Lab-on-a-chip for enzyme activity monitoring in industrial solid-state fermentation processes compatible with R2R fabrication

We present a disposable lab-on-a-chip (LoC) for colorimetric enzyme activity monitoring in solid-state fermentation (SSF) processes. The microfluidic chip structures are fabricated <i>via</i> roll-to-roll (R2R) extrusion coating, which reduces costs and enhances efficiency. The LoC operates on capillary-driven flow microfluidics in which a droplet added at the inlet self-fills the chip by capillary action, reaching the reaction chamber. A capillary pump then removes excess liquid, isolating the detection area where the enzymatic reaction takes place. The selection of the target enzymes (α-amylase and cellulase) was made based on their relevance to the industrial biodetergent production processes. For LoC compatibility, enzymatic assays must deliver a strong signal and must be user-friendly. One-step colorimetric assays meet these criteria by releasing a dye from a substrate through enzymatic action. To make the chip easier to handle, the enzymatic substrates were integrated into its reaction chamber in dryed form. For this purpose, two strategies for integration were tested: drop-casting followed by freeze-drying, and piezoelectric deposition with air-drying. Additionally, storage conditions were optimized to enhance shelf-life and reagent stability. To measure enzymatic activity, a pocket-sized colorimetric reader was developed and adapted to the LoC geometry while an Android app was created to enable smartphone-based control of the reader. Furthermore, validation with commercial enzymes established the limit of detection (LoD), and subsequent tests with SSF samples from an industrial plant confirmed the functionality of the system. The enzymatic activity measurements are completed in under 10 minutes, revealing increasing enzymatic activity as fermentation progresses. In conclusion, the LoC provides a quick and cost-effective solution for detecting α-amylase and cellulase in samples derived from SSF processes.