General strategy for biosensor design and construction employing multifunctional surface-tethered components

Biosensors function by reversibly linking bioreceptor-target analyte binding with closely integrated signal generation and can either continuously monitor analyte concentrations or be returned to baseline readout values by removal of analyte. We present an approach for producing fully reversible, reagentless, self-assembling biosensors on surfaces. In the prototype biosensor, quencher-dye-labeled biotin-linked E. coli maltose binding protein (MBP) bound in a specific orientation to a NeutrAvidin-coated surface is employed as a bioreceptor. To complete sensor formation, a modular tether arm consisting of a flexible biotinylated DNA oligonucleotide, a fluorescence resonance energy-transfer (FRET) donor dye, and a distal beta-cyclodextrin (beta-CD) analyte analogue is bound in an equimolar amount to the same surface by means of DNA-directed immobilization. After self-assembly, a baseline level of FRET quenching is observed due to specific interaction between the beta-CD of the flexible tether arm and the sugar binding site of MBP, which brings the two dyes into proximity. Addition of the target analyte, the nutrient maltose, displaces the linked beta-CD-dye of the DNA-based tether arm, and a concentration-dependent change in FRET results. Biosensor sensitivity and dynamic range can be controlled by either using MBP variants having different binding constants or by binding of modulator DNA oligonucleotides that are complementary to the flexible DNA tether. The sensor can be regenerated and returned to baseline quenching levels by washing away analyte. A complex set of interactions apparently exists on the sensing surface that may contribute to sensor behavior and range. This approach may represent a general way to assemble a wide range of useful biosensors.     

Multiplexed toxin analysis using four colors of quantum dot fluororeagents

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, we prepared bioinorganic conjugates made with highly luminescent semiconductor nanocrystals (CdSe-ZnS core-shell QDs) and antibodies to perform multiplexed fluoroimmunoassays. Sandwich immunoassays for the detection of cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B were performed simultaneously in single wells of a microtiter plate. Initially the assay performance for the detection of each toxin was examined. We then demonstrated the simultaneous detection of the four toxins from a single sample probed with a mixture of all four QD-antibody reagents. Using a simple linear equation-based algorithm, it was possible to deconvolute the signal from mixed toxin samples, which allowed quantitation of all four toxins simultaneously.     

Influence of probe size on the measurement accuracy of non-uniform ELF magnetic fields

The accuracy of extremely low frequency (ELF) magnetic field measurements can be sensitively varied by the meter probe size when the spatial distribution of the magnetic flux density is highly non-uniform, e.g. if the measurement point is close to the field source. The deviation between measured and actual field varies from point to point depending on the probe dimension and on the source configuration, once the other measurement conditions are fixed. The analysis of this effect is developed through a two-dimensional numerical model that enables the evaluation of the actual field value and of the corresponding probe indication. The error distribution, i.e. the deviation between actual and measured value, is computed for magnetic fields generated by industrial three-phase systems under both balanced and unbalanced supply conditions. The analysis shows how, for a given source, the error depends not simply on the distance from the source in relation to the probe size, but on its spatial distribution, which is complex and cannot be a priori predicted without using a computational tool such as the one proposed.     

Analysis of the Electromagnetic Interaction Between a Resonator and a Lossy Dielectric Body as a First Theoretical Approach to Noninvasive Heart Movement Detection

In this paper the electromagnetic interaction between a resonator and a lossy dielectric body is examined by means of a simple model suitable for computer simulation; the body was assumed to have a revolution form and the resonator was electromagnetic coupled to the body by its inductive element constituted by a planar circular or spiral coil. On the basis of the model a computer program was derived which allows a simple numerical evaluation of the variation in the resonant frequency caused by the interaction mentioned above.     

Effect of hypoxemia on the erythropoietic activity of organs during perfusion

Hypoxemia (45-minute) influence in vivo on erythropoietic activity of the kidney, liver, spleen, and sternum was studied by normoxemic perfusion of the isolated organs. The erythropoietic activity proved to increase after 6-hour perfusion of the liver; this confirmed the participation of this organ in the extrarenal secretion of the erythropoietic factor.     

Immunocytochemical study of alpha 1 and beta 2/3 subunits of GABAA receptors in freehand isolated vestibular Deiters' neurons

Vestibular Deiters' neurons have been isolated from bovine brain by the Hydén's freehand dissection technique and challenged with monoclonal antibodies directed toward the alpha 1 and beta 2/3 subunits of the GABAA receptors. Subsequent challenge with fluorescent secondary antibodies and confocal microscopy allowed the study of the cellular distribution of such subunits. In Deiters' neurons the beta 2/3 subunit displayed a clear presence all along the cell body profile and the initial parts of the dendrites. The alpha 1 subunit was found highly present all over the cell interior except the nuclear profiles. The strong presence inside the cells possibly masked its presence on the plasma membrane. However, in part of the cells studied a distinct presence on the plasma membrane was evident. This subunit was visualized also all along the long dendrites of these neurons. The approach we describe here, involving freehand isolated mature neurons from adult animals, may allow a better characterization of the tridimensional distribution of different types of neuronal GABAA receptors in the respect of the approach with brain slices.     

Sensitivity Analysis Methods to Design Optimal Ship Hulls

Automatic procedures for the design of ship hull geometries yielding minimal wave resistance and wave breaking are an attractive opportunity from both the economical and practical standpoints. Estimating the cost function gradient according to the Sensitivity Equation and Adjoint Methods (SEM, AM) instead of using the standard finite difference approximations has the potential of reducing the computational cost of the overall optimization procedure. Aim of this paper is to assess the actual extent of the cost reduction. Speed-up factors of up to 3.3 have been obtained in the evaluation of the cost function gradient and of about 1.6 in the overall optimization procedure applied to an optimal shape design problem of an existing tanker ship. The SEM and AM methods perform better than finite differences mainly because of (i) the smaller number of flow solutions needed to compute the cost function gradient and (ii) the opportunity of using the same LU factored matrix for both the flow solver and the SEM or AM equations, a circumstance arising as a consequence of having chosen a linearized potential flow model of the 3D free-surface problem.