Tony E. Edmonds

Flow injection electrochemical enzyme immunoassay for theophylline using a protein A immunoreactor and p-aminophenyl phosphate–p-aminophenol as the detection system

A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass-protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V versus the saturated calomel electrode. For each sample the antibody-protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.

Direct reductive amperometric determination of nitrate at a copper electrode formed in situ in a capillary-fill sensor device

A method has been developed for determining nitrate amperometrically by direct reduction at a freshly deposited copper electrode surface in a capillary-fill device (CFD). Copper(II) is added to the nitrate sample which is then taken up into the device. The potential of the screen-printed carbon electrode is held at –0.75 V versus the screen-printed silver reference electrode. At this potential, copper is plated onto the carbon electrode forming a freshly prepared copper electrode. At the same time dissolved oxygen is reduced. The potential is then scanned to more negative potentials and the signal at –0.90 V, due to the reduction of the nitrate, is measured. The method for determining nitrate given here is preliminary to the production of CFDs in which chemical reagents, copper sulphate and potassium hydrogen sulphate (used to produce the acidity), are screen-printed or otherwise coated onto the upper plate within the device.

Investigation of decomposition products of microwave digestion of food samples

The involatile residues remaining after closed-vessel microwave digestion of various food samples, using nitric acid, with and without post-digestion treatment with hydrogen peroxide, have been studied. Decomposition products were found to include aliphatic and aromatic acids, nitro-compounds, oxalates and inorganic nitrates and phosphates. Measures of digestion completeness were provided by appearance, carbon content, infrared spectra and thin layer chromatograms of the residues, enabling a comparison of different digestion methods and sample types. Residual carbon levels varied linearly with the relative amounts of carboxylic acid and inorganic nitrate, as measured by infrared spectrometry of the residues. The formation of calcium oxalate was also a function of the degree of decomposition: the carboxylic acid: oxalate ratio increased in a logarithmic fashion with increasing residual carbon content. Particularly high levels of residual carbon from nitric acid digestion of milk powder, due largely to carboxylic acid residues, were substantially decreased by post-digestion treatment with hydrogen peroxide. However, nitrobenzoic acids, which proved major interferents in electrochemical analysis, were only removed by treatment with perchloric acid.

Flow Injection Immunosensor for Theophylline

Abstract A heterogeneous on line electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline using a flow injection analysis system containing a protein A immunoreactor to separate antibody bound and unbound theophylline-alkaline phosphatase. In this assay the antibody, label and sample are sequentially immobilised onto the protein A immunoreactor. The antibody-protein A and antibody-protein A label/sample reaction takes place at near neutral pH, and the complexes are eluted at acid pH. p-Aminophenyl phosphate (PAPP) and p-aminophenol (PAP) have been used as the detection system. Experimental variations such as flow rate and reactant concentration have been studied and serum theophylline has been determined by this method and good r.s.d values and % recoveries have been achieved.

Differential-pulse cathodic stripping voltammetric determination of sodium nitroprusside at a hanging mercury drop electrode aided by copper(II) and poly-L-lysine modification

The nitroprusside anion, Fe(CN)5NO2–, can be accumulated from pH 5 Britton–Robinson buffer solution on to the surface of a hanging mercury drop electrode (HMDE), which has been modified by means of adsorbed poly-L-lysine and which is held on open circuit. This was shown by closing the circuit at –0.2 V versus the Ag–AgCl electrode, after accumulation, and scanning cathodically by use of differential-pulse voltammetry. The voltammetric peak that was observed at about –0.6 V, however, was that for the adsorbed product of the first reduction step of nitroprusside, reported to be Fe(CN)4NO2–, which is the product of the one-electron reduction of the nitroprusside ion [to Fe(CN)5NO3–] followed by the elimination of CN–. When accumulation was carried out at the peak potential of the first polarographic reduction peak of nitroprusside (–0.35 V), the peak current at –0.6 V was enhanced 4-fold, which indicated that the Fe(CN)4NO2– was accumulated on poly-L-lysine more extensively than was the nitroprusside ion. From pH 2 (0.01 mol dm–3 hydrochloric acid) solution, nitroprusside can be accumulated very efficiently between 0 and –0.1 V at an unmodified HMDE in the presence of excess of copper(II). The cathodic stripping peak at about –0.35 V is due to the reduction of an adsorbed copper/reduced nitroprusside species, which has not been fully identified, but is most probably either the copper(I) or copper(II) salt of Fe(CN)4NO2–. This signal was enhanced slightly when accumulation was effected at a poly-L-lysine-modified HMDE. Linear calibration graphs were obtained for up to at least 1 × 10–7 mol dm–3 for the determination of nitroprusside at both pH 5 and pH 2, by using cathodic stripping voltammetric methods based on the above electrode processes, and the limit of detection was calculated to be about 5 × 10–10 mol dm–3 in both instances. Relative standard deviations were typically <2% at the 1 × 10–8 mol dm–3 level.

Adaptation of on-line reactions developed for use with flow injection with amperometric detection for use in disposable sensor devices: reductive determination of phosphate as pre-formed 12-molybdophosphate in a capillary-fill device

Preliminary experiments have been carried out to show that on-line reactions developed for use with flow injection can be adapted for use in capillary-fill devices. The reversibility of the oxidation of a 1 × 10–3M solution of potassium hexacyanoferrate(II) in 1 M HCl solution at a screen-printed carbon electrode in a capillary-fill device (CFD) incorporating a screen-printed silver reference electrode was found to be only slightly less than at a smoothly operating glassy carbon electrode (GCE)[Epa–Epc= 95 mV (CFD), 65 mV (GCE)]. For the determination of phosphate as 12-molybdophosphate by reduction at a screen-printed carbon electrode in a CFD, a device with a screen-printed unchloridised silver reference/counter electrode was preferred to a chloridised silver electrode. A stable measurement potential was obtained when the measured solution was made 0.1 M in potassium chloride. The presence of potassium ion was found to affect markedly the linear sweep voltammograms obtained for the reduction of 12-molybdophosphate particularly at a GCE but also to a lesser extent at a screen-printed carbon electrode; this effect was controlled in further work with the CFD by the inclusion of potassium chloride in the measured solution. A rectilinear response was obtained for 5 × 10–6M(the detection limit)–2 × 10–3M pre-formed 12-molybdophosphate solutions at an applied potential of 0.16 V. The acidity required to carry out the reaction of phosphate can be provided by the addition of potassium hydrogen sulphate. It should be possible, therefore, to produce a satisfactory CFD for the determination of phosphate by screen-printing a mixture of sodium molybdate, potassium hydrogen sulphate and potassium chloride on to the glass face opposite to the screen-printed electrodes.

Development of a multi-sensor system using coated piezoelectric crystal detectors

The development of a multi-sensor piezoelectric crystal detector system for the determination of airborne contaminants such as atmospheric ammonia is described. The system incorporates separate sensors for ammonia, moisture and temperature determination. The effects of variations in operating conditions such as analyte concentrations, humidity, gas flow-rate and coating area and mass were examined with the objective of developing a monitoring system that will operate under ambient conditions. The application of a microcomputer for data handling is described. The microcomputer was used to evaluate ammonia concentrations in air using the differential response from a pair of coated piezoelectric crystals, together with a series of correction routines that compensate for changes in environmental conditions.

Novel instrumentation and biomedical applications of very near infrared fluorescence

A high wavelength fluorescent probe, Nile Red, was added to four proteins, viz., bovine albumin, α1-acid glycoprotein, β-lactoglobulin and ovomucoid. Nile Red showed an enhancement in fluorescence and a shift in emission wavelength, suggesting it was bonding hydrophobically to these proteins. Drug displacement of Nile Red from α1-acid glycoprotein was achieved with both D, L-propranolol and flufenamic acid, showing that the binding site is less electrostatic and more hydrophobic in nature. In order to monitor these interactions, a simple spectrofluorimeter was constructed from solid-state components; the sensitivity of this instrument compared well with that of standard laboratory spectrofluorimeters.

Assessment of on-line nitration reactions as a means of determining nitrate by reverse flow injection with reductive amperometric detection at a glassy carbon electrode

Five compounds were investigated for use as on-line reagents in concentrated sulphuric acid for the reductive reverse flow injection amperometric determination of nitrate as a nitro derivative at a glassy carbon electrode. The sulphuric acid was diluted rapidly on injecting the reagent solution into a sample carrier stream and therefore the nitration reaction, which generally only takes place at sulphuric acid concentrations of greater than about 70%, had to be rapid. Thiophene-2-carboxylic acid was found to be the most suitable reagent of those studied. The nitration reaction was sufficiently rapid and the first of two reduction steps was at –0.19 V versus SCE (as indicated by linear sweep voltammetry); hence the determination was free from interference by dissolved oxygen. Problems associated with contamination of the electrode surface with reduction product, which caused loss of signal after making repeated injections over an extended period, remained.

Communication. Liquid nitrogen cooling in microwave digestion

In an attempt to minimize the delay in opening Teflon pressure vessels following microwave acid digestion, and thus significantly reduce sample preparation time, various approaches to vessel cooling have been investigated. These include the feasibility of carrying out digestions with the pressure vessels immersed in liquid nitrogen and the use of liquid nitrogen as a pre- and post-digestion coolant. Liquid nitrogen cooling in the microwave unit was found to decrease digestion rates considerably, although the prevention of rapid and uncontrollable increases in pressure during digestion of organic material could be useful in some cases. Liquid nitrogen cooling subsequent to or between heating cycles was found to be very effective, especially where several heating, cooling and venting cycles were required. As well as enabling the vessels to be cooled rapidly and opened, it stopped any continuing increase in pressure which could otherwise cause the hot vessels to vent with potential loss of sample. Pre-digestion cooling also helped to delay the onset of rapid increases in pressure.