Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads.
Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the 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 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 was achieved. A lower detection limit for the protein (175nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45nM of thrombin demonstrating the best analytical performances. With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.
Centi
S
S
Dipartimento di Chimica, Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy. sonia.centi@unifi.it
Messina
G
G
Tombelli
S
S
Palchetti
I
I
Mascini
M
M
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018189:Immunomagnetic Separation / Q000295:instrumentation* / Q000379:methods; D008863:Microspheres; D013917:Thrombin / Q000032:analysis* / Q000737:chemistry*
D052157:Aptamers, Nucleotide; D013917:Thrombin