Microscale hemoglobin assay for time-resolved kinetic analysis of nitric oxide release from electrospun hydrogels

Nitric oxide (NO) delivery from diazeniumdiolate donors is widely explored in biomaterial design for wound healing and regenerative medicine. However, accurate quantification of NO release remains challenging due to its gaseous nature, short half-life, and high reactivity. The hemoglobin assay provides a direct, stoichiometric method for NO detection but is typically limited in throughput and temporal resolution. Here, we adapt this assay to a microscale, multiwell spectrophotometric format to monitor NO release from electrospun methacrylated alginate hydrogels, with and without mesoporous silica nanoparticles.

The miniaturized assay enabled continuous, parallel monitoring of NO release over 24 h while reducing reagent consumption. Four diazeniumdiolate donors (commercial and in-house synthesized) were evaluated at 0.05 and 0.1 mg/ml, yielding cumulative NO concentrations of approximately 2-7 μM. Nonlinear exponential modeling (C(t) = A·(1 - e^(-kt)) revealed donor-specific release kinetics consistent with known diazeniumdiolate decomposition behavior. Unprotected donors exhibited rapid initial release (k ≈ 0.2-0.5 1/h), whereas sterically protected analogs showed slower, more sustained release (k < 0.1 1/h). Incorporation of mesoporous silica nanoparticles modulated release behavior in a donor-dependent manner. Statistical analysis identified donor chemistry as the primary determinant of release kinetics, while concentration acted mainly as a scaling factor.

This microscale hemoglobin assay provides a resource-efficient platform for parallel, time-resolved quantification of NO release from biomaterials. It enables comparison of donor chemistry and matrix effects under standardized conditions, facilitating the evaluation of NO-releasing systems for biomedical applications.