Different approaches for the detection of thrombin by an electrochemical

SONIA CENTI; GERMÁN ALEJANDRO MESSINA; SARA TROMBELLI; ILARIA PALCHETTI; MARCO MASCINI

BIOSENSORS & BIOELECTRONICS

Año: 2008 p. 1602 - 1609

0956-5663

AbstractDifferent assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection ofthrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin havebeen developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of theenzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transductionwith the innovative and advantageous use of magnetic beads.The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer.With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430nM for thrombin wasachieved. A lower detection limit for the protein (175 nM) was instead obtained by detecting the product of the enzymatic reaction catalysed bythrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45nM of thrombin demonstratingthe best analytical performances.With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances ofthe best assay configuration has been demonstrated.