RESUMO
Microfluidic devices are typically fabricated in an expensive, multistep process (e.g., photolithography, etching, and bonding). Additive manufacturing (AM) has emerged as a revolutionary technology for simple and inexpensive fabrication of monolithic structures-enabling microfluidic designs that are challenging, if not impossible, to make with existing fabrication techniques. Here, we introduce volumetric stereolithography (vSLA), an AM method in which polymerization is constrained to specific heights within a resin vat, allowing layer-by-layer fabrication without a moving platform. vSLA uses an existing dual-wavelength chemistry that polymerizes under blue light (λ = 458 nm) and inhibits polymerization under UV light (λ = 365 nm). We apply vSLA to fabricate microfluidic channels with different spatial and vertical geometries in less than 10 min. Channel heights ranged from 400 µm to 1 mm and could be controlled with an optical dose, which is a function of blue and UV light intensities and exposure time. Oxygen in the resin was found to significantly increase the amount of dose required for curing (i.e., polymerization to a gelled state), and we recommend that an inert vSLA system is used for rapid and reproducible microfluidic fabrication. Furthermore, we recommend polymerizing far beyond the gel point to form more rigid structures that are less susceptible to damage during post-processing, which can be done by simultaneously increasing the blue and UV light absorbance of the resin with light intensities. We believe that vSLA can simplify the fabrication of complex multilevel microfluidic devices, extending microfluidic innovation and availability to a broader community.
RESUMO
Phenotype-based screening of a fungal extract library yielded an active sample from a Penicillium sp. isolate that impaired zebrafish motility. Bioassay-guided purification led to the identification of 14 meroterpenoids including six new metabolites, arisugacins L-Q (4, 5, 8, and 12-14), seven known arisugacins (1-3, 6, 7, 9, and 10), and one known terreulactone (11). Their structures were determined using a combination of NMR and HRESIMS data, evidence secured from theoretical and experimental ECD spectra, and the modified Mosher's method. The purified compounds were tested in zebrafish embryos, as well as in vitro for cholinesterase inhibition activities. Compound 12 produced defects in myotome structure (metameric muscle, which is critical for locomotion) in vivo and showed the most potent and selective acetylcholinesterase inhibitory activity with an IC50 of 191 nM in vitro. The phenotype assay was also used to reveal bioactivities for several previously reported arisugacins, which had failed to show activity in prior cell-based and in vitro testing. This study demonstrates that utilization of the zebrafish phenotype assay is an effective approach for the identification of bioactive extracts, is compatible with the bioassay-guided compound purification strategies, and offers a valuable tool for probing complex natural product sources to detect bioactive small molecules with potential therapeutic or other commercial applications.
