Advances in nanotechnology have significant impacts in the field of biodiagnostics. Highly sensitive methods are urgently needed for measuring tiny amount of DNA/protein for forensics, early disease detection, etc. A novel signal amplification technology based on fluorescein diacetate (FDA) nanocrystal as the fluorescent label which contains 2.6 × 10⁶ fluorescein molecules was developed with a minimum self-quenching problem. ⁴...[ Read more ]

Advances in nanotechnology have significant impacts in the field of biodiagnostics. Highly sensitive methods are urgently needed for measuring tiny amount of DNA/protein for forensics, early disease detection, etc. A novel signal amplification technology based on fluorescein diacetate (FDA) nanocrystal as the fluorescent label which contains 2.6 × 10⁶ fluorescein molecules was developed with a minimum self-quenching problem.

This thesis presents applications of FDA nanocrystals for the sensitive detection of biomolecules. The FDA nanocrystal was first encapsulated by a amine modified polymer [DSPE-(2000)Amine] to provide an interface for the coupling of antibodies. A high molar ratio of fluorochrome to biomolecules (2.8 × 10⁴) was achieved for the nanocrystal biolabel system.

The applicability of FDA-antibody conjugates to be served as a label in immunoassay was demonstrated for detection of immunoglobin in the solid-phase sandwich fluorescence immunoassay and a newly developed fluorescence ‘barcode-style’ lateral-flow test. The utilization of FDA nanocrystals as a label in solid-phase sandwich fluorescence immunoassay offers a highly sensitive detection. The limit of detection was lowered by a factor of 5 to 28 and sensitivity was 400 to 2700-fold higher compared with state of the art immunoassay using direct fluorescent labelled antibodies. The novel fluorescence ‘barcode-style’ lateral-flow test offers a semi-quantitative measurement in a short time without the usage of expensive reading instruments. FDA-nanocrystal conjugates were further applied in real clinical samples detection including C-reactive protein (CRP) and pathogen deoxyribonucleic acid - Human papillomavirus (HPV).

Different strategies and techniques were also described to further optimize the FDA nanocrystals labeling system to fully explore the potential signal amplification ability to achieve a much higher sensitivity for the detection of biomolecules.