A highly sensitive sandwich-type electrochemical aptasensor using a pair of novel truncated aptamers for the detection of vaspin.

• A novel nonessentials-guided in situ aptamer truncation was successfully developed. • This novel strategy was applied to a pair of aptamers, elucidating the binding sites. • A pair of novel truncated aptamers showed improved binding affinity and specificity. • This truncated pair was first realized in a sandwich-type electrochemical aptasensor. • This new aptasensor showed highly sensitive detection of vaspin in buffer and serum. Visceral adipose tissue-derived serpin (vaspin) is a biomarker associated with obesity and type 2 diabetes, both of which are prevalent and increasing in numbers worldwide. However, the sensitivity of existing aptasensors for vaspin detection does not yet match the reported serum levels. Aptamer sequences could contain nonessential nucleotides, which can be truncated out to potentially enhance their functionality and applicability. In this study, we developed a novel truncation method, a nonessentials-guided in situ truncation strategy to generate high-affinity aptamers by directly removing the nonessential portions of the aptamer sequences using exonucleases from the aptamer-protein complex kept stably. We applied this novel strategy to the original vaspin-binding aptamers, V1 and V49, resulting in two truncated aptamers, V1(3′-5′)–26 and V49(3′-5′)–30. These truncated aptamers showed enhanced binding affinity and specificity compared to their original aptamers. Confirmed to have different binding sites, this pair of truncated aptamers was successfully incorporated into a sandwich-type electrochemical aptasensor. This aptasensor achieved 16-fold higher sensitivity than the one using untruncated aptamers and enabled specific detection of vaspin in a buffer and even sensitive detection in serum within the physiological range. The reduced size of the aptamers could potentially cover the surface of the sensing platform, blocking nonspecific binding, thereby lowering the blank value, and improving the signal-to-noise ratio and limit of detection. This approach highlights the potential of the nonessentials-guided in situ truncation strategy for developing improved truncated aptamers, examining aptamer binding sites, and enhancing the analytical performance of biosensing platforms. [ABSTRACT FROM AUTHOR]