Simple and sensitive colorimetric assay system for horseradish peroxidase using microfluidic paper-based devices

Abstract This article describes the development of a simple, portable assay system using microfluidic paper-based analytical devices (μPADs) coupled with colorimetric detection for rapid measurements. The properties of different paper substrates were first investigated to determine which type of paper would be the most suitable for the fabrication of the μPADs. Simultaneous detection of horseradish peroxidase (HRP) utilizing a 5 μL sample analytical volume was demonstrated using a single μPAD. Hydrophilic test regions were separated by hydrophobic barriers, which were fabricated through photolithography. These test regions were immobilized with 10 mM of 3,3′,5,5′-tetramethylbenzidine for HRP assay. The detection range obtained with the proposed system covered HRP concentrations from 0.37 to 124 fmol (or 3–1000 ng mL−1). The detection limit (blank + 3σ) for HRP was calculated to be 0.69 fmol (or 5.58 ng mL−1) through a 4-parameter logistic nonlinear regression using results obtained within a 15 min assay time. The findings obtained using the developed system suggest that μPAD assay systems for simple but highly sensitive measurements can be designed to give on-site determinations of target compounds using peroxidase-conjugated molecules.