A Nanoplasmonic‐Fluorescent Ruler for Detection of Site‐Specific Protein Binding to Composite DNA of Multiple Sites

Abstract

A gold or silver nanoparticles (AuNPs and AgNPs)‐supported fluorescent intercalator displacement (FID) assay, termed nanoplasmonic‐fluorescent ruler, for site‐specific detection of protein binding to composite DNA of multiple sites is constructed. A 20 nm, 100 nm AuNPs, or 80 nm AgNPs is introduced to one end of double‐stranded DNA (dsDNA). The dsDNA–AuNPs conjugates are saturated with a DNA intercalator, thiazole orange (TO). The distance‐determined fluorescent quenching and enhancement by mNPs give each intercalated TO its own contribution to the overall fluorescent ­intensity, according to their distance to the particles. Protein binding at a specific DNA site displaces the respective TO molecules, causing relative fluorescent intensity drop that is of a function of distance. Proteins Sp1 and ERα and their composite DNA, containing two identical Sp1 sites and one ERα site, are studied. With the smaller particle (i.e., 20 nm AuNPs) and larger particles (i.e., 100 nm AuNPs and 80 nm AgNPs) as quenching and enhancement ­profilers, respectively, it is possible to detect preferential binding of Sp1 to one of its sites, identify protein bound, and determine the cooperative binding of Sp1 and ERα where conventional FID fails unless a comprehensive set of DNA with respective protein sites being mutated is involved.

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A centrifugation-assisted visual detection of SNP in circulating tumor DNA using gold nanoparticles coupled with isothermal amplification

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