Influence of crosslinker on aptamer immobilization and aptasensor sensing response for non-metallic surfaces

Aptasensors transduce the specific binding of the target biomolecules with an aptamer into a measurable signal and enable rapid detection of biomolecules. Aptamer immobilization on the sensing surface through covalent bonding is preferred as it ensures stable and repeatable attachment of the aptamers in the desired configuration. Previous sensing reports have utilized homo-bifunctional crosslinkers like glutaraldehyde to immobilize aptamers to amine-terminated surfaces, but the efficacy of attachment chemistries and their influence on sensor response have not been evaluated. Here, we compare sensing and immobilization density results for four separate crosslinkers - one homo-bifunctional and three hetero-bifunctional - to immobilize the Ebola soluble Glycoprotein (sGP) binding DNA aptamer on the amine-terminated non-metallic surfaces - nanoporous anodized alumina oxide (NAAO) membranes and polystyrene beads. In each case, the aptamer-functionalized NAAO membranes were exposed to a range of Ebola virus sGP concentrations to obtain sensing responses. Measured sensing responses were fit to the Langmuir-Hill's equation. The maximum possible sensor response, F_max, obtained for each attachment chemistry is compared to determine the effectiveness of the different attachment chemistries. We also used crosslinkers to attach radiolabeled aptamers to amine modified polystyrene particles to determine the attachment efficiency based on the acquired aptamer density on the particle for each crosslinker. Sensing and attachment efficiency results show that aptamer attachment on the sensor surface using a homo-bifunctional crosslinker led to inconsistent sensing response for sensing of sGP across experiments and lower aptamer densities on polystyrene particles. In contrast, the results from aptasensors with aptamers attached through hetero-bifunctional crosslinkers provided consistent sensing responses across experiments and higher aptamer densities on the surface.