Protocol for Model-Based Response-Corrected Fluorescence Quantification with Low-Cost Spectral Sensors

Fluorescent reporters are a key element of synthetic biology, as they enable rapid and noninvasive evaluation of gene expression in engineered constructs. Due to the technical and cost limitations of existing commercial platforms, several open-source bioreactor designs have been developed that allow the simultaneous control of growth, gene expression, and induction conditions. To fully exploit these platforms, robust calibration strategies are required to define their dynamic range and to quantify the limitations imposed by compact, low-cost optical designs. The OSRAM AS7341 spectral sensor is employed in mini-bioreactor platforms such as Chi.Bio and Pioreactor. In this chapter, we present a calibration framework for the AS7341 to enable quantitative measurement of green fluorescence. Using yeast cells and fluorescein, a fluorophore with spectral properties similar to those of green fluorescent protein, we systematically characterize the sensor response across controlled fluorophore and biomass concentrations. To facilitate these measurements, we adapted a 3D-printed bioreactor holder to integrate the sensor in a configuration suitable for fluorescence measurements and generated calibration datasets spanning relevant operating conditions. From these data, we developed a model of the sensor response, identified the concentration range over which measurements are reliable, and proposed an approach to improve fluorophore quantification in low-cost systems. A calibration protocol is provided together with MATLAB code, 3D design files, and component specifications to ensure reproducibility and to facilitate application of the framework to future bioreactor designs.