human thrombin detection through a sandwich aptamer microarray interaction analysis in solution and in solid phase人类凝血酶检测通过三明治适体和固相微阵列交互分析的解决方案.pdfVIP

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human thrombin detection through a sandwich aptamer microarray interaction analysis in solution and in solid phase人类凝血酶检测通过三明治适体和固相微阵列交互分析的解决方案.pdf

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human thrombin detection through a sandwich aptamer microarray interaction analysis in solution and in solid phase人类凝血酶检测通过三明治适体和固相微阵列交互分析的解决方案

Sensors 2011, 11, 9426-9441; doi:10.3390/s111009426 OPEN ACCESS sensors ISSN 1424-8220 /journal/sensors Article Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase Alice Sosic 1,2, Anna Meneghello 2, Erica Cretaio 2 and Barbara Gatto 1,* 1 Dipartimento di Scienze Farmaceutiche, University of Padova, via F. Marzolo 5, I-35131 Padova, Italy; E-Mail: alice.sosic@studenti.unipd.it 2 Associazione CIVEN, via delle industrie 9, I-30175 Venezia-Marghera, Italy; E-Mails: meneghello@ (A.M.); cretaio@ (E.C.) * Author to whom correspondence should be addressed; E-Mail: barbara.gatto@unipd.it; Tel.: +39-049-827-5717; Fax: +39-049-827-5366. Received: 29 July 2011; in revised form: 27 September 2011 / Accepted: 28 September 2011 / Published: 3 October 2011 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 (TBA 1) 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