Characterizing the Incorporation of DNA into Single NIPAm Hydrogel Nanoparticles with Surface Plasmon Resonance Imaging Measurements

N-Isopropylacrylamide-based hydrogel nanoparticles (HNPs) that incorporate 30 mer single-stranded DNA (ssDNA) oligonucleotides were synthesized and characterized with single-nanoparticle surface plasmon resonance imaging (SPRI) microscopy, dynamic light scattering, transmission electron microscopy, and fluorescence measurements. The synthesized HNPs had an averaged diameter of 230 nm and exhibited a large (5–10×) increase in the average single-nanoparticle SPRI refractive index (Δ% RNP), as compared with HNPs without DNA. A combination of SPRI and fluorescence measurements was used to measure the uptake of approximately 20 000 complementary ssDNA into each HNP, resulting in a Langmuir isotherm adsorption coefficient of 4.89 × 108 M–1. Single-nanoparticle SPRI measurements also showed that approximately 35% of the incorporated ssDNA was accessible for both hybridization uptake and enzymatic hydrolysis using exonuclease I. We attribute the presence of an inactive ssDNA population in the nanoparticle to a combination of acrylamide Michael addition reactions with adenine, cytosine, and guanine nucleotides, as well as the possible formation of self-complementary secondary structures in the polymerized ssDNA