Analysis of Non‐Covalent Bioconjugation of Colloidal Nanoparticles by Means of Atomic Force Microscopy and Data Clustering

AFM topography data of non‐covalently assembled biomolecule–nanoparticle hybrids is examined by statistical cluster analysis (see figure). As exemplified for fluorescent‐protein‐modified quantum dots and DNA/protein‐conjugated gold nanoparticles, this analytical approach provides insights into the formation of bioconjugate populations. Non‐covalent modification of nanoparticles with biomolecules is frequently applied in the generation of hybrid nanomaterials. Herein, we report on the analysis of non‐covalently assembled hybrid nanoparticles on surfaces, using AFM measurements and examination of the resulting topography data by means of statistical cluster analysis. Two different model systems are investigated. On the one hand, we apply the combined AFM–cluster analysis to CdSe/ZnS core/shell quantum dots, bioconjugated with varying amounts of the hexahistidine‐tagged enhanced yellow fluorescent protein (EYFP). On the other hand, gold nanoparticles modified with single‐stranded DNA oligonucleotides were further functionalized by Watson–Crick base pairing, using complementary DNA oligonucleotides or covalent conjugates of DNA and streptavidin or EYFP. The results clearly indicate that cluster analysis of AFM data allows for the identification of sub‐populations in complex reaction mixtures. Therefore, this analytical approach is useful to elucidate bioconjugate species, formed in the course of non‐covalent synthesis of nanoparticle–biomolecule hybrid systems. AFM topography data of non‐covalently assembled biomolecule–nanoparticle hybrids is examined by statistical cluster analysis (see figure). As exemplified for fluorescent‐protein‐modified quantum dots and DNA/protein‐conjugated gold nanoparticles, this analytical approach provides insights into the formation of bioconjugate populations.