Immobilization of redox-labeled hairpin DNA aptamers on gold: Electrochemical quantitation of epithelial tumor marker mucin 1

We report herein an aptamer-based electrochemical biosensor for the quantitative determination of mucin 1 (MUC1), a glycoprotein expressed on most epithelial cell surfaces, based on the analyte-binding induced, global-scale conformational change of electrode-bound anti-MUC1 DNA aptamers. The modified electrode was readily prepared by directly immobilizing thiolated, single-stranded anti-MUC1 DNA aptamers on gold; they were also tethered at the distal end with methylene blue (MB) for electrochemical measurements. In the absence of MUC1, these DNA single strands fold into a thermodynamically stable “hairpin” conformation, which facilities direct electron transfer between MB and the gold electrode. Upon MUC1 binding, the aptamer no longer retains its inherent hairpin conformation, which relocates the redox center (MB) further away from the electrode surface. The surface density of the DNA aptamer probes on gold measured by MB voltammetric response is consistent with that of electrostatically bound [Ru(NH3)6]3+, indicating that every MB molecule is electroactive at low scan rates; yet, the electroactivity of MB decreases remarkably upon binding MUC1. The detection limit (50 nM) and dynamic response range (up to 1.5 μM) are superior to the commercially available enzyme-linked immunosorbent assay (ELISA) kits and our previously developed quantum dot-based fluorescence protocol. The sensing principle pertaining to the specific folding conformation described in this paper has the potential to serve as a general approach of using hairpin DNA aptamers to construct biosensors for other molecular analytes.