Dextran鈥揕ipase Conjugates as Tools for Low Molecular Weight Ligand Immobilization in Microarray Development

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• 作者：Sonia Herranz ; Marzia Marciello ; David Olea ; Margarita Hern谩ndez ; Concepci贸n Domingo ; Marisela V茅lez ; Levi A. Gheber ; Jose M. Guis谩n ; Mar铆a Cruz Moreno-Bondi
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：August 6, 2013
• 年：2013
• 卷：85
• 期：15
• 页码：7060-7068
• 全文大小：454K
• 年卷期：v.85,no.15(August 6, 2013)
• ISSN：1520-6882

文摘

The development of effective array biosensors relies heavily on careful control of the density of surface-immobilized ligands on the transducing platform. In this paper we describe the synthesis of new dextran鈥搇ipase conjugates for use in immobilizing low molecular weight haptens onto glass planar waveguides for immunosensor development. The conjugates were synthesized by immobilizing bacterial thermoalkalophilic lipases (Geobacillus thermocatenulatus lipase 2, BTL2) on agarose macroporous beads, followed by covalent coupling to dextran networks of variable molecular weight (1500鈥?0000). The chimeras were immobilized via nonspecific hydrophobic interactions onto glass planar waveguides modified with 1,1,1,3,3,3-hexamethyldisilazane to obtain highly ordered and homogeneous molecular architectures as confirmed by atomic force microscopy. Microcystin LR (MCLR) was covalently bound to the dextran鈥揃TL2 conjugates. The usefulness of this approach in immunosensor development was demonstrated by determining amounts of MCLR down to a few picograms per liter with an automated array biosensor and evanescent wave excitation for fluorescence measurements of attached DyLight649-labeled secondary antibody. Modifying BTL2 with dextrans of an increased molecular weight (>6000) provided surfaces with an increased loading capacity that was ascribed to the production of three-dimensional surfaces by the effect of analyte binding deep in the volume, leading to expanded dynamic ranges (0.09鈥?36.56 ng L^鈥?), lower limits of detection (0.007 卤 0.001 ng L^鈥?), and lower IC₅₀ values (4.4 卤 0.7 ng L^鈥?). These results confirm the effectiveness of our approach for the development of high-performance biosensing platforms.