Nichola J. Seare

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.

Research and development topics in Analytical Chemistry

Summaries of seven of the papers presented at a Meeting of the Analytical Division held on July 13-14, 1993, in the University of Bradford.

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.

Determination of Arsenic in a Nickel-based Alloy by Flow Injection Hydride Generation Atomic Absorption Spectrometry Incorporating Continuous-flow Matrix Isolation and Stopped-flow Pre-reduction Procedures

A flow system incorporating a microcolumn of strongly acidic cation-exchange resin (Dowex 50W), to achieve continuous-flow matrix isolation, was used to eliminate nickel interference in the determination of arsenic in a nickel-based alloy by flow injection hydride generation atomic absorption spectrometry. A stopped-flow iodide pre-reduction procedure within the matrix isolation unit converted all arsenic present in the sample into AsIII prior to determination with a tube-in-flame atomizer. The flow injection valve interface between the matrix isolation and hydride generation manifolds allowed separate optimization of each chemistry. After removal of the nickel, the sample stream, flowing at 2.2 ml min–1, was merged first with a stream of 12 mol dm–3 hydrochloric acid flowing at 2.2 ml min–1 and then with a stream of potassium iodide solution (30% m/v) flowing at 1.4 ml min–1. For stop times between 5 and 30 s, an average recovery of 97% with respect to AsIII was obtained. A 400 µl volume of solution was then injected into a water carrier stream (flowing at 11.0 ml min–1), merged with 3.6 mol dm–3 hydrochloric acid flowing at 3.2 ml min–1 and a stream of 1.0% m/v sodium tetrahydroborate solution. After passage through a 600 mm open-tubular reactor argon was merged at 400 dm3 min–1 and the arsine separated in a glass U-tube separator. A 0.2 µm polytetrafluoroethylene membrane filter in the gas transfer line removed aerosol droplets with consequent improvement in the performance of the tube atomizer. The procedure was successfully applied to the determination of arsenic in a nickel-based alloy reference material (BCS-346) containing 50 µg g–1 of arsenic for which a sealed-vessel microwave digestion procedure, involving nitric and hydrofluoric acids, was found to produce arsenic in the + 5 oxidation state. A characteristic concentration of 2.0 ng ml–1 of arsenic and a limit of detection of 3.9 ng ml–1 of arsenic were obtained with the pre-reduction procedure (which diluted the samples by a factor of 2.7) for a set of operating parameters optimized with respect to interference tolerance and throughput in addition to sensitivity. Under these conditions a sample throughput of 54 h–1 was obtained.

An investigation of the use of Nile Red as a long-wavelength fluorescent probe for the study of α1-acid glycoprotein-drug interactions

Spectrofluorimetry in the long-wavelength region of the electromagnetic spectrum (600-1000 nm) is a fairly recent development in photoluminescence spectroscopy, which has numerous advantages over measurements in the more conventional ultraviolet and visible spectral region. 9-Diethylamino-5H-benzophenoxazine-5-one (Nile Red) is an unchanged, hydrophobic molecule, and long-wavelength fluorescence of which is strongly influenced by the polarity of its environment. When Nile Red was added to solutions of alpha 1-acid glycoprotein (Orosomucoid. OMD), it showed an enhancement in fluorescence intensity and a shift to blue in emission wavelength, suggesting it was binding hydrophobically to a non-polar site on the protein. The association constant (12,261,000 +/- 900,000 M-1) and number of binding sites (0.746 +/- 0.044) were calculated for the probe. Upon addition of both acidic and basic drugs, the Nile Red fluorescence reverted to its unbound form, indicating that OMD probably has one high-affinity, wide and flexible binding area for such drugs. Possible enantiomeric selectivity was shown with ephedrine, and the association constant determined for a racemic mixture of propranolol was found to be comparable to other values obtained with alternative, more conventional techniques.

Elimination of Copper Interference by Continuous Flow Matrix Isolation in the Determination of Selenium by Flow Injection Hydride Generation Atomic Absorption Spectrometry

A flow system incorporating a microcolumn of strongly acidic cation-exchange resin (Dowex 50W) to achieve continuous flow matrix isolation was used to eliminate copper interference in the determination of selenium by flow injection hydride generation atomic absorption spectrometry. The microcolumn manifold used for the selective retention of the copper interferent was interfaced with the hydride generation manifold through a flow injection sample injection valve. The two manifolds were made independent of each other in order to achieve optimum performance characteristics for both the matrix isolation and hydride generation. Following removal of the copper, a 400 µl sample was injected into a water carrier stream. This was merged with hydrochloric acid and subsequently with sodium tetrahydroborate soution. After introduction of argon, the selenium hydride was separated by a glass U-tube separator and atomized by a tube-in-flame atomizer. The intermittent regeneration of the column with 1 mol dm–3 HCl enabled repeated matrix isolation without any loss in resin efficiency or the need for column repacking and gives the option for total automation. The procedure was validated through accurate analyses of two copper metal reference materials, National Institute of Standards and Technology Standard Reference Material 454 Unalloyed Copper XI and Bundesanstalt fur Materialforschung und -prufung, Germany, Certified Reference Material 361 Copper, containing 479 and 36 µg g–1 of SeIV, respectively. The system was found to have a characteristic concentration of 1.0 ng ml–1, limit of detection of 2.1 ng ml–1, relative standard deviation of 1.5%(10 ng ml–1 SeIV, n= 12) and sample throughput of 51 h–1.

Heterogeneous fluorescence immunoassays using flow-injection analysis with protein G solid phase reactors

Abstract Immunoreactors containing immobilized protein G were used in the development of flow injection-based heterogeneous fluorescence immunoassays. Insulin was used as the model anlyte and rhodamine isothiocyanate (RITC), whose fluorescence properties are appropriate to the pH changes used in the assays, as the fluorescent label. RITC—insulin conjugate bound to anti-insulin antibodies combined with the protein G column at pH 8.8, and was eluted at pH 2.5. Insulin concentrations of 50 pg ml −1 could be determined with good repeatability and reproducibility (R.S.d. = 4%), and the protein G reactors lasted for many consecutive immunoassays.

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.

Use of Nile Red as a long-wavelength fluorophore in dual-probe studies of ligand-protein interactions.

The long-wavelength fluorescence probe, Nile Red, has polarity-dependent fluorescence intensity and wavelength properties that can be used to monitor the binding of drugs and other ligands to plasma proteins such as albumin and α-1 acid glycoprotein. This paper shows that it can be used in tandem with another fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, to study two or more types of ligand binding sites simultaneously. Some ligands displace one or the other probe from the protein/dual-probe complex, other ligands displace both probes. In each case the resulting decrease in fluorescence can be used to estimate the numbers of binding sites and their association constants.

On-line liquid–liquid extraction with near-infrared fluorescence detection

A flow injection liquid–liquid extraction procedure is described using near-infrared (long wavelength) fluoresence as a means of detection. The fluorescence signal of Methylene Blue (λex= 655 nm; λem= 671 nm) upon the addition of warfarin and flufenamic acid, and after extraction with chloroform, was shown to be enhanced. The liquid–liquid flow injection extraction procedure is compared with the manual extraction procedure.