A first-generation amperometric galactose biosensor has been systematically developed utilizing layer-by-layer (LbL) construction of xerogels, polymers, and carbon nanotubes toward a greater fundamental understanding of sensor design with these materials and the potential development of a more efficient galactosemia diagnostic tool for clinical application. The effect of several parameters (xerogel silane precursor, buffer pH, enzyme concentration, drying time and the inclusion of a polyurethane (PU) outer layer) on galactose sensitivity were investigated with the critical nature of xerogel selection being demonstrated. Xerogels formed from silanes with medium, aliphatic side chains were shown to exhibit significant enhancements in sensitivity with the addition of PU due to decreased enzyme leaching. Semi-permeable membranes of diaminobenzene and resorcinol copolymer and Nafion were used for selective discrimination against interferent species and the accompanying loss of sensitivity with adding layers was countered using functionalized, single-walled carbon nanotubes (CNTs). Optimized sensor performance included effective galactose sensitivity (0.037 μA/mM) across a useful diagnostic concentration range (0.5 mM to 7 mM), fast response time (~30 s), and low limits of detection (~80 μM) comparable to literature reports on galactose sensors. Additional modification with anionic polymer layers and/or nanoparticles allowed for galactose detection in blood serum samples and additional selectivity effectiveness.
Keywords: 1st generation biosensor; amperometric sensor; carbon nanotube; galactose; galactose oxidase; galactosemia; xerogel.