Dextran–Lipase Conjugates as Tools for Low Molecular Weight Ligand Immobilization in Microarray Development

Abstract

The development of effective array biosensorsrelies heavily on careful control of the density of surface-immobilized ligands on the transducing platform. In this paperwe describe the synthesis of new dextran−lipase conjugates foruse in immobilizing low molecular weight haptens onto glassplanar waveguides for immunosensor development. Theconjugates were synthesized by immobilizing bacterial thermoalkalophilic lipases (Geobacillus thermocatenulatuslipase 2,BTL2) on agarose macroporous beads, followed by covalent coupling to dextran networks of variable molecular weight (1500−40000). The chimeras were immobilized via nonspecific hydrophobic interactions onto glass planar waveguides modified with1,1,1,3,3,3-hexamethyldisilazane to obtain highly ordered and homogeneous molecular architectures as confirmed by atomic forcemicroscopy. Microcystin LR (MCLR) was covalently bound to the dextran−BTL2 conjugates. The usefulness of this approach inimmunosensor development was demonstrated by determining amounts of MCLR down to a few picograms per liter with anautomated array biosensor and evanescent wave excitation forfluorescence measurements of attached DyLight649-labeledsecondary antibody. Modifying BTL2 with dextrans of an increased molecular weight (>6000) provided surfaces with anincreased loading capacity that was ascribed to the production of three-dimensional surfaces by the effect of analyte binding deepin the volume, leading to expanded dynamic ranges (0.09−136.56 ng L−1), lower limits of detection (0.007±0.001 ng L−1), andlower IC50values (4.4±0.7 ng L−1). These results confirm the effectiveness of our approach for the development of high-performance biosensing platforms.