We propose a method for measuring protease activity utilizing fluorescent charge-changing peptide substrates. The central mechanism of detection relies on the cleavage of a fluorescent peptide by trypsin. Upon cleavage, this peptide changes from a net -1 charge to a net +1 charge.

In the LFA, this positively charged product can be pushed through the paper using a buffer agent like Tris Borate (TB) buffer and can be captured by a negatively charged capture agent embedded in the paper. Our final design iteration features Poly(sodium 4-styrenesulfonate) (PSSA-Na) (6% WT) as the capturing agent. Due to its high molecular weight (~10^6 g/mol), PSSA-Na travels through the paper at a negligible speed during the relevant time scale of ~10 minutes. Thus, it forms a stationary capture zone.

The envisioned design of the LFA follows the demonstrated formats in literature closely. The current paper prototype will be sandwiched in a plastic casing that creates a stable and separated internal environment for the flow reactions to occur. The blood or plasma sample will be premixed with the charge changing substrate for a designated amount of time. The mixture of product, uncleaved substrate, and media (buffer/plasma/whole) will be applied at the proximal end of the paper (green) and wick through the cellulose paper. The distal end of the paper acts as an absorbent sink that draws the fluid media through the body of the paper, carrying the mixture over the capture zone. The PSSA-Na (blue) embedded within the paper captures the positive product and will emit a concentrated signal.