Development and Optimization of a Thrombin Sandwich Aptamer Microarray.
A sandwich microarray employing two distinct aptamers for human thrombin has been optimized for the detection of subnanomolar concentrations of the protein. The aptamer microarray demonstrates high specificity for thrombin, proving that a two-site binding assay with the TBA1 aptamer as capture layer and the TBA2 aptamer as detection layer can ensure great specificity at times and conditions compatible with standard routine analysis of biological samples. Aptamer microarray sensitivity was evaluated directly by fluorescent analysis employing Cy5-labeled TBA2 and indirectly by the use of TBA2-biotin followed by detection with fluorescent streptavidin. Sub-nanomolar LODs were reached in all cases and in the presence of serum, demonstrating that the optimized aptamer microarray can identify thrombin by a low-cost, sensitive and specific method.
Associazione CIVEN, Via delle Industrie 9, I-30175 Venezia-Marghera, Italy. meneghello@civen.org.
Anna
A
Meneghello
Dipartimento di Scienze del Farmaco, University of Padova, Via F. Marzolo 5, I-35131 Padova, Italy. alice.sosic@studenti.unipd.it.
Alice
A
Sosic
Veneto Nanotech S.C.p.A., Via S. Crispino 106, I -35129 Padova, Italy. agnese.antognoli@venetonanotech.it.
Agnese
A
Antognoli
Associazione CIVEN, Via delle Industrie 9, I-30175 Venezia-Marghera, Italy. cretaio@civen.org.
Erica
E
Cretaio
Dipartimento di Scienze del Farmaco, University of Padova, Via F. Marzolo 5, I-35131 Padova, Italy. barbara.gatto@unipd.it.
Barbara
B
Gatto
aptamer; bioassay; fluorescence; multiplexing microarray; thrombin