: forelimb placing behavior and cliff avoidance behavior; and (d) learning and memory impairments. Daily injections of SNV to ApoE-deficient newborn pups resulted in increased cholinergic activity and marked improvements in the time of acquisition of behavioral milestones, with peptide-treated animals developing as fast as control animals and exhibiting improved cognitive functions after cessation of peptide treatment. Specificity was demonstrated in that treatment with a related peptide (PACAP), pituitary adenylate cyclase-activating peptide, produced only limited amelioration. As certain genotypes of ApoE increase the probability of Alzheimers disease, early counseling and preventive treatments may now offer an important route for therapeutics design.

Probing Antigen–Antibody Interactions on Electrode Supports by the Biocatalyzed Precipitation of an Insoluble Product

The amplified sensing of an antibody by an antigen monolayer-functionalized transducer by using a biocatalyzed precipitation of an insoluble product on the transducer is addressed. Faradaic impedance spectroscopy, cyclic voltammetry and microgravimetric, quartz crystal microbalance analyses are used to probe the precipitation of the insoluble product on the transducer. A dinitrophenyl, DNP, antigen monolayer is assembled on a Au-electrode, or a Au-quartz crystal, as a sensing interface for the dinitrophenyl-antibody, DNP-Ab. An anti-Fc-antibody-HRP conjugate is used as a biocatalytic probe for the formation of the antigen/DNP-Ab complex on the transducer. Biocatalyzed oxidation of 4-chloronaphthol by H2O2 in the presence of the anti-Fc-antibody-HRP conjugate yields an insoluble product on the transducer. Formation of the insoluble film on the electrode results in the increase of interfacial electron-transfer resistances detected by impedance spectroscopy or cyclic voltammetry. The precipitate formation also results in the mass increase of the modified Au-quartz crystal detected as a frequency change of the piezoelectric transducer. The DNP-Ab is easily sensed at a sensitivity that corresponds to 0.5 ng mL–1 (3×10–12 M).

Fully integrated biocatalytic electrodes based on bioaffinity interactions.

Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers. A catalyst-NAD(+)-dyad for the binding of the NAD(+)-dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems. NAD(+)-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD+ monolayer. The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N6-(2-aminoethyl)-NAD+ to the monolayer. The interface modified with the PQQ-NAD(+)-dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer. The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH. Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C. 1.9.6.1 from E. coli) or CoII-protoporphyrin IX reconstituted myoglobin (CoII-Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode. The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules. The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and CoII-Mb show catalytic activities for NO3- reduction and acetylene-dicarboxylic acid hydrogenation, respectively. Two FeIII-protoporphyrin IX units are reconstituted into a four alpha-helix bundle de novo protein assembled as a monolayer on a Au-electrode. Vectorial electron transfer proceeds in the synthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the FeIII-protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate.

Amplified microgravimetric quartz-crystal-microbalance analyses of oligonucleotide complexes: a route to a Tay–Sachs biosensor device

An oligonucleotide monolayer acts as an active interface for the microgravimetric, quartz-crystal-microbalance analysis of the complementary oligonucleotide.

Amplified electronic transduction of oligonucleotide interactions: novel routes for Tay–Sachs biosensors

Abstract Two different configurations for the amplified microgravimetric analysis of oligonucleotides (DNA) are presented. A thiol-tagged oligonucleotide, ( 1 ), is assembled on a Au-quartz electrode, and the complementary oligonucleotide ( 2 ) is microgravimetrically sensed by the crystal. Formation of the ds-oligonucleotide assembly is amplified using mouse anti-dsDNA antibody and goat anti-mouse Fc antibody. Alternatively, a biotin-labeled oligonucleotide, ( 4 ), is used to form the ds-oligonucleotide. Biotin is used to amplify the formation of the ds-complex. An electrochemical method, using cytochrome c as redox probe, to assay the formation of the ds-oligonucleotide assembly, is described. The 5′-carboxylic acid-functionalized oligonucleotide ( 5 ) is coupled to a cystamine monolayer associated with an Au-electrode. The electrostatic attraction of cytochrome c to the ds-oligonucleotide complex facilitates the interfacial electron transfer to cytochrome c . Formation of the ds-oligonucleotide assembly is detected electrochemically using differential pulse voltammetry.

Patterning Gradient Properties from Sub-Micrometers to Millimeters by Magnetolithography

A new method is presented for patterning surfaces with gradient properties. The method is based on magnetolithography in which the surface patterning is performed by applying a gradient of a magnetic field on the substrate, using paramagnetic metal masks in the presence of a constant magnetic field. Superparamagnetic nanoparticles (NPs) are deposited on the substrate, and they assemble according to the field and its gradients induced by the mask. Once they pattern the substrate, they protect their sites on the substrate from interacting with any other species. The areas not protected by the NPs can be covered by molecules that chemically bind to the substrate. After these molecules are bound, the NPs are removed, and other molecules may be adsorbed on the newly exposed area. The new technique is based on a parallel process that can be carried out on a full wafer. It provides high resolution, it creates gradient continuously from sub-micrometers to millimeters, and it can be performed on surfaces that are not flat and that are even on the inside of a tube. The gradient that is formed is not limited to a specific property or type of substrate.

Magnetolithographic patterning of inner walls of a tube: a new dimension in microfluidics and sequential microreactors.

By applying magnetolithography it is possible to chemically pattern the inside of tubes. This new capability allows one to perform sequential processes within the tubes. Several enzymatic reactions are demonstrated.

Protection Against Developmental Deficiencies by a Lipophilic VIP Analogue

Stearyl-Nle-VIP (SNV) is a novel agonist of vasoactive intestinal peptide (VIP) exhibiting a 100-fold greater potency than the parent molecule and specificity for a receptor associated with neuronal survival. Here, the developmental and protective effects of SNV were investigated in vivo using two models of developmental retardation, hypoxia and cholinergic blockade. In both cases chronic administration of SNV during development provided protective effects. Water maze experiments on the weaned animals have demonstrated a prophylactic action for SNV and enhancement of spatial memory in animals exposed to a cholinotoxin. SNV may act by providing neuroprotection, thereby improving cognitive functions. This work is dedicated to Prof. R.J. Wurtman whose inspiration and leadership in the field of neuroscience and cognition is beyond comparison.