Seesawed fluorescence nano-aptasensor based on highly vertical ZnO nanorods and three-dimensional quantitative fluorescence imaging for enhanced detection accuracy of ATP

Probe-mediated fluorescence biosensing methods based on spectrophotometry still have limitations such as detection inaccuracy caused by the occurrence of false signals and lack of simultaneous qualitative and quantitative read-outs with an ultra-low detection limit. Herein, we describe a novel seesawed fluorescence detection strategy based on dual-colour imaging-based quantitation in which the green fluorescence of the capture aptamer decreases and the red fluorescence of the detection aptamer increases simultaneously upon their respective interactions with the target biomolecule. This approach enhances detection accuracy through facilitating identification of probable false-positives in biological samples. Furthermore, combining the seesawed detection scheme with three-dimensional imaging of fluorescence signal enhanced by highly vertical ZnO nanorods increases signal-to-noise ratio, which addresses the limited performance of digital cameras and, in turn, enhances sensitivity and dynamic range. This simple, robust, scalable, imaging-based and label-free fluorescence method allows highly specific and sensitive quantification of biomolecules with excellent reliability.