Kinetics of Plant‐derived Heteropolysaccharide Bioabsorption Characterized by Fluorescence‐based Microfluidics System

Plant-derived structural polymer, pectin, is the "glue" between plant cells, a biomaterial with promising performance as a sealant on visceral organs. However, the nature of bioabsorption and biodurability of pectin remains unclear. Here, we investigate the feasibility of a quantitative microfluidic perfusion assay based on photochemical quenching, to define the kinetics of pectin bioabsorption. We hypothesize that fluorescent tracers embedded into pectin biopolymers will reliably reflect predictable kinetics patterns of bioabsorption including predictable trends when modifying the chemical species used in fluorescence quenching experiments. A fluorescent tracer, Fluorescein Sodium, was embedded into high methoxyl citrus pectin. Dried Fluorescein pectin films were placed into 24-well plates, to monitor degradation via parallel microfluidics perfusion. This perfusion was performed via micro-perfused exogenous 0.4% Trypan Blue, an anionic hydrophilic azo dye, which was delivered into porous matrix interfacing on pectin in each well. To assess biodurability, the fluorescence of perfused pectin under quenching by dye was monitored over time using a multi-well plate fluorescence reader (Cytofluor 4000, PerSeptive Biosystems). Our data show that consistent trends in fluorescence result during quenching experiments. When un-perfused, Fluorescein-embedded pectin exhibited statistically insignificant change in quantum yield over 1 week (p=0.457), suggesting that results were not impacted by substantial photobleaching over the course of experimentation. To minimize interfacial flow artifact, 6µm porous matrix provided an effective interface for observable perfusion of azo dye into pectin. During micro-perfusion, serial measurements of fluorescence in Fluorescein pectin demonstrated exponential decline in fluorescence intensity. This quenching of fluorescent signal from Fluorescein correlated with the progressive bioabsorption of the pectin polymer. Absorption was feasibly modulated by adjusting dye volumes used in perfusion. In this in vitro assay, plant-derived pectin exhibits predictable decay characteristics and bioabsorption kinetics when incorporated with fluorescent tracers. These tracers not only provide a quantitative measure of biodurability, but also an opportunity for guided biochemical tuning of the complex biomaterial, to improve its performance as a potential therapeutic sealant.