Development and Optimization of a Thrombin Sandwich Aptamer Microarray

Abstract

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.

Keywords: aptamer; bioassay; fluorescence; multiplexing microarray; thrombin.

Figure 1

Specificity of aptasensor for thrombin. Fluorescence signal (635 nm) recorded on thrombin aptamer microarray after the incubation of the different indicated proteins with TBA2-Cy5 (protein and TBA2 final concentration: 500 nM and 1 mM, respectively); (a) thrombin, blank (TBA2-Cy5 only), BSA, lysozyme and VEGF and (b) corresponding microarray images.

Figure 2

Effect of incubation time. Fluorescence signal (635 nm) recorded on TBA1 microarray (a) after the incubation of thrombin and TBA2-Cy5 for different times (180–120–90–60–30–15–5 min) and (b) corresponding microarray images.

Figure 3

Effect of serum. Fluorescence signal (635 nm) recorded on TBA1 microarray (a) after the incubation of thrombin and TBA2-Cy5 complexes in presence of different Fetal Bovine Serum (FBS) dilutions (0%–10%–20%–30%–40%–50%–65%) and (b) corresponding microarray images.

Figure 4

Direct and indirect method for thrombin detection. (a) Fluorescence signal (635 or 532 nm) plot of thrombin aptamer microarray after the incubation of thrombin and TBA2-Cy5 (direct method, red squares, 635 nm) or thrombin and TBA2-biotin plus Cy3-streptavidin (indirect method, green dots, 532 nm) and (b) relative microarray images. Tested thrombin dilutions were: 200 nM–100 nM–50 nM–10 nM–5 nM–1 nM–0 nM. TBA2-Cy5 (red squares) or TBA2-biotin (green dots) concentration was 400 nM. For the indirect method the microarray slide was incubated for 1 h with Cy3-streptavidin, for a total incubation time of two hours in all cases.

Figure 5

Thrombin calibration curves in FBS solution. (a) fluorescence signal (532 nm) plot of TBA1 microarray in 20% FBS solution after the incubation of thrombin and TBA2-biotin plus Cy3-streptavidin and (b) corresponding microarray images. Tested thrombin dilutions were: 50 nM–10 nM–5 nM–1 nM–0.5 nM–0 nM, with a fixed TBA-2 biotin concentration of 200 nM.