Richard F. Taylor

An acetylcholine receptor-based biosensor for the detection of cholinergic agents

Abstract A practical, acetylcholine receptor-based prototype biosensor is reported. The biosensor utilizes a polymeric film containing the receptor and appropriate stabilizers to coat an interdigitated electrode transducer. Upon binding of specific cholinergic ligands to the immobilized receptor, changes occur in the electrical field of the electrodes proportional to the amount of cholinergic agent present. The biosensor can detect micro- to nano-gram quantities of cholinergic ligands per ml within 1–5 s. The biosensor can be recycled with acetylcholine and is stable for at least 72 h during use and over 6 months in storage at normal temperatures. The cholinergic biosensor represents the first instance of a biosensor based on a physiologically active receptor for the detection of a class of chemical compounds.

Antibody- and receptor-based biosensors for detection and process control

The extension of a generic immobilization technology for the fabrication of antibody- and receptor-based biosensors is reported. The biosensors utilize polymerized protein films containing the antibody or receptor and appropriate stabilizers to coat an interdigitated electrode transducer. Changes in the electrical field of the coated electrodes are related to specific interactions between the immobilized biomolecule and the analyte to be measured. A portable electronics module has been developed into which the coated electrode chips are inserted and challenged with analyte. Within 5–10 s, an output reading (change in impedance, phase angle, current or voltage) is obtained that is related to analyte concentration. The use of parallel, non-antibody or non-receptor containing control chips allows automatic correction for background and non-specific binding. The biosensors can detect low ng ml−1 levels of analyte in serum or other liquids. Such antibody- and receptor-based biosensors are applicable to the determination of a wide range of analytes for analytical, clinical, environmental and process control applications.

A comparison of various commercially-available liquid chromatographic supports for immobilization of enzymes and immunoglobulins

Abstract A number of commercially-available, activated supports were evaluated and compared for the immobilization of enzymes (alkaline phosphatase, glucose oxidase and peroxidase) and human immunoglobulin G (IgG). The supports studied included pressure-stable, epoxy-activated acrylate-based supports (Separon HEMA 1000 and Eupergit C); agarosebased, epoxy-, cyanogen bromide-, glutaraldehyde- or N-hydroxysuccimide-activated supports (epoxy-activated or cyanogen bromide-activated Sepharose, ACT-Ultrogel AcA 22, Reacti-Gel 6X and Affi-Gel 10); and glass bead-based, activated supports (CDI- and NHS-Glycophase). As expected, the pH required for maximum protein immobilization and retention of activity varied with both protein and support. For example, the amount of alkaline phosphatase coupled was maximum at pH 3 or 5 for most supports, but retention of activity was greatest for immobilization at pH 7, 9 or 11. Glucose oxidase and peroxidase coupling and activity retention in general showed less variation in optimal coupling pH. Coupling of IgG and retention of anti-IgG binding activity were both optimal at a coupling pH of 9 or 11. The Separon HEMA-IgG support made in these studies was also utilized for rapid h.p.l.c, purification of anti-IgG from serum.

The solubilization of carotenogenic enzymes of phycomyces blakesleeanus

The effect of nine ionic and nine non-ionic detergents, over a 0.3–3.0% (w/v) concentration range, on the activity of the enzymes which convert [2-14C]mevalonic acid into phytoene (7,8,11,12,7′,8′,11′,12′-ψ,ψ-carotene) and β-carotene (β,β-carotene) has been investigated with cell extracts of the C115 carS42 mad-107(−) (β-carotene-accumulating) strain of Phycomyces blakesleeanus. The enzymes catalyzing the conversion of mevalonic acid into phytoene in the C115 and the C5 carB10(−) (phytoene-accumulating) strains of Phycomyces could be released from membranes with high molarity Tris-HCl buffer, but the other carotenogenic enzymes required solubilization with detergents. Enzymic activity was retained with only two ionic detergents (Zwittergents 3–8 and 3–10), whilst Tweens 40 and 60 were the least inhibitory of the non-ionic surfactants. Both Tween 60 and Zwittergent 3–08 solubilized almost 50% of the enzymic activities for the conversion of phytoene to β-carotene, but the former preparation was significantly more stable on storage at −70°C.

Comparison of low- and high-pressure affinity chromatography for the purification of serine and sulfhydryl esterases

A series of affinity chromatography packings for the purification of serine and sulfhydryl esterases (acetylcholinesterase, alkaline phosphatase, urokinase and papain) have been synthesized using commercially available agarose, glass and acrylate parent matrices. Two ligands were coupled to the matrices by utilizing carbodiimide or reaction with active groups already present on the matrix: the quaternary ammonium compound trimethyl(p-aminophenyl)ammonium chloride and the serine esterase inhibitor analog p-aminobenzamidine. It was found that enzyme purification on the agarose- or acrylate-based packings was most successful, resulting in as much as fifty-fold purification over starting material. The pressure stability of the acrylate packing allowed purification by high-pressure affinity chromatography and decreased purification times as much as six-fold in comparison to agarose columns.

Advances in HPLC and HPLC-MS of Carotenoids and Retinoids

High performance liquid chromatography (HPLC) has been established during the past 10 years as one of the primary methods for the separation and purification of carotenoids and retinoids. The rapid analysis times (10 to 45 min), high sensitivity (low ng), high resolving power, high recovery and non-destructive conditions of HPLC make it an ideal method for carotenoid and retinoid analysis.