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Work Package 5

Uncertainties in Biotechnology Assays using fluorescent nanoparticles

The aim of this work package is to investigate the barriers to the use of fluorescent nanoparticles in biotechnology assays such as immunoassays. Immunoassays are frequently used in biotechnology, clinical and research laboratories to measure the concentrations of biological analytes, often at very low (nanomolar) concentrations. Compared to small-molecule fluorescent probes, fluorescent nanoparticles (such as quantum dots or polymeric nanoparticles) have many advantages including brightness and photostability. However, additional concerns and uncertainties arise as a result of phenomena such as blinking, photobrightening or annealing, oxidation, and problems with colloidal stability. This work package will act as a case study to investigate the magnitude of these problems and, by incorporating the results of the traceable optical measurements in WP3, to develop an understanding of the uncertainties involved.

Assay design

The aim of this task is to select assay designs to be developed within subsequent tasks. The assay design will consist of the analytes to be measured; the fluorescent particles to be used; the type of sample (e.g. buffer, culture medium, serum); the assay type (ELISA, lateral flow) and format (single analyte or multiplex). This will take into account advice from the project’s stakeholders and the availability of suitable standards and reagents.

Assay optimisation and validation

The aim of this task is to optimise the performance of the assay in buffer (with respect to both uncertainties and sensitivity) and to validate its performance with analytes in appropriate biological matrices such as serum. It will include work to minimise the influence of any artefacts or uncertainties due to the fluorescent nanoparticles identified in the preceding task.