An Aptamer‐Based Bound/Free Separation System for Protein Detection

Abstract

Aptamer hybridizes with its complementary strand. However, the complementary strand has difficulties to hybridize with the aptamer bound to a target because the aptamer forms higher-order structures. Exploiting this property, we developed simple bound/free separation systems for thrombin and IgE detection. The complementary strand was immobilized onto beads and the aptamer was labeled with pyrroquinoline quinone glucose dehydrogenase (PQQGDH). In the absence of a target, the aptamer is trapped by beads, whereas in the presence of a target, the aptamer bound to the target is not trapped. Thus the aptamer-target complexes can be recovered easily and detected by PQQGDH activity. This system allow the detection of 270 pM thrombin and 1 nM IgE.

Electrochemical Detection of Vascular Endothelial Growth Factor by an Aptamer-Based Bound/Free Separation System

“…However, we observed slight increase of electric current in the presence of BSA with concentration dependency. Since we observed little increase of electric current for thrombin detection system, 14 it might indicate that electric current increase in the presence of BSA causes nonspecific binding to the aptamer. Therefore we need to improve binding specificity of this aptamer.…”

“…Avidin-PQQGDH conjugate was prepared using glutalaldehyde crosslinking, as has been described elsewhere, to label the biotin- 14 The activity of the PQQGDHavidin conjugate was measured using 1 mM 2,6-dichlorophenolindophenol (DCIP), 6 mM 1-methoxy-5-methylphe-nazinium methylsulfate (m-PMS), 60 mM glucose, and we defined 1 unit of GDH activity as the amount of enzyme required to reduce 1.0 µmol DCIP per min in the presence of glucose. To prepare the PQQGDHlabeled VEGF aptamer, 1 nmol PQQGDH-avidin conjugate and 0.5 nmol biotin-labeled VEGF aptamer were mixed in 500 µL of reaction buffer [10 mM Tris-HCl (pH 7.0), 100 mM NaCl] and incubated for 30 min at 4°C.…”

“…13 We have already reported a simple bound/free (B/F) separation system based on the structural change of an aptamer for protein detection, which consists of an aptamer and its part of the complementary strand. 14 The sensing system is shown in Fig. 1.…”

“…We previously reported thrombin detection and IgE detection using this system. 14 Thrombin aptamer and IgE aptamer have been characterized in depth by many researchers. In this study, to demonstrate the versatility of this strategy, we constructed a simple B/F separation system using aptamers that we selected against VEGF165.…”

Aptamer Sensors Combined with Enzymes for Highly Sensitive Detection

“…This system consists of two parts: an aptamer and its complementary DNA. We previously described two types of single aptamer-based B/F separation systems (Abe et al, 2011;Fukasawa et al, 2009;Ogasawara et al, 2009) (Fig. 3).…”