Development of a multiplex sandwich aptamer microarray for the detection of VEGF165 and thrombin

Abstract

In this work we have developed a multiplex microarray system capable of detecting VEGF165 and thrombin. We recently described a Sandwich Aptamer Microarray (SAM) for thrombin detection feasible for use in multiplex microarrays; here we describe a new aptasensor for VEGF165 detection employing Vap7 and VEa5, two DNA aptamers recognizing different sites of the protein. The aptamers were modified to be adapted to the solid phase platform of SAM and their capability to simultaneously recognize VEGF165 by forming a ternary complex was analyzed in solution. Having so defined the best tandem arrangement of modified aptamers, we set up the aptasensor for VEGF165, and finally analyzed the multiplex system with the two aptasensors for the simultaneous detection of VEGF165 and thrombin. The results indicate that each sandwich is specific, even when the two proteins are mixed. The system performance is consistent with the behavior evidenced by the biochemical analysis, which proves to be valuable to drive the evaluation and refinement of aptamers prior to or along the development of a detection platform. Since thrombin upregulates VEGF expression, the simultaneous recognition of these two proteins could be useful in the analysis of biomarkers in pathologies characterized by neo-angiogenesis.

Figure 1.

Scheme of the microarray printed glass slide. A1: detection of VEGF555; A2: detection of VEGF. B1: detection of VEGF555 mixed with thrombin555; B2: detection of VEGF mixed with thrombin. C1: detection of thrombin555; C2: detection of thrombin.

Figure 2.

Electrophoretic Mobility Supershift Assay of Vap7-NH₂ + VEGF₁₆₅ + VEa5-FAM (a) and of VEa5-NH₂ + VEGF₁₆₅ + Vap7-FAM (b). In Panel (a) the Vap7-NH₂-VEGF₁₆₅-VEa5-FAM interaction was analyzed, while in Panel (b) the VEa5-NH₂-VEGF₁₆₅-Vap7-FAM interaction was analyzed. Each aptamer was incubated separately or simultaneously with the protein under the described conditions. The respective aptamer and VEGF₁₆₅ concentrations are indicated in the figure. Binding reactions were applied on a 12% non-denaturing PAA gel containing glycine buffer and 10 mM KCl. The mobility of free and complexed aptamers, detected by the FAM fluorescence, was analyzed using the Geliance 600 Imaging System.

Figure 3.

Images of the microarray slide after the simultaneous incubation of Alexa555-VEGF₁₆₅ and Alexa647-thrombin (each 500 nM). On the same cell, Vap7(12T)NH₂ was anchored as capture layer for VEGF₁₆₅ (left) and TBA1(12T)NH₂ was anchored as capture layer for thrombin (right). Green fluorescence (λ^em: 532 nm) represents labeled VEGF₁₆₅ (Alexa 555) and red fluorescence (λ^em: 635 nm) represents labeled thrombin (Alexa 647).

Figure 4.

Images of the microarray slides reporting the multiplex SAM for VEGF₁₆₅ and thrombin detection. The six spots printed with Vap7(12T)NH₂ are on the left of each subarray and the six spots printed with TBA1(12T)NH₂ on the right. Panel A reports the detection of human VEGF₁₆₅, Panel C the detection of human thrombin. Panel B reports the simultaneous detection by each aptasensor of the specific target mixed in solution. Each protein, at the same concentration, was then incubated separately with its own specific detection aptamer (VEa5-Cy5 or TBA2-Cy5) and analyzed on the glass slide. Cy-5 detection aptamers, namely VEa5-Cy5 for VEGF₁₆₅ or TBA2-Cy5 for thrombin, were 1 μM. Glass slides A1, B1 and C1 were incubated with Alexa-555 labeled proteins, while slides A2, B2 and C2 were incubated with unlabeled proteins.