Optofluidic lab-on-chip for absorbance and fluorescence analysis in protein aggregation studies

Abstract

In recent years, kinetics of aggregation and liquid-liquid phase separation (LLPS) of certain proteins have been extensively studied due to their link to neurodegenerative diseases such as amyotrophic lateral sclerosis and Parkinson's disease. Optical techniques including absorbance and fluorescence spectroscopy are widely used for these investigations. However, most commercial devices suffer from high sample volume ( 100 μL/test) requirements. Optofluidic lab-on-chips (LOCs) provide accurate sampling and mixing in nanoliter scale sample volumes. State-of-the-art optofluidic chips have fabrication tolerances as low as the fiber core diameter, requiring monolithic fabrication and limiting the microfluidic design. We present the design and prototype fabrication of an optofluidic chip to monitor the absorbance and fluorescence of phase separating and aggregating protein samples. The design offers tolerances wide enough for assembly with a variety of microfluidic chips. The prototype LOC is compared with a commercial plate reader (BioTek Synergy MX) through internal validation of polystyrene beads (absorbance) and propidium iodide (fluorescence) dilution series. The LOC either matches or exceeds the plate reader in the limit of detection, linearity, and precision. Furthermore, LLPS and aggregation of G3BP1, a protein linked with neurodegenerative diseases, are demonstrated through absorbance and fluorescence (with thioflavin T dye), using the LOC and the commercial plate reader.