Paper-based flow sensor for detection of chymotrypsin and its inhibitors via viscosity change of gelatin

Chymotrypsin (CHT) is an important proteolytic enzyme that can quickly decompose denatured proteins. Herein, we reported a new, simple, rapid method for the detection of CHT and its inhibitors by a paper-based flow sensor for the first time. The principle of the sensor is the interconversion of solidification and dissolution of gelatin. Generally, the 3D structure of gelatin can be enzymatically hydrolyzed by CHT releasing internal water, leading to a gradual increase in the water mark coverage area. Conversely, water cannot be released and the water mark coverage area is 0 in the absence of CHT, which is attributed to the water being trapped in the gelatin hydrogel. The limit of detection of CHT is determined to be 0.8 U/mL. In addition, the detection of CHT in milk, screening of CHT inhibitors and determination of Bowman-Birk inhibitors in soymilk were successfully achieved. Interestingly, the sensor only requires gelatin hydrogel, pH indicator strips, and PVC plates. In summary, a rapid, high-throughput, low-cost strategy for the detection of CHT and screening of CHT inhibitors was developed. Moreover, this method also allows the detection of CHT in non-transparent samples such as milk and soymilk. Paper-based flow sensors are highly promising for applications in food quality control, new medicine discovery and disease diagnosis, which also have a high potential for commercialization.