Label-free fluorescent detection of ions, proteins, and small molecules using structure-switching aptamers, SYBR Gold, and exonuclease I.

We have demonstrated a label-free sensing strategy employing structure-switching aptamers (SSAs), SYBR Gold, and exonuclease I to detect a broad range of targets including inorganic ions, proteins, and small molecules. This nearly universal biosensor approach is based on the observation that SSAs at binding state with their targets, which fold into secondary structures such as quadruplex structure or Y shape structure, show more resistance to nuclease digestion than SSAs at unfolded states. The amount of aptamer left after nuclease reaction is proportional to the concentrations of the targets and in turn is proportional to the fluorescence intensities from SYBR Gold that can only stain nucleic acids but not their digestion products, nucleoside monophosphates (dNMPs). Fluorescent assays employing this mechanism for the detection of potassium ion (K(+)) are sensitive, selective, and convenient. Twenty μM K(+) is readily detected even at the presence of a 500-fold excess of Na(+). Likewise, we have generalized the approach to the specific and convenient detection of proteins (thrombin) and small molecules (cocaine). The assays were then validated by detecting K(+), cocaine, and thrombin in urine and serum or cutting and masking adulterants with good agreements with the true values. Compared to other reported approaches, most limited to G-quadruplex structures, the demonstrated method has less structure requirements of both the SSAs and their complexes with targets, therefore rending its wilder applications for various targets. The detection scheme could be easily modified and extended to detection platforms to further improve the detection sensitivity or for other applications as well as being useful in high-throughput and paralleled analysis of multiple targets.