A dual-mode fluorescence sensing platform based on reversible i-motif switching and FRET for reliable pH detection

A dual-mode fluorescent sensing platform based on reversible i-motif DNA conformational switching and fluorescence resonance energy transfer (FRET) was developed for pH detection using complementary fluorescence outputs. Two cytosine rich probes, a single stranded turn-on probe and a double stranded turn-off probe, generated complementary fluorescence responses through pH dependent i-motif folding and unfolding. Both probes showed sensitive responses around the i-motif transition region near pH 6.5. The platform was applied to monitor storage associated pH changes in bass fish and minced mutton and to detect urease activity. For urease detection, both modes showed good linearity over 1-4 U/L, with detection limits of 0.02 and 0.11 U/L, respectively. The opposite fluorescence outputs provide complementary evidence for cross-checking pH-related signals, offering potential to improve analytical reliability. However, further validation is needed to establish the effectiveness of this approach in complex food matrices.