Silk-fibroin film as enzyme stabilizing material and optical signal transducer for developing alcohol oxidase-based μPAD methanol biosensor

Abstract

The commercial success of paper-based microfluidic biosensor devices with enzyme as recognition elements may be achieved greatly by improving the enzyme stability, averting the coffee-ring effect on the detection zone, and making it a stand-alone system. We have addressed these issues here by introducing silk-fibroin (SF) film for immobilizing enzymes and chromogenic reagent (ABTS), and wave-design microfluidic channels in the chromatographic paper for developing a hybrid microfluidic paper-based analytical device (μPAD), for methanol detection through peroxidase reaction. The activity of alcohol oxidase (AOx), used as a biorecognition element, could be wholly retained in a non-dissolvable SF film (pore size <3.5 μm) until 40 days of storage at room temperature (RT) by reducing oxygen permeability to the film. Similarly, ABTS in a dissolvable SF film was protected from air-oxidation even up to two months of storage at RT. The detection approach exploited purple color as a high contrast response signal generated from ABTS di-cation formed from the reaction of SF protein with the ABTS radicals generated from the substrate-dependent peroxidase reaction. The wave-designed microfluidic channels could significantly reduce the coffee ring effect in the detection film. The bi-enzyme SF films prepared by co-immobilizing AOx with peroxidase (HRP) offered high sensitivity to the device with a detection limit (LoD) of 1 ± 0.05 mM and a dynamic range of 1 mM–2 M for methanol. The stand-alone hybrid μPAD biosensor for methanol has a high potential for commercial applications.