An universal biolabel, fluorescein diacetate (FDA) covalently conjugated with NeutrAvidin, for ultrasensitive detection has been developed. The FDA nanoparticles provide the highest possible packing density, and thus achieve an exceptional high signal generating molecules to analyte (protein or DNA) ratio. The use of FDA nanoparticles as a label for signal amplification has been demonstrated in HPV DNA detection....[ Read more ]

An universal biolabel, fluorescein diacetate (FDA) covalently conjugated with NeutrAvidin, for ultrasensitive detection has been developed. The FDA nanoparticles provide the highest possible packing density, and thus achieve an exceptional high signal generating molecules to analyte (protein or DNA) ratio. The use of FDA nanoparticles as a label for signal amplification has been demonstrated in HPV DNA detection.

Fabrication of FDA nanoparticle down to 120 nm has been achieved by using ultrasonication. A narrow size distribution ranged from 114 nm to 126 nm has been obtained. Yield of FDA nanoparticle has found to be proportional to the intensity of the ultrasonication but inversely proportional to viscosity of the solution. The size of the FDA nanoparticle depends on concentration of FDA, the percentage of surfactant and the intensity of ultrasonication. Functionalized FDA nanoparticle with amino groups has been successfully fabricated.

A stable universal NeutrAvidin-FDA conjugate with low non-specific binding has been achieved. The covalent NeutrAvidin-FDA conjugate showed a higher sensitivity than the physical NeutrAvidin-FDA conjugate. There was less than 20% in signal for the assay using the covalent conjugate after storage at 37°C for 30 days.

The application of the universal NeutrAvidin-FDA conjugate in DNA assay for detection of high risk genotype HPV 16 has been successfully demonstrated. Optimal conditions for coupling DNA capture probe to the plate and hybridization between DNA capture probe, HPV DNA and universal biotinylated probe are essential for efficient HPV DNA hybridization. Optimal size of FDA nanoparticle as a label for sandwich-type HPV DNA assay has been optimized. FDA cluster, composed of NeutrAvidin-FDA and Biotin-BSA-FDA conjugates, as a biolabel in sandwich-type HPV DNA assay, offer a great potential in enhancing assay sensitivity when compared with NeutrAvidin-HRP and NeutrAvidin-FDA as biolabels. The in-house sandwich-type HPV DNA assay could detect down to 1000 DNA copies.