ac300690r_si_001.pdf (5.23 MB)
Label-Free Fluorescent Detection of Ions, Proteins, and Small Molecules Using Structure-Switching Aptamers, SYBR Gold, and Exonuclease I
journal contribution
posted on 2012-04-17, 00:00 authored by Dongmei Zheng, Ruxing Zou, Xinhui LouWe 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.