Fluorescein sodium salt

The following reagents were used: 2,20-azobis-2-amidinopropane (AAPH) and fluorescein sodium salt were purchased by Merck (Merck, Germany). Organic solvent for analysis from Merck. Ultra-pure water was prepared by using a PW-Ultra Water System (Heal Force, Shanghai, China).

At the end of each experiment, we created a “mask” image to characterize the porous structure. To this end, we saturated the porous medium with a florescent dye (fluorescein sodium salt, from Merck, visible through a GFP filter cube) and recorded a large image of the entire structure. After thresholding, binarizing the mask image (pore-space: 1; solid-space: 0), we geometrically discretized the two pore classes, TPs and DEPs, using the maximum inscribed circle method described in^{23 (link)}. From the binary image we obtained then the skeleton, a 1-pixel width representation of the pore space (Supplementary Fig. S7a). We assigned a segregation index (ζ) to the pore-regions based on the number of neighboring grains. Hence, a segregation index (ζ) of 1 has distinguished the dead-end pores from the transmitting pores with ζ > 1. This characterization allowed to generate a “mapped mask”, where grains have been attributed a pixel value of 0, dead-end pores a pixel value of 1, and transmitting pore a pixel value of 2 (Fig. 1d, gray, red and cyan, respectively). The medium is quite homogeneous, such that the statistical distribution of λ is narrow and has a strong peak close to the mean pore-size λ_m = 0.04 mm (Supplementary Fig. S7b).

The Parastacus pugnax crayfish exoskeletons were provided by “Cooperativa Agricola Cacike Juan Currimil de Lolocura Ltd.a.” (Carahue, La Araucanía, Chile). Commercial chitosan was purchased from a local supplier (Citrex, La Cruz, Chile) and used as a control in this study. NaOH, HCl, ethanol, NaCl, ascorbic acid and acetic acid were acquired from Sigma-Aldrich (São Paulo, Brazil), while trichloroacetic acid, Trolox reagent, fluorescein sodium salt, Bradford reagent, Methyl orange reagent were purchased from Merck (Darmstadt, Germany). Na-phosphate buffer was purchased from J.T. Baker (Deventer, The Netherlands); AAPH from Cayman Chemical (Ann Arbor, MI, USA); DPPH reagent from Calbiochem (Darmstadt, Germany); Lactic acid from Sabores (Santiago, Chile); L-ascorbic acid from Mallinckrodt (Phillipsburg, NJ, USA); Na-phosphate buffer from J T Baker (Deventer, The Netherlands); and technical Acetone from HES (Santiago, Chile).

Zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O ≥ 99%,Acros) and citric acid anhydrous (C₆H₈O ≥ 99.5%, Fisher scientific), Dyes: Rhodamine B ≥ 95% (HPLC), Merck) and Fluorescein sodium salt ≥ 97.5% zHPLC),Merck).The solvent used is deionized (DI) Milli-Q water. The UV–Vis absorption spectra of the prepared samples was measured using a double beam spectrophotometer (Cary 5000 UV–Vis-NIR, Agilent Technologies). The FTIR spectra were collected using a FTIR spectrometer (Vertex 70, Bruker, Germany). XRD of the as-prepared Zinc-Citrate precursor and ZnO samples were characterized using a Malvern Panalytical Empyrean 3 diffractometer. The morphology and particle size of the samples were determined by FESEM, (Quattro S, Thermo Scientific).

The performance of the fabricated MCD was validated using epifluorescence microscopy (Nikon Ti-E) with an aqueous fluorescent dye (fluorescein sodium salt, Sigma) in various concentrations (described below). Images of the dye distribution were captured at the midplane of the channels with a sCMOS camera (Zyla 5.5; Andor Technology). Fluorescein was chosen due to its similar diffusion coefficient with the chemostimulant serine (Altindal et al., 2011 (link)). Minor deviations in the performance of the MCD from the original circuit design (Figure 1—figure supplement 1) are likely due to variations in the fabricated channel mold heights (Appendix 1). Such variations impact the hydraulic resistances (Oh et al., 2012 (link)) and symmetry of the cell solution (Figure 1—figure supplement 3).

Folin–Ciocalteu reagent solution (2 N), 1,1-diphenyl-2-picrylhydrazine (DPPH, 95%) (powder), Trolox (97%), 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH, 95%) (powder), and ethanol solution (99.5%) were purchased from Wako Chemicals Ltd. (Osaka, Japan). Fluorescein sodium salt (1 mg/mL in pure water) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Catechin (CTN) (>98%, powder) was purchased from Funakoshi Corporation (Tokyo, Japan). STC, DPPH, and hydrophilic oxygen radical absorbance capacity (H-ORAC) values of the bread samples were determined. Samples were extracted in 60% ethanol at 40 °C for 2 h while being subjected to shaking. The STC of the extracts was measured using the Folin–Ciocalteu method [9 (link)]. The STC was expressed as mg equivalent/100 g of dry matter using CTN as the standard (mg CTN eq/100 g DW). The antioxidant activity of the extracts was analyzed using the DPPH [22 (link)] and H-ORAC assays [23 (link)]. The DPPH and H-ORAC values were expressed in µmol Trolox equivalent/g of DW (µmol TE/g DW). For STC and all antioxidant activity assays, extraction was performed twice per treatment, and measurements were obtained in triplicate per extraction.