Label-Free Quantification of DNA Loading on Centrifugation-Resistant Spherical Nucleic Acids

Abstract

The accurate determination of DNA loading on spherical nucleic acids (SNAs) is critical for biosensing and therapeutic applications, as DNA loading directly affects target binding, cellular uptake, and therapeutic efficacy. Traditional methods involve centrifugation to separate unbound DNA from SNAs, followed by DNA quantification to calculate the number of strands per particle. However, this approach is ineffective for centrifugation-resistant SNAs. Alternative methods, such as centrifugal filtration, require multiple washing steps, often resulting in sample loss, and can be labor- and time-intensive, taking several hours. Additionally, many existing techniques rely on expensive labels or toxic reagents. Here, we present a simple, rapid, and label-free method for determining DNA loading using diethylaminoethyl (DEAE)-functionalized beads. We demonstrate its effectiveness on centrifugation-resistant SNAs, including those with metallic or protein-based cores and various DNA lengths, sequences, and densities. This approach exploits the different binding strengths of free and nanoparticle-bound DNA to DEAE, allowing the selective elution of free DNA by adjusting the ionic strength. The eluted DNA is quantified to determine the unbound fraction, which is then used to calculate the number of bound DNA strands per nanoparticle. Requiring only standard laboratory equipment─such as a benchtop centrifuge, a shaker, and either a UV–vis spectrophotometer or a commonly available plate reader─this method provides a fast and reliable alternative to conventional approaches, delivering results in under 15 min. Its versatility and broad applicability across diverse SNA core materials and sizes make it a valuable tool for DNA quantification in a wide range of nanoparticle-based platforms.