4. Different approaches for detection of a plasma protein by an electrochemical aptamer-based affinity biosensor coupled to magnetic beads

S. CENTI; G. A. MESSINA; I. PALCHETTI; S. TOMBELLI; M. MASCINI

Napoli. Italia

Congreso; XX Congresso Nazionale di Chimica Analitica. S. Martino al Cimino (Viterbo). La Chimica Analitica per l'ambiente e gli alimenti. Napoli. Italia; 2007

Universita' degli studu della Tuscia

DIFFERENT APPROACHES FOR THE DETECTION OF THROMBIN BY ANELECTROCHEMICAL APTAMER-BASED ASSAY COUPLED TO MAGNETICBEADSS. Centi, G. Messina, S. Tombelli, I. Palchetti, M. MasciniDipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, ItaliaDifferent assay formats based on the coupling of magnetic beads with electrochemicaltransduction were here compared for the detection of thrombin by using a thrombin specificaptamer. This with the aim of demonstrating that when developing aptamer-based assays wellestablished operating conditions have not been created yet and each individual aptamer-basedassay has to be carefully optimised in order to find the optimum operating protocol to obtainthe best analytical performances.By using the thrombin binding aptamer, a direct and an indirect competitive assay forthrombin have been developed by immobilising the aptamer or the protein, respectively.Moreover, another strategy was based on the direct measurement of the enzymatic product ofthrombin captured by the immobilised aptamer. All the assays were developed by couplingthe electrochemical transduction with the innovative and advantageous use of magnetic beads.The assays based on the immobilisation of the protein were not successful since no bindingwas recorded between thrombin and its aptamer. With the direct competitive assay, when theaptamer was immobilised onto the magnetic beads, a detection limit of 430 nM for thrombinwas achieved. A lower detection limit for the protein (175 nM) was instead obtained bydetecting the product of the enzymatic reaction catalysed by thrombin. All these assays werefinally compared with a sandwich assay which reached a detection limit of 0.45 nM ofthrombin demonstrating the best analytical performances1.With this comparison the importance of a deep study on the different analytical approachesfor thrombin detection to reach the performances of the best assay configuration (sandwichassay) has been demonstrated.Bibliography:1. S. Centi, S. Tombelli, M. Minunni, M. Mascini; Analytical Chemistry 2007, 79, 1466-1473.