Information-driven nanozyme screening enabled sensitive detection of seminal neutral α-glucosidase activity reveals clinical infertility relevance.

Seminal neutral α-glucosidase (NAG) emerges as a promising diagnostic marker for epididymal function; however, its accurate quantification is hindered by low abundance and limited sample volume per ejaculate, necessitating highly sensitive analytical methods. Herein, we established a nanozyme-enhanced colorimetric assay for the sensitive detection of seminal NAG activity via an information-driven nanozyme screening workflow. Machine learning-based screening prioritized Mn(Ⅳ)-containing materials and, together with considerations of synthetic feasibility and chemical stability, guided the selection of MnO2 as an experimentally accessible candidate. A large language model-assisted retrieval-augmented generation framework was subsequently used to generate literature-grounded MnO2 synthesis routes. Highly active oxidase-mimicking α-MnO2 was synthesized following the predicted protocol, confirming efficient catalysis of the 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction. In the established assay, NAG specifically hydrolyzes α-arbutin to release hydroquinone, which acts as a reducing agent to suppress the α-MnO2-mediated oxidation of TMB, yielding a concentration-dependent absorbance response. This principle enabled quantitative analysis of NAG activity with a detection limit down to 0.97 mU/mL, substantially outperforming previous approaches and permitting point-of-care testing with microliter-level semen samples. The reliability of our nanozyme-based assay was rigorously validated using clinical specimens. Notably, clinical testing revealed a significant correlation between seminal NAG activity and male fertility status, underscoring the practical utility of our biosensing platform for robust male reproductive assessment in infertility clinics.