Highly sensitive and selective ratiometric fluorescence detection of ATP using aptamer-functionalized Fe₃O₄ nanozymes...

Herein, a highly sensitive and selective ratiometric fluorescence platform was constructed for probing adenosine triphosphate (ATP) for the first time by using magnetic Fe₃O₄ nanozymes that were modified by ATP-specific aptamer labeled with 7-Amino-4-methyl-3-coumarinylacetic acid (Apt-AMCA) to yield Fe₃O₄@Apt-AMCA. It was discovered that the incorporation of Apt-AMCA greatly enhance the peroxidase-like activity of Fe₃O₄ nanozymes, which might catalyze more o-phenylenediamine (OPD) substrates into fluorescent oxidized OPD (DAP) with a emission of 560 nm. More importantly, upon ATP addition, Apt-AMCA could potentially be released from Fe₃O₄@Apt-AMCA because of Apt-target binding and the competition between ATP and Apt. The rational increase in the levels of free Apt-AMCA could thereby be attained in the reaction solutions so as to induce an enhancement of fluorescence at 450 nm. Meanwhile, the desorption of Apt-AMCA from the Fe₃O₄@Apt-AMCA surface would decrease the peroxidase-like activity of Fe₃O₄@Apt-AMCA, thereby reducing its ability to catalyze OPD into fluorescent DAP and thus decreasing fluorescence at 560 nm. A ratiometric fluorescence method was accordingly formulated to permit the sensitive and selective identification of ATP, featuring a linear dynamic range that ranges from 0.10 μM to 80 μM. Accurate measurements of ATP in practical blood samples were also confirmed, promising a new detection method for ATP analysis that can widely applied in clinical laboratories.