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A highly sensitive fluorescent immunoassay based on avidin labeled nanocrystals

K-K Sin, C.P-Y Chan, T-H Pang, M. Seydack and R. Renneberg
The Hong Kong University of Science and Technology, HK

Keywords:
FDA, avidin, neutravidin, biotin, labeled avidin-biotin technique, immunoassay, fluorescent nanocrystals

Abstract:
Nanocrystals of the fluorogenic precursor fluorescein diacetate (FDA) were applied as labels to enhance assay sensitivity in our previous studies. Each FDA nanocrystal can be converted into ~2.6 x 106 fluorescein molecules, which is useful for improving sensitivity and limits of detection of immunoassays. NeutrAvidin was simply adsorbed on the surface of the FDA nanocrystals which was coated with distearoylglycerophosphoethanolamine (DSPE) modified with amino(poly(ethylene glycol))(PEG(2000)-Amine) as an interface for coupling biomolecules. It can be applied to detect different kinds of analytes which are captured by corresponding biotinylated biomolecules in different bioanalytical applications. The applicability of the NeutrAvidin-labeled nanocrystals was demonstrated in an immunoassay using the labeled avidin-biotin technique. Biotinylated antibody and FDA-labeled avidin were applied to the assay sequentially. The performance was compared with the traditional sandwich type assay for mouse immunoglobulin G detection. Following the immunoreaction, the nanocrystals were released by hydrolysis and dissolution instigated by adding a large volume of organic solvent/sodium hydroxide mixture. The limit of detection of 0.0256 g/L mouse immunoglobulin G was achieved, which was lowered by a factor of 2.5–21 compared with the immunoassay using commercial labeling systems, i.e. FITC and peroxidase. In addition, the sensitivity was 3.5–30-fold higher. This study shows that the fluorescent nanocrystals combined with the avidin-biotin technique can enhance the assay sensitivity and achieve a lower limit of detection without requiring long incubation times as in enzyme-based labels.

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