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New approaches are required to understand the complex processes taking place in the smallest unit of life. Recent years have seen an increasing activity in the use of optical devices and techniques for the investigation of the properties of single cells and also populations of cells including cell to cell communication. This article reviews relevant optical technologies to date as well as new advances in biosensor development, and goes on to explore reported applications in detection of biotargets and cellular signalling pathways inside individual living cells.

A flow-injection immunoassay (FI-IA) method with amperometric detection for aflatoxin M1 (AFM1) determination in milk has been developed. The first step consists in an incubation of the sample containing AFM1 (Ag) with fixed amounts of anti-AFM1 antibody (Ab) and of the tracer (Ag*, AFM1 covalently coupled to HRP) until equilibrium is reached. In this mixture a competition occurs between Ag and Ag* for the Ab. The mixture is then injected into a flow system where the separation of the free tracer (Ag*) and the antibody-bound tracer (AbAg*) is performed in a column with immobilized Protein G. The antigen-antibody complexes are retained in the column due to the high affinity of the Protein G for the antibody. The activity of the eluted enzyme label is then amperometrically detected. The immunoassay was optimised relative to conditions for antibody-antigen incubation (pH, incubation time, ionic strength, temperature) and enzymatic label detection. This method showed a dynamic concentration range between 20 and 500 ppt AFM1, a low detection limit (11 ppt), good reproducibility (RSD < 8%) and a high throughput (six samples per hour in triplicate). Different milk samples were analysed and the results were in good agreement with those obtained by HPLC using the AOAC 2000.08 method. © 2004 Elsevier B.V. All rights reserved

Traditional methods of monitoring health changes in animals are based entirely on the human senses. However, in modern dairy production systems humans are rarely present, this is particularly the case with the introduction of robotic milking. In these systems all the functions of milking are automated and cows visit at times of their own choosing. Systems of automatic health monitoring are therefore a priority for research to ensure that the health and reproductive status of the animals can be assessed for management purposes. These systems must be automatic, work in ¢eld conditions without technical support and cost a few pence per analysis. The ¢rst task is to obtain representative biological samples automatically and non-invasively. As milk is £owing into the milking machine from the cow this can be achieved with ease, except that milk is non-homogeneous with a changing lipid fraction during milking. Lipid soluble components such as progesterone and vitamin A are a¡ected by this change and a model has to be established to determine thresholds at di¡erent times during milking. Our main interests in dairy cows are in predicting ovulation, detecting metabolic imbalance and detecting preclinical mastitis in£am-matory response. Our team is developing a fully automated ovulation prediction system based on the screen-printed carbon electrode biosensor for progesterone demonstrated by Pemberton et al. (1998). In recent experiments the automated system was able to detect concentrations of progesterone between 2 and 30 ng/ml in stored milk samples (r 2 = 0.96). The results of ¢eld tests are presented showing a good correlation between ELISA and the biosensor (r 2 = 0.91) on samples of fresh milk. The results of the recent ¢eld tests show the ability of the biosensor to characterise ovulation cycles of cows and to detect pregnancy. We have identi¢ed a major lack of other biological models to detect disease with on-line sensors. Our next objective is to create an integrated system for biological research with sensor systems for urea, ketones, lipids and enzymes in milk. This will allow the development of diagnostic models based on analysing numerical sensor-derived data rather than human visual observations for signs of ill health in dairy cows.

Novel, reproducible, and accurate LC/MS/MS methods were developed for the determination of the urinary content of O(6)CMG and O(6)CMdG, and of the possible formation of O(6)MeG and O(6)MedG by decarboxylation. Clinical samples from volunteers on different diets were analysed. Further studies are required to discover a link between the presence of these biomarkers in urine and red meat consumption.

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Heparanase is an enzyme involved in extracellular matrix remodelling and heparan sulphate proteoglycan catabolism. It is secreted by metastatic tumour cells, allowing them to penetrate the endothelial cell layer and basement membrane to invade target organs. The release of growth factors at the site of cleaved heparan sulphate chains further enhance the potential of the tumour by encouraging the process of angiogenesis. This leads to increased survival and further proliferation of the tumour. Aptamers are single or double stranded oligonucleotides that recognise specific small molecules, peptides, proteins, or even cells or tissues and have shown great potential over the years as diagnostic and therapeutic agents in anticancer treatment. For the first time, single stranded DNA aptamers were successfully generated against the active heterodimer form of heparanase using a modified SELEX protocol, and eluted based on increasing affinity for the target. Sandwich ELISA assays showed recognition of heparanase by the aptamers at a site distinct from that of a polyclonal HPSE1 antibody. The binding affinities of aptamer to immobilised enzyme were high (7×10 7 to 8×10 7 M −1 ) as measured by fluorescence spectroscopy. Immunohistochemistry and immunofluorescence studies demonstrated that the aptamers were able to recognise heparanase with staining comparable or in some cases superior to that of the HPSE1 antibody control. Finally, matrigel assay demonstrated that aptamers were able to inhibit heparanase. This study provides clear proof of principle concept that nucleic acid aptamers can be generated against heparanase. These reagents may serve as useful tools to explore the functional role of the enzyme and in the future development of diagnostic assays or therapeutic reagents.