Dual-signal electrochemical strategy for urinary albumin detection using Ag+-labeled AuNP immunoprobe and Cu2+ enhancement

Abstract

Monitoring human serum albumin (HSA) levels in urine is crucial for the early detection and management of kidney diseases, including chronic kidney disease and diabetic nephropathy. In this work, the novel direct-type electrochemical immunoassay for quantitative determination of human serum albumin in urine (HSA) was proposed. This platform utilizes a silver ion-labeled, PEGylated gold nanoparticle immunoprobe (Ag⁺-labeled AuNP immunoprobe), which generates a strong electrochemical signal at −0.1 and 0.01 V (vs. Ag/AgCl) for Cu²⁺ and Ag⁺ signal, respectively. The detection process involves specific recognition of HSA in the presence of Cu²⁺ ions, enabling dual-signal amplification from Ag⁺ and Cu²⁺ ions. By drop-casting the immunoassay complex onto unmodified screen-printed carbon electrode (SPCE), HSA concentration was quantified through two linear detection ranges for lower (0.5 to 50 mg/dL) and higher (2 to 50 mg/dL) concentrations, by monitoring the current signal of Ag⁺ and Cu²⁺ ions, respectively. The method demonstrated excellent sensitivity, achieving limits of detection (LOD) as low as 1.26, and 10.54 mg/dL for Cu²⁺ and Ag⁺ signals, respectively. Validation with HSA-spiked real urine samples showed consistent linearity and accuracy, confirming its potential for clinical diagnostics. This dual-ion electrochemical system offers a promising platform for advanced protein detection in biomedical applications. Moreover, the proposed immunosensor operates on a bare SPCE without electrode preparation, offering a clear advantage over methods requiring multiple electrode modification steps.