Photonic Stop Band - Fluorophore Overlap in Inverse Structure Enhances E. coli Detection via Fluorescence Imaging

Enhancing fluorescence image brightness is critical for improving the detection sensitivity in analytical applications. In this study, we evaluated the respective contributions of (i) the overlap between the photonic stop band (PSB) wavelength (λ_PSB) of a hydrogel-based inverse opal photonic crystal (IOPC) and the emission wavelength (λ_emi) of a fluorescent dye, and (ii) the pore diameter (d) of the IOPC, which governs the fluorophore loading capacity, to the overall fluorescence intensity. The overlap was selected by employing a consistent d and a specific dye, Alexa Fluor™ 488 (A488). Meanwhile, the variation in fluorescence intensity (I_fluor.) with respect to d was normalized, revealing that although d reached 90% of the value at which wavelength overlap occurred (d_overl.), I_fluor. increased to only 59%. However, when d increased to 110% and 125% of d_overl., I_fluor reached only 75.6% and 77.1%, respectively. This suggested that the overlap effect was the key factor contributing to the enhancement of the I_fluor. compared with the contribution from an increase in d. In addition, this result highlighted the potential application of the IOPC with an overlap between λ_PSB and λ_emi for the detection of Escherichia coli (E. coli). Although IOPC samples with d value reached 110% and 125% of d_overl were used, I_fluor. only reached 71.6% and 78.4%, respectively, of the value at which the overlap occurred. The limit of detection (LOD) and linear range were 10 cfu/mL and 10 ÷ 10⁵ cfu/mL (R² = 0.91), respectively. This indicated the potential application of the hydrogel IOPC exhibiting the overlap effect for analytical detection based on fluorescence imaging.