James N. Miller

Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer

Relative fluorescence quantum yields are determined using a computer-controlled luminescence spectrometer. The relative absorbances of the standards and unknowns are measured using the same instrument as for the fluorescence measurements. Relative quantum yields are presented for a wide range of compounds at room temperature.

Recent developments in fluorescence and chemiluminescence analysis. Plenary lecture

Recent developments in photoluminescence and chemiluminescence spectroscopy are summarised, with particular reference to methods for improving the selectivity of luminescence analyses. The acquisition and manipulation of contour spectra and the problems and benefits of generally available corrected fluorescence spectra are discussed. Fluorescence and chemiluminescence immunoassays are evaluated and some probable future developments in these areas are indicated.

Tutorial review—Outliers in experimental data and their treatment

This review summarizes critically the approaches available to the treatment of suspect outlying results in sets of experimental measurements. It covers the use of parametric methods such as the Dixon test (with comments on the problems of multiple outliers); the application of non-parametric statistics based on the median to by-pass outlier problems; and the application of robust statistical methods, which down-weight the importance of outliers. The extension of these approaches to outliers occurring in regression problems is also surveyed.

Fluorescence energy transfer methods in bioanalysis

Energy transfer phenomena, in which excited fluorophores transfer energy to neighbouring chromophores, are well characterised in photochemistry and have found a wide range of applications in analytical biochemistry. The transfer of energy from a donor to an acceptor group is only significant over distances of a few nm, so it can be used as a spectroscopic ruler and as a means of detecting molecular interactions and conformational changes. Such methods usually retain the great sensitivity and sample handling flexibility of conventional fluorescence techniques. As a result many assays involving enzymes, antibodies and nucleotides utilise energy transfer measurement principles. This article outlines these principles for the main types of energy transfer, and summarises some of their most important areas of application.

Automation of an energy-transfer immunoassay by using stopped-flow injection analysis with merging zones

Abstract A homogeneous fluorescence energy-transfer immunoassay for serum albumin has been automated by using flow-injection analysis. Application of the merging-zone and stopped-flow principles permits low consumption of labelled reagents and samples, and sub-micromolar concentrations of albumin can be rapidly and precisely determined. Studies on individual serum samples show good agreement with other techniques, including a fluorimetric dye-binding assay that has also been automated by using mergingzone flow-injection analysis.

Determination of pH with acid—base indicators: Implications for optical fibre probes

Results are presented which clearly illustrate the possibilities and limitations of the use of indicators immobilized on optical fibres, in the determination of pH.

Thiophilic gels: applications in flow-injection immunoassays for macromolecules and haptens

Abstract Simple fluorescence flow-injection immunoassays have been developed for theophylline (a hapten) and albumin (a macromolecule) using a thiophilic affinity matrix (T-gel), This gel is far superior to the previously used bacterial immunoglobulin binding proteins (e.g., proteins A and G) in terms of cost, elution conditions, non-specific binding and specificity for immunoglobulins regardless of type or subclass. Both preincubation and on-line immunoassay formats were investigated. Human serum albumin and theophylline were determined by this method and good R.S.D. and %recoveries were achieved.

A model on-line flow injection fluorescence immunoassay using a protein a immunoreactor and lucifer yellow

Abstract A heterogeneous on-line fluorescence immunoassay has been developed for a model analyte (transferrin) using a flow injection analysis system containing a controlled pore glass protein A immunoreactor. Lucifer yellow VS (LYVS) a 4-aminonaphthalimide with a large stokes shift and pH independence was the fluorophore. For each assay the antibody-protein A reaction takes place at near neutral pH, and the complexes are eluted at acid pH. Transferrin levels in human serum has been determined by this method, and good within assay variations have been achieved.

Flow injection analysis (FIA) — a personal view

Abstract 36 authors from 10 countries express their viewpoints on the merits of flow injection analysis. Alison Macdonald has contributed invaluably to the development of FIA through critical evaluation of work of all the above authors, by editing manuscripts, and by carefully resolving differences between authors and referees. Therefore Analytica Chimica Acta, together with its Editor, may be regarded as the Alma Mater of FIA. It is our intention to honour (and humour). Dr. Macdonald by publishing this nonorchestrated and, by us, unedited patchwork of individual papers which reflect a rich mixture of applications, modifications and interpretations of FIA, together with a variety of personal opinions. We are most grateful to our colleagues for endorsing this unconventional way of celebrating the 25-year anniversary of our Editor (J.R. and E.H.H.).

Energy-transfer immunoassay: a study of the experimental parameters in an assay for human serum albumin.

Abstract The use of fluorescein and rhodamine as fluorescent groups in energy-transfer immunoassays has been evaluated by developing an assay for human serum albumin. The sensitivity of the assay is found to depend on several factors, including the degrees of labeling of antigen and antibody, concentrations of labeled antigen and antibody, and the fluorimeter spectral bandwidth. Although fluorescein and rhodamine are far from ideal as energy-donor andacceptor labels, the assay is capable of detecting nanomolar concentrations of albumin. Comparative studies on serum samples show that the assay gives results that compare well with those obtained using the slower electroimmunoassay method.