期刊
TALANTA
卷 197, 期 -, 页码 406-412出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.talanta.2019.01.057
关键词
Aptamer characterization; NGAL; Electrochemical detection; Microscale thermophoresis; Rolling circle amplification
资金
- Spanish Ministerio de Educacion, Cultura y Deporte [FPU16/05670]
- Spanish Ministerio de Economia, Industria y Competitividad [CTQ2015-63567-R]
- FEDER
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.
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