Base-modified aptamer-DAPI probe for label-free detection of doxorubicin in dairy and serum samples

Abstract

Doxorubicin is an important chemotherapeutic agent, making its sensitive detection essential for therapeutic monitoring and food safety assessment. Although DNA aptamers exhibit high affinity and specificity toward doxorubicin, the absence of well-defined structural motifs often hampers the development of rapid, label-free sensing strategies. Here, we report a structure-engineered aptamer biosensor (ATP-3), in which the native doxorubicin aptamer was redesigned to incorporate an AT-rich duplex region capable of binding the fluorescent dye 4’,6-diamidino-2-phenylindole (DAPI), thereby endowing the aptamer with an intrinsic fluorescence reporting function. Upon DAPI binding, ATP-3 generates a strong fluorescence signal. Upon target binding, it disrupts the duplex structure, releases DAPI, and results in pronounced fluorescence quenching with a 15.25-fold signal change. This dynamic signal-switching mechanism enables rapid and highly sensitive label-free quantification of doxorubicin. The ATP-3 sensor exhibits an excellent linear response (R 2 = 0.9946) over the range of 5–200 nM, with a detection limit of 5 nM. Collectively, this work provides a rapid, reliable, and cost-effective platform for doxorubicin detection while establishing a general strategy for transforming aptamers lacking intrinsic signaling capability into label-free fluorescent biosensors.