Unravelling the lipocalin 2 interaction with aptamers: May rolling circle amplification improve their functional affinity?

Publication date: 15 May 2019

Source: Talanta, Volume 197

Author(s): Ramón Lorenzo-Gómez, Noelia Fernández-Alonso, Rebeca Miranda-Castro, Noemí de-los-Santos-Álvarez, María Jesús Lobo-Castañón

Abstract

Cancer diagnosis based on serum biomarkers requires receptors of extreme sensitivity and selectivity. Tunability of aptamer selection makes them ideal for that challenge. However, aptamer characterization is a time-consuming task, not always thoroughly addressed, leading to suboptimal aptamer performance. In this work, we report on the affinity characterization and potential usage of two aptamers against a candidate cancer biomarker, the neutrophil gelatinase–associated lipocalin (NGAL). Electrochemical sandwich assays on Au electrodes and SPR experiments showed a restricted capture ability of one of the aptamers (LCN2–4) and a small detectability of the other (LCN2-2). Interestingly, a truncated version of the signaling aptamer LCN2-2 selectively binds to NGAL covalently linked to magnetic beads due to high local protein concentration. The functional affinity of this aptamer is enhanced by three-orders of magnitude using rolling circle amplification (RCA), completed in only 15 min, followed by hybridization with short complementary fluorescein-tag probes, enzyme labeling and chronoamperometric measurement. Microscale thermophoresis experiments show a poor affinity for the protein in solution, which urges the importance of a full and in-depth characterization of aptamers to be used as diagnostic reagents.

Graphical abstract

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