SPLASH: a benchtop platform for accessible, ultrasensitive quantification of plasma biomarkers in Alzheimer’s disease

Blood-based biomarkers have emerged as a promising tool for the detection and monitoring of neurodegenerative diseases such as Alzheimer’s disease (AD), yet broad implementation of ultrasensitive protein quantification remains constrained by reliance on specialized instrumentation and centralized laboratory infrastructure. Here we present SPLASH (Solid Phase Ligation Assay with Single wasH), an ultrasensitive proximity ligation assay platform that achieves sub-pg/mL sensitivity using only standard benchtop qPCR equipment. We developed five assays targeting Alzheimer’s disease biomarkers – pTau-217, Aβ1–40, Aβ1–42, neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP) – with limits of detection ranging from 0.0005 to 0.119 pg/mL. In a cohort of 101 participants characterized for amyloid status by PET, SPLASH quantification of plasma pTau-217 discriminated amyloid-positive from amyloid-negative individuals (AUC = 0.839) with performance equivalent to three established pTau-217 assays. We further established compatibility with dried plasma spot samples, demonstrating the potential for decentralized collection and analysis without cold-chain storage. A multiplexed five-analyte panel was applied to 69 plasma samples, revealing heterogeneous biomarker profiles consistent with AD-associated patterns. By eliminating dependencies on proprietary instrumentation, SPLASH facilitates broad implementation of ultrasensitive protein quantification for neurodegenerative disease research and diagnostics.