Clicking Fluorometric Probes on Micropatterned Glass Microfiber Filters for ppb-Level Copper Quantitation

In this work, we demonstrated the efficacy of combining covalent modification via the click reaction and benchtop machine-cutting of glass microfiber filters (GMFs) to fabricate microfluidic paper-based analytical devices (μPADs) for performing ultrasensitive fluorometric assays. This has been accomplished by activating the GMF surface through the self-assembly of propargyl-PEG3-triethoxysilane (PPTES) and the click reaction to immobilize fluorometric probes (i.e., 3-azido-7-hydroxycoumarin, A-7-HC). Specifically for making the device, mini-discs of GMF (<i>d</i> = 0.5 cm or any other shape and size) are machine-cut and treated with a low concentration of PPTES in dichloromethane, while the remainder of the original GMF substrate is rendered superhydrophobic by reacting with octadecyltrimethoxysilane (OTMS). Following the reassembly of the device, the Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC)-based assay is performed by introducing A-7-HC in the presence of different concentrations of copper standards. Coupling with smartphone imaging under customized LED irradiation, we showed that the assay sites (discs) are distinct and uniform (free of coffee-ring effect); the achieved limits of detection (9.2 ± 0.8 ppb) for copper are impressive in comparison with traditionally prepared devices (drop-casting reagents on photolithographically patterned GMF/paper). As we have validated the device with the results obtained with atomic absorption spectroscopy of commercial copper diary supplements, this work reveals the potential of combining covalent surface modification with benchtop patterning techniques to create flexible μPADs for performing quantitative colorimetric and fluorometric assays.