[4S03e] 08. Functional Molecules and Materials for Biological Systems - Part 6

5:10 PM - 5:30 PM JST(8:10 AM - 8:30 AM UTC)

[4FMBS-10] DNA-Mediated On-Membrane Assembly Strategy of Conjugated Polymer Nanoparticles for Sensitive Detection of Cell Surface Markers

Yuki Maeda¹, Noriko Nakamura¹, ○Seiichi Ohta¹ (1. The University of Tokyo (Japan))

Keywords:

Cell Surface Markers,Conjugated Polymer Nanoparticles,DNA Nanotechnology

Cell surface markers have been used as diagnostic targets for various diseases, with flow cytometry as the gold standard for detection. However, because of its limited sensitivity to fluorescent signals, detection of low-expression surface markers using flow cytometry remains challenging. To address this problem, the antibody modification with synthetic polymers copolymerized with fluorescent dyes have been investigated. However, flexible polymer backbones often induce close fluorophore proximity, leading to concentration-dependent quenching. Another approach is to use large fluorescent beads for antibody labeling. However, steric hindrance between the beads resulted in incomplete coverage of the markers. In this study, we investigated a DNA-mediated, on-membrane assembly of conjugated polymer nanoparticles (Pdots) that amplifies the fluorescence signal from cell surface markers for sensitive detection via flow cytometry. Single-stranded DNA (ssDNA)-conjugated antibodies are first bound to cell surface markers, from which ssDNA-modified Pdots are sequentially assembled using DNA hybridization. The use of DNA as a linker enables the distance-controlled assembly of Pdots to prevent fluorescence quenching, whereas their on-membrane sequential assembly allows amplification of the fluorescence signal without reducing the binding ability of antibodies. Thus, two rounds of Pdot assembly achieve 36-fold amplification of the fluorescence signal from CD19 on Nalm-6 cells, which is 125-fold brighter than that obtained using the conventional fluorescent dye-based method. Whereas the sequential assembly of Pdots achieved the successful fluorescence signal amplification, the requirement for multiple assembly steps poses challenges in terms of time and cost. Therefore, we next employed the hybridization chain reaction (HCR) to achieve simpler and efficient signal amplification. HCR is an enzyme-free, isothermal, one-pot reaction initiated by a short single-stranded DNA trigger that sequentially opens hairpin DNAs to generate long double-stranded DNA polymers. Two types of DNA scaffolds, linear and branch, were polymerized from antibodies bound to cell surface markers, followed by incorporation of DNA-modified Pdots via hybridization. The flow cytometry analysis confirmed the successful fluorescence signal amplification via HCR-guided Pdot assembly. Furthermore, the optimal Pdot size was found to depend on the DNA scaffold structure, reflecting a balance between higher individual particle brightness and reduced incorporation efficiency associated with increased particle size. Under the optimized conditions, detection of a low-abundance marker, CD63, in a basophil activation test (BAT)-mimicking assay was demonstrated, which is difficult to achieve using conventional fluorophore-modified antibodies. The proposed method is expected to contribute to the sensitive detection of low-expression cell surface markers for early and accurate diagnosis.