title: Human thrombin detection through a sandwich aptamer microarray: interaction analysis in solution and in solid phase.
abstract: We have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM) and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized.
authors.affiliation: Dipartimento di Scienze Farmaceutiche, University of Padova, via F. Marzolo 5, I-35131 Padova, Italy. alice.sosic@studenti.unipd.it
authors.forename: Alice
authors.initials: A
authors.lastname: Sosic
authors.forename: Anna
authors.initials: A
authors.lastname: Meneghello
authors.forename: Erica
authors.initials: E
authors.lastname: Cretaio
authors.forename: Barbara
authors.initials: B
authors.lastname: Gatto
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007477:Ions; D046228:Microarray Analysis / Q000379:methods*; D011188:Potassium; D011485:Protein Binding; D060327:Solid-Phase Synthesis Techniques / Q000379:methods*; D012996:Solutions; D013917:Thrombin / Q000032:analysis*
chemical_list: D052157:Aptamers, Nucleotide; D007477:Ions; D012996:Solutions; C081676:thrombin aptamer; D013917:Thrombin; D011188:Potassium