Elaine T. Vandenberg

In vivo probes: Problems and perspectives

Devices constructed for potential use as invasive bioprobes incorporate a selective receiving site for molecular or ionic recognition, and a transducer which is capable of translating a perturbation of physical chemistry of the determinant-site reaction (interaction) into a usable signal. Four types are envisioned--implants for general hospital use, transient-use probes to replace classical blood tests, short-term implantable probes and the long-term variety. Performance criteria are selectivity, sensitivity, fast response, site-reversible, small, rugged, inexpensive, biocompatible, calibratible, facile use by non-expert personnel and ease of telemetry. These demands, not surprisingly, create enormous challenges to the sensor specialist. With respect to biocompatibility the sensor must not be involved in infection, clot formation or antigenic response, and, furthermore, protein adsorption, etc., which can affect the sensor response should be avoided. Calibration remains a problem of monumental proportions. Many devices drift from calibrated levels even in in vitro experiments, let alone in the implanted milieu. One solution has been to carry out on-line switching between patient blood and standard solutions. However, this type of approach leaves a lot to be desired with respect to portability. Another method which is attracting increasing attention is the chemometric or artificial intelligence system involving compensation by multi-sensor array configurations. Sensitivity and limit-of-detection have attracted little research due to the overwhelming nature of other difficulties. In the present paper we evaluate a number of these technical problems and discuss the architecture of devices that are currently available. Finally, some thoughts as to priorities for re-directing sensor research in the bioprobe area are presented.

Langmuir-Blodgett film characteristics and phospholipid membrane ion conduction : Part 1. Modification by Cholesterol and Oxidized Derivatives

Abstract The energy barrier to inorganic ion conduction through bilayer lipid membranes (BLM) is investigated as a function of molecular packing and dipolar potential characteristics. Arrhenius energy barrier information is derived from temperature-dependent electrochemical experiments with phosphatidyl choline/steroid BLM. The steroids studied at 0.65 mole fraction in phospholipid were 5-cholesten-3β-ol, 5,7-cholestadien-3β-ol, 5-cholesten-3β,7α-diol, 5α-cholestan-3β,5α,6β-triol, 5α-cholestan-5α,6α-epoxy-3β-ol, 5-cholesten-3β-ol-7-one and 5α-cholestan-3-one. Correlation of the barrier magnitude with molecular packing characteristics, obtained by collecting monolayer data from a Langmuir-Blodgett trough, indicates that the BLM ion current is almost completely controlled by molecular density. The sensitivity of the energy barrier as a function of molecular packing is as great as 0.1 eV for a 0.01-nm 2 adjustment.

Selective electrochemical biosensors from state-switching of bilayer and monolayer lipid membranes by lectin-polysaccharide complexes

Interaction of the lectin concanavalin A with the polysaccharide glycogen can provide rapid spontaneous transients of the surface potential at bilayer and monolayer lipid membranes. The selective binding process can cause large, rapid potassium ion current fluctuations across bilayer membranes in a manner that is periodic and reproducible. The frequency of these transient ion current signals was shown to be related to sub-nanomolar concentrations of the reactive agents in aqueous solution. The physical mechanism responsible for ion current modulation was investigated by fluorescence methods using lipid vesicles, by the thermal dependence of the potassium ion current across planar bilayers and by pressure-area and dipolar potential measurements of lipid monolayers at an air-water interface. The mechanism is primarily associated with physical perturbations of lipid membranes by lectin-polysaccharide aggregates, resulting in the formation of localised domains of variable electrostatic potential and conductivity.

The primary events in chemical sensory perception: Olfaction as a model for selective chemical sensing

Abstract Early theories of olfaction are summarized after brief introductions to the anatomy of the human olfactory system and direct electrical measurements on receptor cells (electro-olfactography). The problems of odourant convection-diffusion, and transduction are discussed in terms of the physical chemistry of interfacial partitioning and membrane-receptor behaviour, respectively. Speculation is presented regarding the significant features of receptor structure required for analytical selectivity. Stimulant—receptor interaction at the membrane surface can lead to changes in membrane permeability to ions in a number of distinct ways. Current knowledge regarding the structure and membrane electrochemistry of the important neurotransmitter receptor for acetylcholine, is examined as a role model for the biological activity of olfactory receptors. Finally the olfactory system is compared concisely to existing chemical sensor technology. Although parallels exist, significant differences are obvious; an example is the particular chemistry exhibited by molecular receptors and the hybrid of digital and analogue coding for transduction.

Chemical transduction with fluorescent lipid membranes using selective interactions of acetylcholine receptor with agonist/antagonist and acetylcholinesterase with substrate

Alterations in the physical structure of vesicles and monolayers of phospholipids and soybean lecithin were monitored by measurement on the average fluorescence intensity changes from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-a-phosphatidyl ethanolamine (NBD-PE) located in the lipid matrices. This probe was intimately dispersed at a concentration of 1-2 mol-% in lipid membranes and had an emission sensitive to local environmental structure. Alterations in the structure of soybean lecithin vesicles were induced by the selective interaction of acetylcholine receptor with the agonist carbamylcholine and the antagonist alpha-bungarotoxin. Structural changes in vesicles with a 7:3 mole ratio of dipalmitoylphosphatidyl choline to dipalmitoylphosphatidic acid were observed for selective interactions between acetylcholinesterase and acetylcholine. Enhancement of fluorescence emission from the lipid membranes provided transduction of the selective binding events of the receptor and enzyme. A maximum sensitivity of about a 30% enhancement per micromole of carbamylcholine and a detection limit for the toxin of 10 nM were observed for the receptor. Fluorescence microscopy was used to establish that protein could be incorporated in monolayer lipid membranes and to provide information about potential mechanisms of fluorescence enhancement. These studies show that lipid membranes containing NBD-PE can be used as generic transducers of protein-ligand interactions.

Fluorescence wavelength, intensity and lifetime for multidimensional transduction of selective interactions of acetylcholine receptor by lipid membranes

Concurrent analysis of the fluorescence intensity, at different emission wavelengths, of lipid vesicles containing acetylcholine receptor (AChR) labelled with a nitrobenzoxadiazole (NBD) moiety shows that selective interactions with the agonist carbamylcholine can be detected reproducibly by a self-calibration method with muM detection limits. Concurrent analysis of the fluorescence intensity and lifetime of the new probe 4-dicyanomethylene-1,2,3,4-tetrahydromethylquinoline (DCQ) shows that general alterations of lipid membrane structure induced by temperature variation in the head-group region of lipid vesicles can be determined. A general approach to detection of selective interactions is introduced by observation of fluorescence intensity and lifetime changes of the probe NBD-phosphatidyl ethanolamine dispersed in lipid membranes containing unlabelled AChR. Detection and differentiation of selective interactions between carbamylcholine and the antagonist alpha-bungarotoxin are possible by correlation with intensity and lifetime at different emission wavelengths.

Immobilization of Proteins for Biosensor Development

There is considerable interest in the permanent attachment of bio-molecules to solid surfaces because of the many uses of immobilized proteins: for purification of molecules that bind to antibodies, lectins or other binding proteins; in solid-phase analytical assays; for improved biocompatibility of materials; and to provide selectivity to biosensors. In such applications, the amount of protein immobilized and the retention of physiological activity of the immobilized protein (such as binding of a second molecule) are of interest. This work will report the amount and activity of immobilized proteins as a function of the method of immobilization, using instrumental analytical techniques. The results are of interest to all whose work involves immobilized proteins, but the study was inspired by its application to the field of biosensors, as evidenced by the choice of solid surfaces (quartz and silicon), and the types of proteins employed (those which bind a second molecule).

Surfactant characterization at an air-water interface by direct reflection ellipsometry

Abstract Lipid monolayers of phosphatidyl choline/cholesterol at an air/water interface are evaluated for thickness, refractive index and average molecular area by direct reflection ellipsometry. Results indicate that changes in chemical composition of monolayers can be readily detected, and that average molecular area can be reliably established. The latter results are contrasted with values obtained by monolayer-compression methods and indicate that the techniques provide data of similar accuracy and precision.

The prevention of adsorption of interferents to radiolabelled protein by Tween 20

Radiolabels are often used to quantitatively determine the amount of protein immobilized on chromatographic supports, immunochemical plates and biosensor surfaces. Bovine serum albumin (BSA) was chosen as a model protein for quantitative deposition studies. BSA was radioiodinated (125I-) or fluorescently labelled (fluorescein), then incubated with the following surfaces: quartz, quartz derivatized by 3-aminopropyltriethoxysilane (Qz-APTES), and Qz-APTES reacted with glutaraldehyde or tresyl chloride. The amounts of BSA immobilized to the different surfaces were compared using data from radioactivity and fluorescence assays. Irreproducible results were obtained with radioiodinated BSA due to adsorption/desorption behaviour of an unidentified radioactive species. When the non-ionic detergent Tween 20 was added to the protein/surface incubation mixture, radiolabelled BSA gave reproducible protein binding results which agreed with fluorescent protein binding patterns. The effect of Tween 20 was due to either the binding to BSA displacing the interferent and/or the solubilization of the interferent.

Phase Structure Modulation Of Fluorescent Lipid Membranes For Selective Chemical Transduction

Lipid monolayers have been studied by pressure-area relationships and fluorescence microscopy, revealing unique phase domain distribution patterns. Acetylcholine receptor and acetylcholinesterase were incorpoated into these membranes, Producing a change in lipid phase structure. Deposition of these membranes onto various substrates was successfully performed.