Enzymatic and Enzyme Inhibitory Activity on a Paper-Based Lateral Flow Device

In this work it was demonstrated for the first time that Alkaline Phosphatase (ALP) enzymes spotted and dried onto a nitrocellulose membrane remained active. This was accomplished by passing a fixed volume of the BCIP/NBT substrate solution over the immobilized enzymes and measuring the resulting color density at the spot where the enzymes were spotted. A dose response curve was produced of the concentration of the enzymes spotted on the test area versus the color density measured at the spot after the substrate had flowed past it indicating that the assay produces quantitative as well as qualitative results. This experiment was conducted in paper based lateral flow devices. In a second experiment, prior to the flowing of the substrate over the test spot where ALP enzymes were immobilized, a solution of Sodium Orthovanadate (Na3VO4) was introduced to the system. Sodium Orthovanadate (Na3VO4) is an inhibitor to ALP. By varying the concentration of Na3VO4 in the solution that flowed past the enzymes, a certain number of the enzymes were deactivated. When subsequently the BCIP/NBT substrate solution was flowed over the enzymes, the intensity of the color produced depended on the concentration of Na3VO4. It was then possible to generate again a dose response curve, of the concentration of the Na3VO4 inhibitor in the solution versus the color density at the spot where the enzymes were dried. This is a novel result in that it is again the first time where an inhibitory activity assay was run on a paper based lateral flow device. This experiment also demonstrated that the bond between the Na3VO4 molecules and the immobilized ALP molecules is strong enough so that at least not all of the inhibitor molecules were washed away by the substrate solution