Hierarchically ordered ERGO-L-Cys flower-like nanospheres on screen-printed electrodes for electrochemical immunosensing of cow's milk specific IgE with machine learning-assisted calibration.

Cow's milk-specific IgE (sIgE) is a crucial serum biomarker for cow's milk allergy (CMA) in children. However, its low abundance and the scarcity of reference standards have made it challenging to develop effective electrochemical detection platforms for clinical use. This study reports, to the best of our knowledge, the first electrochemical immunosensing platform for the quantitative detection of cow's milk sIgE (CM-sIgE) in pediatric serum. Through the l-cysteine (L-Cys)-mediated electrochemical reduction of graphene oxide, a hierarchically ordered three-dimensional electrochemically reduced graphene oxide functionalized with L-Cys (ERGO-L-Cys) flower-like nanosphere interface was in situ constructed on a screen-printed carbon electrode, distinguishing it from conventional two-dimensional ERGO films. Combined with a biotin-streptavidin bridged alkaline phosphatase/1-naphthyl phosphate (ALP/1-NPP) enzyme amplification system, the quantitative detection of serum sIgE was achieved using differential pulse voltammetry. This platform exhibits a linear range of 0.56-54.52 IU/mL for serum sIgE, with a detection limit of 0.29 IU/mL, demonstrating good selectivity, reproducibility, and storage stability. To overcome the challenge of the lack of sIgE reference standards, pediatric serum samples calibrated against hospital chemiluminescence assay values were utilized. Moreover, random forest (RF) regression was applied for data-driven calibration, further enhancing the quantitative robustness in complex serum matrices. The analysis of clinical samples showed good agreement with the results from hospital chemiluminescence assays. This work provides an eco-friendly, reproducible approach for constructing three-dimensional biofunctionalized interfaces and offers a feasible solution for point-of-care electrochemical detection of CMA in children.