Paper-based enzyme-linked biosensor combined with smartphone for simultaneous colorimetric sensing of xanthines and sarcosine

A portable paper-based enzyme-linked biosensor that integrates a multi-enzyme cascade reaction, natural enzyme, and nanozyme with filter paper, combined with smartphones, is proposed for the detection of xanthine (XA) and sarcosine (SA) in urine. The primary biological component of the sensor is horseradish peroxidase (HRP). By chemically modifying cellulose filter paper (CFP) the HRP in horseradish crude extract is specifically immobilized through boron affinity and metal chelation techniques. Furthermore, the incorporation of UiO-66 on CFP had a good synergistic effect on the electron transfer of HRP and the detection of H2O2, significantly increasing the sensor's sensitivity. Under optimized experimental conditions, the sensor demonstrated a dynamic response range of 8–400 μmol L-1 for SA with a minimum detection limit (LOD) of 5 μmol L-1, and a dynamic response range of 10–400 μmol L-1 with an LOD of 8 μmol L-1 for XA. Additionally, the well-designed detection area of the portable test strip simplifies the detection operation, reduces the detection time, and enables rapid on-site analysis. UV-Vis analysis further confirmed the sensor's accuracy in detecting disease markers in urine. The sensing platform has good reproducibility, specificity and stability, suitable for the detection of SA and XA in real human urine samples, and the sensor has the advantages of simple manufacturing, easy operation, strong portability, low reagent consumption, and low cost benefit. Therefore, the development of this paper-based enzyme-linked biosensor offers a novel approach to the detection of disease markers in urine and has potential applications in medical testing.