Microfluidic Fabrication and Analysis of Biocompatible, Monodisperse DNA-Hydrogels with Tunable Swelling and Dissolution Kinetics

Stimulus-responsive DNA-hydrogels with swelling capabilities are a promising class of materials for biomedical applications such as drug delivery and biosensing. Designing these systems remains challenging because fabrication methods must be simultaneously biocompatible and conserve scarce DNA materials, even at the microscale. Moreover, stimulus-induced swelling must be precisely controlled and shown to drive measurable changes in molecular properties. We present a biocompatible fabrication and characterization method for micron-scale DNA-hydrogels with tunable isotropic swelling and dissolving properties. We first developed a fabrication method demonstrating that both the hydrogel composition and the fabrication process itself are biocompatible, while also minimizing the consumption of valuable DNA reagents. We then demonstrated modular control over isotropic swelling in micron-scale DNA microgels, achieving up to a two-fold size increase with tunable swelling through defined design parameters. We further established a quantitative workflow to measure structural changes of spherical, swollen and unswelled microgels leveraging the diffusive properties of a DNA-binding dye. Finally, we demonstrate tunable dissolving of microgels and quantitatively reveal various experimental factors that influence dissolution rates beyond what is traditionally considered in microgel experiments. Together, these advances establish a biocompatible platform for the fabrication and analysis of stimulus-responsive DNA micro-hydrogels, providing a foundation for their future use in drug delivery, biosensing, and related biomedical technologies.

💡 Research Summary

This paper presents a comprehensive, biocompatible microfluidic platform for producing and characterizing micron‑scale DNA‑hydrogel particles (µSDs) with tunable swelling and dissolution behavior. The authors first designed a three‑inlet flow‑focusing droplet generator (channel height ~60 µm, width 40 µm, length 25 mm) that merges two aqueous pre‑gel streams (pre‑gel 1 and pre‑gel 2) with a fluorinated oil phase (HFE7500 + 2 % surfactant). Surface treatment with Rain‑X and oil pre‑wetting rendered the channel walls highly hydrophobic, preventing aqueous adhesion and ensuring stable droplet formation at flow rates of 30 µL h⁻¹ (pre‑gel 1), 8 µL h⁻¹ (pre‑gel 2) and 425 µL h⁻¹ (oil). The resulting droplets are monodisperse (average diameter ~15 µm, CV < 5 %).

Pre‑gel formulations contain 1.2× PBS, 4 % acrylamide, 5’‑acrydite‑modified oligonucleotides (S1‑C, S1‑C′) and a Cy3‑labeled poly‑T strand. Polymerization is initiated with TEMED and ammonium persulfate, followed by a brief vacuum degassing step to reduce viscosity. After mixing equal volumes of the two pre‑gels, DNA cross‑linking proceeds at room temperature, yielding a network that incorporates programmable “hairpin” sequences (H1, H2) and their terminators. These hairpins act as stimulus‑responsive switches: upon exposure to a trigger (e.g., specific ion concentration or pH), they open, extending the DNA strands and expanding the mesh, which translates into isotropic swelling of the particle.

Swelling is quantified using the intercalating dye YOYO‑1. Because YOYO‑1 fluorescence diminishes as the dye diffuses out of the hydrogel, the authors infer the diffusion coefficient of free YOYO‑1 from the decay of bound fluorescence, providing a non‑invasive, high‑throughput proxy for mesh size changes without single‑molecule tracking. By varying hairpin design and trigger conditions, they achieve up to a two‑fold increase in particle volume, surpassing previous micron‑scale reports.

Dissolution kinetics are controlled through a specially designed “dissolver strand” (DS). By adjusting DS concentration from 0 to 300 µM, the authors modulate the rate at which the DNA network is enzymatically cleaved, effectively tuning particle degradation. Importantly, they compare dissolution in confined (microfluidic channel) versus non‑confined (bulk tube) environments, demonstrating that diffusion limitations in the surrounding fluid dramatically affect degradation rates—a factor often overlooked in prior studies.

Biocompatibility is validated by encapsulating K562‑F cells (5 × 10⁶ cells mL⁻¹) within pre‑gel 1 before droplet formation. After 24 h culture, live/dead assays show >95 % cell viability, confirming that the fabrication process (no UV exposure, low‑temperature polymerization) and the final hydrogel composition are non‑toxic.

Overall, the work delivers four major advances: (1) a low‑loss, fully biocompatible microfluidic workflow for producing monodisperse DNA‑hydrogel microgels; (2) a modular strategy for precise, isotropic swelling using programmable DNA hairpins; (3) a diffusion‑based, dye‑intercalation assay for rapid structural characterization of swollen versus unswollen particles; and (4) a tunable dissolution system that accounts for external diffusion constraints. These capabilities position µSDs as ready‑to‑use building blocks for drug delivery, biosensing, and single‑cell studies, bridging the gap between DNA‑nanotechnology and practical biomedical applications.