Optically encoded nanoparticles for detecting single biomolecules and viruses: Rapid analysis of two-color colocalization data by high-speed computing

Nanometer-sized particles such as luminescent quantum dots (QDs) and energy-transfer nanoparticles have unique optical, electronic, and structural properties (e.g., signal brightness, photostability, and multicolor light emission) that are not available from traditional organic dyes and fluorescent proteins. Here we report the use of color-coded nanoparticles and dual-color fluorescence correlation for real-time detection of single native biomolecules and viruses in a microfluidic channel. Using green and red nanoparticles to simultaneously recognize two binding sites on a single target, we show that individual molecules of genes, proteins, and intact viruses can be detected and identified in complex mixtures without target amplification or probe/target separation. When combined with high-speed computing such as image segmentation and parallel computing, these nanoparticle probes raise new opportunities in molecular diagnostics, bioterrorism agent detection, and intracellular single-molecule imaging.