Riboflavin 5 monophosphate sodium salt hydrate

The addition of poly(ethylene oxide) (PEO, M_v ~900,000, Sigma-Aldrich) to silk solutions generated a viscosity and surface tension suitable for electrospinning. The base solution was made by mixing the 5 wt% PEO solution and prepared 8 wt% silk solution at a ratio of 1:1. Then, 10 mM of active dye was mixed with the base solution. In our experiments, 4 types of dye, including stilbene (Stilbene 420, Exciton), sodium fluorescein (Fluorescein sodium salt, Sigma-Aldrich), riboflavin (Riboflavin 5′-monophosphate sodium salt hydrate, Sigma-Aldrich), and Rhodamine B (Rhodamine B, Sigma-Aldrich), were used. The prepared silk bio-ink was injected in the syringe and mounted on the electrospinning machine. An aluminum foil was used as the collection screen. The distance between the tip and the collector was 20 cm, and flow rate of the bio-ink was set to 10 μL/min. The 10 kV of electric potential was applied between 21 G nozzle tip and collector during electrospinning time between 30 minutes and 2 hours. Finally, the generated FSNs had a uniform diameter of 300–350 nm.

The degree to which the collagen matrix was crosslinked was altered through riboflavin mediated photoactivation and crosslinking [Fig. 3(b)].³⁸ (link)^,39 (link) Riboflavin 5′-monophosphate sodium salt hydrate (Sigma Aldrich, St. Louis, Missouri, United States) at concentrations of 0.5 or 1 mM was added to the collagen mixture followed by 60 s of $150\mathrm{mW}/{\mathrm{cm}}^2$ UV light exposure before self-assembly. Increased crosslinking was confirmed by observing an increased linear modulus during quasi-static uniaxial tensile testing to failure in crosslinked gels compared with controls [Fig. 3(e)].

The FMN concentration was determined by fluorescence spectroscopy. Briefly, 5 mg of Riboflavin 5′-monophosphate sodium salt hydrate (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were dissolved in either 0.9% sodium chloride or Celsior. Thereafter, these solutions were serially diluted to obtain different standard FMN solutions with concentration ranging from 31.25 to 1000.00 ng/mL (Supplementary Figure S2). Effluent samples were then dispensed in triplicate in black microplates, together with the standard samples. Fluorescence readings were performed using a multi-mode microplate reader (Synergy HTX, Biotek U.S, Winooski, VT, USA). A monochrome light with excitation wavelength of 460/40 nm was used, while fluorescence emission was revealed with 100% gain at 528/20 nm. Finally, the average fluorescence readings of effluent samples were plotted against the appropriate standard curve to calculate sample FMN concentration.