Electrochemical biosensor using direct electron transfer and an antibody–aptamer hybrid sandwich for target detection in complex biological samples

Direct electron transfer (DET) between an electrode and redox labels is feasible in electrochemical biosensors using small aptamer–aptamer sandwiches; however, its application is limited in biosensors that rely on larger antibody–antibody sandwiches. The development of sandwich-type biosensors utilizing DET is challenged by the scarcity of aptamer–aptamer sandwich pairs with high affinity in complex biological samples. Here, we introduce an electrochemical biosensor using an antibody–aptamer hybrid sandwich for detecting thrombin in human serum. The biosensor enables rapid DET through an antibody–aptamer hybrid configuration comprising (i) an antibody capture probe that provides high and specific affinity to the target in human serum, (ii) the target thrombin, and (iii) an aptamer detection probe that facilitates convenient terminal conjugation with long flexible spacer DNA and polylinker peptide containing multiple amine-reactive phenazine ethosulfate (arPES) redox labels, allowing the conjugated labels to easily approach the electrode. Rapid repeated DET using arPES-catalyzed NADH oxidation strongly enhanced the electrochemical signals. Properly sized spacer and polylinker provided low nonspecific adsorption of the aptamer probe conjugated with multiple arPESs and low interference with the binding of the aptamer probe. Methods for immobilizing thiol-terminated antibodies on Au electrodes were compared and optimized. The developed biosensor using the antibody–aptamer hybrid sandwich exhibited high sensitivity and selectivity in detecting thrombin, surpassing the limitations of an aptamer–aptamer sandwich owing to the low affinity of thrombin aptamers in human serum. The calculated detection limit of the biosensor was ∼1.5 pM in buffer and ∼2.7 nM in human serum.