Construction of hollow double shell NiMn PBA nanozymes for sensitive sarcosine detection via a cascade strategy

As sarcosine (SA) is a significant biomarker for the early development of prostate cancer (PCa), its sensitive detection in urine can serve as an effective non-invasive early warning method. Herein, we developed a novel hollow double shell NiMn PBA (NiMn-PBA-DSNB) with outstanding peroxidase-like activity through a facile cation exchange reaction according to the distinct solubility product constants (K_sp) of different Prussian blue analogues (PBAs). Moreover, we innovatively proposed an "enzyme-nanozyme" cascade strategy to realize ultrasensitive SA sensing. This system utilized the enzymatic reaction between SA and sarcosine oxidase (SOX) to generate H₂O₂in situ. In the presence of the generated H₂O₂, the NiMn-PBA-DSNB nanozyme can efficiently catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to produce blue oxTMB. Consequently, the concentration of SA directly correlated with the absorbance value of oxTMB, providing quantitative detection. Based on this dual-enzyme cascade mechanism, a highly sensitive colorimetric assay was developed for the precise analysis of SA. The assay achieved a wide linear range from 8 to 500 μM, with a low detection limit of 1.75 μM, fully fulfilling the requirements for SA sensing in the urine of PCa patients. The method was also successfully applied to the analysis of real human urine samples, achieving recoveries ranging from 102.15% to 104.35% with relative standard deviations of ≤5.03%, which demonstrates its strong potential for clinical application.