Simultaneous Electrochemical Detection of NGF and proNGF Under Native Conditions Using Molecularly Imprinted Polymers: Toward Point‐of‐Care Diagnosis of Alzheimer's Disease

In the brain, proNGF, the NGF precursor, is in a homeostatic equilibrium with its processing product, mature NGF. Dysregulation of the NGF/proNGF ratio has been associated with neurodegeneration in Alzheimer's disease (AD), positioning these neurotrophins as promising diagnostic biomarkers. Yet, their clinical validation as biomarkers has been hindered by the lack of analytical methods capable of discriminating and quantifying both isoforms under native conditions. Here, we introduce a dual electrochemical sensor based on Molecularly Imprinted Polymers (MIPs) that enables the simultaneous, selective, and label-free quantification of NGF and proNGF. The sensors were fabricated via electropolymerization of o-phenylenediamine on platinum microelectrodes, yielding highly specific recognition sites for each isoform. The MIP-based platform demonstrates remarkable selectivity, reproducibility, and isoform discrimination, achieving picomolar detection limits even for NGF, which is typically present at low concentration in cerebrospinal fluid (CSF). Validated on clinical CSF samples from AD and control patients, this system successfully quantifies both NGF and proNGF without antibodies or sample denaturation. To the best of our knowledge, this represents the first quantitative and simultaneous detection of NGF and proNGF under native conditions. This technology paves the way toward cost-effective, high-throughput, and point-of-care diagnostics for Alzheimer's and other neurodegenerative diseases.