An origami microfluidic paper-based analytical device for colorimetric determination of the albumin-creatinine ratio using Mn-ZnS quantum dots coated with molecularly imprinted polymers.

Herein, we report a novel, highly selective, and cost-effective Albumin-to-Creatinine Ratio microfluidic paper-based analytical device (ACR-μPAD) for rapid point-of-care testing (POCT) of chronic kidney disease (CKD) biomarkers. We pioneered the use of manganese-doped zinc sulfide quantum dots (Mn-ZnS QDs) as a catalytic core for synthesizing two distinct molecularly imprinted polymers (MIPs): Mn-ZnS QD-MIP-Alb and Mn-ZnS QD-MIP-Cre, specific for albumin (Alb) and creatinine (Cre), respectively. The biosensing mechanism is based on the competitive inhibition of the Mn-ZnS QDs-MIP peroxidase-mimicking activity. In the presence of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), the nano-catalysts produce a vibrant blue color. When the target analytes (Alb or Cre) bind to the specific recognition sites on the MIP shell, the catalytic activity is sterically hindered, causing a concentration-dependent decrease in blue color intensity. This simple, single-reagent colorimetric readout is integrated into a compact, screen-printed μPAD, which allows for the simultaneous, naked-eye detection of both biomarkers. The optimized sensor exhibited high sensitivity and selectivity, providing broad linear detection ranges for both biomarkers. For Cre, the detection ranges were 1-60 mg dL-1 and 60-1000 mg dL-1. For Alb, two distinct ranges, 0.1-10 mg L-1 and 10-100 mg L-1, were established. Critically for early diagnosis, the sensor demonstrated superior sensitivity within the lower concentration range for both Cre and Alb, aligning perfectly with the levels needed to detect initial stages of nephropathy. The calculated limit of detection (LOD) were 0.06 mg dL-1 for Cre and 0.23 mg L-1 for Alb (based on the 3 SD/slope method). Furthermore, the ACR-μPAD exhibited high accuracy, with no significant interference observed during analysis of real human urine samples. The combination of simple screen-printing fabrication, instrumental simplicity, and rapid response time makes this ACR-μPAD an exceptionally well-suited platform for mass-producible, field-based diagnostics, offering a crucial and accessible tool for early kidney disease detection and monitoring.