Rapid Determination of Ligand Density on DNA-Functionalized Nanoparticles

The accurate determination of ligand density on DNA-functionalized nanoparticles (DNPs) is critical for biosensing and therapeutic applications, as DNA density directly affects target binding, cellular uptake, and therapeutic efficacy. Traditional methods involve centrifugation to separate unbound DNA from DNPs, followed by DNA quantification to calculate the number of strands per particle. However, this approach is ineffective for centrifugation-resistant DNPs. 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 various DNPs, including those with metallic or protein-based cores and various DNA lengths, sequences, and densities. This approach exploits the dif-ferent 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 stand-ard laboratory equipment—such as a benchtop centrifuge, a shaker, and either a UV-Vis spectrophotome-ter or a commonly available plate reader—this method provides a fast and reliable alternative to conven-tional approaches, delivering results within 5 minutes. Its versatility and broad applicability across diverse DNP core materials and sizes make it a valuable tool for DNA quantification in a wide range of nanoparti-cle-based platforms.