Microsyringe-assisted visual volume detection based on phase separation: the case of chymosin milk-clotting activity study

The constantly diverse demand scenarios for rapid on-site analysis have put forward high requirements for developing low-cost and user-friendly visual detection methods. Therefore, developing a visual detection method with simple operation and intuitive results has important practical value in the field of analysis and detection, but it is also challenging. In this work, we propose a microsyringe-assisted visual volume detection method based on phase separation, and apply it to analyze the milk-clotting activity of chymosin. Chymosin can cause phase separation of milk with whey in the mobile phase and curd in the gel state. The network structures of casein in curd can trap water molecules, resulting in separation of whey from curd gradually. Therefore, the analysis of chymosin milk-clotting activity can be realized according to the volume of whey measured using a portable microsyringe. This method shows a good linear correlation when the concentration of chymosin ranges from 1.02 U L⁻¹ to 1020 U L⁻¹ and the limit of detection of this method for chymosin is calculated to be 0.03 U mL⁻¹. This work successfully realizes the visual analysis of chymosin milk-clotting activity based on the enzyme-triggered phase separation. It also shows great promise to be applied in other phase separation-based detection systems with the advantages of high accuracy, great portability and user-friendliness.