Competitive protection of aptamer-functionalized gold nanoparticles by controlling the DNA assembly

Functionalization and control of nanoparticle surface are critical to the desirable application of nanoparticles. We describe a DNA assembly method that dynamically protects aptamer-functionalized gold nanoparticles (apt-AuNP) from nondesirable binding to interfering molecules. Competitive protection of the apt-AuNP was achieved by using a carefully designed DNA that served two primary functions: reducing cross-reaction by competitive hybridization to the aptamer and overcoming nonspecific adsorption by restricted access to the AuNP surface. Tuning the length and sequence of the hybridization and the overhang components of the protection DNA allowed for control of the selectivity of apt-AuNP probes for the target protein. Alternating application of the protection DNA containing a polyA and polyT overhang enabled layer-by-layer formation of the supermolecular assembly. This directed assembly of apt-AuNP resulted in enhancement of signal for visual detection of minute amounts of proteins. This method was successfully demonstrated for Western blot analysis of thrombin, with an improved detection limit of 6 ng. This study demonstrates a unique dynamic protection strategy for achieving high specificity and sensitivity in the detection of protein.