Blood, sweat, and tears: developing clinically relevant protein biosensors for integrated body fluid analysis.

Biosensors are being developed to provide rapid, quantitative, diagnostic information to clinicians in order to help guide patient treatment, without the need for centralised laboratory assays. The success of glucose monitoring is a key example of where technology innovation has met a clinical need at multiple levels ­ from the pathology laboratory all the way to the patient's home. However, few other biosensor devices are currently in routine use. Here we review the challenges and opportunities regarding the integration of biosensor techniques into body fluid sampling approaches, with emphasis on the point-of-care setting.

Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories.