Experimental comparison of surface chemistries for biomolecule immobilization on paper-based microfluidic devices

Biomolecules (e.g. proteins and nucleic acids) as target analytes of microfluidic paper-based analytical devices (μPADs) are usually immobilized on a cellulose paper substrate (with intrinsically anionic surface) through physical adsorptions by van der Waals forces and electrostatic interaction thanks to cationic patches on the biomolecule. However, the physical adsorption could lead to weak biomolecule-substrate binding strength and thus low biosensing performance. Benefitting from the abundance of hydroxyl groups on the cellulose paper, chemical modification based on specific surface chemistries is capable of biofunctionalization on the μPADs by providing functional groups for covalent bindings with the target biomolecule. There are many previous reports on chemical modifications of cellulose surface for improvement of biomolecule immobilization. Nevertheless, no study has been performed on experimental evaluation of modification efficiencies of various biofunctionalization methods in the context of biosensing applications. In this paper, we compare five surface chemistries for protein immobilization on μPADs made from pure cellulose paper. For each chemical modification method, surface analyses were first conducted to monitor the surface modification process. Then, paper-based fluorometric experiments and colorimetric enzyme-linked immunosorbent assays (ELISA) were carried out on paper substrates modified by the five surface chemistries to compare their efficiencies of covalent protein immobilization. Finally, a stability experiment was carried out on the five types of surface-modified paper after 30 d storage. It was demonstrated that the potassium periodate (KIO4)-modified cellulose paper has the best performance with 53% increase in the signal output and 59% decrease in background noise of the colorimetric ELISA, and only 13% bioactivity loss after the 30 d storage. The comparison results provide a valuable experimental guideline for selecting the suitable surface chemistry for protein immobilization on μPADs.