Nanoconfinement-Driven Solid-State Ratiometric Fluorescent Aptasensor for 17β-Estradiol Detection in Complex Matrices

Precise quantitative monitoring of 17β-estradiol (E2) is important for reproductive management in precision livestock farming. However, E2 determination in complex biological matrices remains challenging because of matrix-derived background and signal variability. Here, we developed a nanoconfinement-assisted solid-state ratiometric fluorescent aptasensor integrating target-induced strand displacement (TISD), magnetic separation, and anodic aluminum oxide (AAO) nanochannel confinement. The sensing probe consisted of streptavidin-coated magnetic nanoparticles (MNPs) carrying a FAM-labeled cDNA internal reference and a Texas Red-labeled E2 aptamer reporter. E2 binding promoted dissociation of the Texas Red-labeled aptamer from the magnetic probe. Magnetic separation and washing reduced soluble matrix-derived interference, while subsequent deposition of the sensing complexes onto an AAO membrane mitigated coffee-ring-associated nonuniformity and produced a more spatially uniform dual-color fluorescence distribution for ratiometric analysis. Under matrix-matched calibration conditions, linear ranges of 5.0–50.0 pM were obtained in tap water and sow saliva, 5.0–40.0 pM in whole milk, and 5.0–15.0 pM in post-estrus sow urine. The LOD determined in tap water was 3.62 pM. The different calibration slopes obtained among the four matrices indicated that residual matrix-dependent effects remained and that matrix-specific calibration was required for quantitative analysis. Matrix-matched spike recoveries ranged from 86.92% to 119.54% across the investigated matrices. The aptasensor exhibited the strongest response toward 17β-E2 among the tested compounds; however, cross-reactivities of 77.3% for E3 and 47.3% for 17α-E2 indicated preferential rather than exclusive recognition. Molecular docking suggested a putative binding pose but did not experimentally establish the molecular recognition mechanism. Overall, the platform demonstrated laboratory-scale analytical feasibility in pretreated tap water, sow saliva, whole milk, and post-estrus sow urine. Further development of sample preparation, magnetic handling, membrane loading, probe selectivity, and portable fluorescence readout will be required before in situ or on-site application.