Enclosure-Free Smartphone Detection of Enrofloxacin Using an AlphaFold-Engineered Nanobody Biosensor with Active Illumination Imaging and Multidimensional Color Analysis.

Smartphone-based colorimetric assays are attractive for on-site analysis but often suffer from measurement instability caused by ambient illumination and imaging conditions, as well as limited sensitivity from conventional single-channel color readout. Herein, an AlphaFold-engineered nanobody biosensor was developed for enrofloxacin (ENR) by integrating active-illumination imaging with smartphone-based multidimensional color analysis to enable interference-resistant, enclosure-free detection. A specially engineered light source generates intense, spatially uniform diffuse illumination that dominates image acquisition, effectively suppressing ambient-light interference and eliminating the need for a light-shielding enclosure. Multidimensional descriptors extracted from RGB, HSV, and CIELAB (Lab) color spaces were fused to improve discrimination of subtle color changes and enhance readout sensitivity. Under variable lighting, the coefficient of variation was stabilized at 8-10%, compared with 66-97% without active illumination. The biosensor achieved an LOD of 2.93 ng/mL for ENR, which was further reduced to 1.91 ng/mL using a fused parameter (Senh), representing a 34.8% improvement in sensitivity. The assay also showed good performance in spiked marine fish samples, supporting its on-site applicability. Overall, this study presents a strategy whereby active illumination minimizes environmental interference and data-fusion-based multidimensional analysis enhances readout sensitivity for portable and adaptable detection.