Influence of antibody immobilization strategy on carbon electrode immunoarrays

We report here the influence of antibody immobilization strategy for protein immunosensors on screen printed carbon electrode arrays in terms of antibody binding activity, analytical sensitivity, limit of detection, and stability. Horseradish peroxidase (HRP) was the model analyte with anti-HRP immobilized on the sensors, and HRP activity was used for detection. Covalently immobilized anti-HRP antibodies on electrodes coated with chitosan, electrochemically reduced graphene oxide (rGO), and dense gold nanoparticle (AuNP) films had only 20-30% of the total immobilized antibodies active for binding. Active antibodies increased to 60% with passively adsorbed antibodies on bare electrodes, to 85% with oriented antibodies using protein A covalently immobilized on AuNP-coated carbon electrode, and to 98% when attached to protein A passively adsorbed onto bare electrodes. Passively adsorbed antibodies on bare electrodes lost activity in 1-2 days, but antibodies immobilized using other strategies remained relatively stable after 5 days. Covalent immobilization gave limits of detection (LOD) of 40 fg mL^-1, while passively adsorbed antibodies or protein A on carbon electrodes had LODs 4-8 fg mL^-1, but were unstable. Sensitivity was highest for antibodies covalently attached to AuNP electrodes (2.40 nA per log pg per mL) that also had highest antibody coverage, and decreased slightly when protein A on AuNP was used to orient antibodies. Passively adsorbed antibodies and oriented antibodies on protein A gave slightly lower sensitivities. Immobilization strategy or antibody orientation did not have a significant effect on LOD, but dynamic range increased as the number of active antibodies on sensor surfaces increased.