A length-band fluorescence-based paper analytical device for detecting dipicolinic acid via ofloxacin complexation with Cu2+

Dipicolinic acid (DPA) is a key biomarker of bacterial spores. In this study, we present a novel distance-based paper analytical device (d-PAD) for the fluorescence sensing of DPA. The detection mechanism relies on the complexation of ofloxacin (OFL) with Cu²⁺ ions, where Cu²⁺ quenches the fluorescence of OFL via static quenching. Upon the introduction of DPA, it interacts with the OFL-Cu²⁺ complex, resulting in an enhanced fluorescence signal from OFL. The assay demonstrated a limit of detection (LOD) of 0.08 μM over a range of 0.6–120 μM, as measured using a spectrofluorometer. The d-PAD was designed for efficient reagent transport through capillary action on paper substrates, allowing for rapid on-site DPA analysis without requiring advanced laboratory equipment. The length of the fluorescent bands on the d-PADs was proportional to the concentration of DPA, providing a simple and effective readout method. With a sensitivity of 0.6 μM, the device shows a strong response to varying DPA concentrations. This distance-based platform offers a straightforward and quantitative approach to result interpretation, making it a promising tool for detecting bacterial spores in real samples. The development and optimization of this paper-based microfluidic assay represent a significant step forward in portable diagnostic technologies.