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  • A majority of aptamer based biosensors termed

    2022-05-19

    A majority of aptamer-based biosensors (termed aptasensors) is based on structure-switching process of aptamer probes upon target binding [[12], [13], [14], [15]]. And canonical structure-switching aptamer probes are strand-displacement aptamer probes, aptamer beacons and split aptamers [[16], [17], [18]]. Through the mechanism of structure-switching, the aptamers have been adopted to different signal transduction methods such as fluorescent [[19], [20], [21], [22], [23], [24]], electrochemical [[25], [26], [27]], colorimetric [28,29] and chemiluminescence [30] sensing platforms to output the signals. The designability nature of nucleic aminopeptidase inhibitor allows designing probes with different structures [[31], [32], [33]], thus accommodate aptamers for constructing enormous aptamer probes, which could achieve highly specific, sensitive and rapid detection both in homogenous and heterogenous bioassays [7,34]. However, the process of the incorporation of an aptamer into a structure-switching aptasensor often relies on trial-and-error without general design principles [18]. Particularly, the design of structure-switching aptasensors may be hurdled by the intrinsic complexation structure of aptamers [18]. For example, the complexation structure of aptamer may render the aptamer probe hard to form the predesigned aptamer beacon structure and also inevitably interfere the stability of strand-displacement aptamer probe. Therefore, the elegant design strategy for aptamer probes may lie in the utilization of the intrinsic structure of aptamer itself, such as the commonly existed G-quadruplex structure. G-quadruplex specific dyes such as thioflavin T (4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,Ndimethylaniline, ThT), thiazole orange and N-methylmorpholine are powerful tools for probing nucleic acid structures [[35], [36], [37], [38], [39]]. They could specifically bind to G-quadruplex structures rather than other forms in DNA or RNA sequences, and confer in a remarkable enhancement or decrease of fluorescence [36,40]. At present, the G-quadruplex specific ThT, a water-soluble fluorogenic benzothiazole dye, has attracted increasing interest due to its high specificity and high fluorescence enhancement (>1700-fold) when binding to G-quadruplex [41]. The intramolecular rotations around the C-C bond between benzothiazole and dimethylaniline of ThT would suppress its fluorescence property. And ThT could induce G-rich DNA to fold into the G-quadruplex, and endowed with a significant fluorescence enhancement because of the rotation restriction [40] when binding to G-quadruplex. ThT was firstly used as a light-up fluorescence probe for monitoring the G-quadruplex [40]. Recently, ThT has been involved in aptasensor for small molecules and proteins, allowing specific, homogeneous and fast detection [[41], [42], [43]]. Nevertheless, in their designs, the sensing systems are mostly based on single signal output and the signal output is usually aminopeptidase inhibitor in “turn-off” mode [[42], [43], [44], [45]]. The “turn-off” bioassays may be potentially more susceptible to environmental interference which would be seriously detrimental to the accuracy of these assays [46,47]. Herein, based on G-quadruplex specific dye ThT, a ratiometric fluorescence resonance energy transfer (FRET) aptasensor has been developed. The key design of ratiometric aptasensor lies in the FRET process between G-quadruplex bound ThT and terminal-labeled dyes. As a proof of concept, ochratoxin A (OTA) was chosen as the target. OTA is nephrotoxic, carcinogenic and would exert a serious threat to the health of humans [3]. The quantification of OTA is accomplished via FRET signals. Remarkably, the FRET mechanism would eliminate the signal fluctuation resulted from varied probe concentration, thus benefiting the robustness of the assay. And the FRET aptasensor would confer a remarkable enhancement of the signal to background ratio compared to the ThT-based non-FRET aptasensor. We applied this aptasensor in the detection of OTA, the result showed that it was highly sensitive, rapid, easy to operate. And it could be applied to detect OTA in complex samples such as coffee and oat. Thus, G-quadruplex specific dye-based probing and FRET method would be a compelling design strategy for aptasensor and may facilitate practical application in food safety and environmental screening.