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. J.S.Y. receives funding from NIH-NIA R01AG057234, P30AG062422, P01AG019724, and U19AG079774; NIH-NINDS U54NS123985; the Rainwater Charitable Foundation; the Alzheimer’s Association; the Global Brain Health Institute; and the Mary Oakley Foundation. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

These authors contributed equally: Noah Elder and Hien Nguyen. Noah Elder, Hien Nguyen, Jason Wan, Marissa Lee, Christopher Ng & Robert Lin Edward and Pearl Fein Memory and Aging Center, Department of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA Taylor P. Yokoyama Department of Radiology and Biomedical Imaging, University of California, San Francisco, CA, USA N.E., H.N., J.W., M.L., C.N., and R.L. are employees of Taudia, which has filed patent applications related to the SPLASH platform described in this study.

These authors may hold equity in the company. J.S.Y. serves on the scientific advisory board for the Epstein Family Alzheimer’s Research Collaboration, the Charleston Conference on Alzheimer’s Disease, and Taudia, Inc., and is the editor-in-chief of npj Dementia. T.P.J. declares no competing interests.

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