(341b) An Electrokinetic Platform for the Detection of Alzheimer’s Disease from Colocalized Surface Proteins on Exosomes

Alzheimer’s is one of the most prevalent neurodegenerative diseases affecting more than 6 million people, with 1 in 3 seniors dying with Alzheimer’s or another form of dementia. Alzheimer’s disease is notable for the buildup of Amyloid Beta plaques and neurofibrillary tangles in neurons causing loss of neuron function and cell death. Over the years methods of Alzheimer’s detection have developed from post mortem positron emission tomography (PET) to cognitive tests, like MMSE and MoCA, and cerebral spinal fluid (CSF) protein screening. While easily repeatable, cognitive tests require a large amount of direct patient time with a provider and their results can be subjective. CSF testing can be potentially dangerous and not as easily repeated for progression or drug efficacy studies. There is great need for a safe and rapid clinical test that can screen Alzheimer’s progression. Recent studies have found that neuron derived exosomes (NDEs) can cross the blood-brain barrier and are found in plasma in sufficient concentration for diagnostics. Groups have shown that expression levels of hallmark Alzheimer’s markers, like glial fibrillary acidic protein (GFAP), neurofilament light (NfL) and phosphorylated tau (pTau), in plasma correlate with those for the same markers in CSF and that there is true potential in neurodegenerative diagnosis from a plasma sample. We looked for these targets on the surface of NDEs using an ion exchange membrane-based electrokinetic sensor. As the Alzheimer markers are also released by non-neurons, the key challenges are to identify the NDEs from the large exosome population in blood and to detect the low-concentration (single-copy) markers on them. We identify markers on NEDs with a sandwich assay that uses surface capture antibodies (for the Alzeheimer markers) functionalized to a nanoporous ion-selective membrane and a reporter antibody (for neuron markers) conjugated to a charged silica nanoparticle reporter. To achieve the requisite sensitivity, we use electric-field induced ion-depletion action of the membrane to reduce Debye screening of the charged reporters amplify their effect on the ion current through the membrane, without electrochemical reaction (see [1, 2] for a description of the sensor). We are able to measure picomolar concentrations of exosomes with a dynamic range of three decades, corresponding to 0.1% to 100% of the exosome concentration in plasma. The ion-depletion action not only amplifies the charge signal but also enhances reproducibility by eliminating sensitivity to ionic strength variations, which is a key issue with Field-Effect Transistor charge sensors. Capture proteins included neurodegenerative markers NfL, GFAP, pTau181, pTau217, pTau231 and Amyloid Beta using L1 cell adhesion molecule ( L1CAM) as a reporter. NfL, GFAP and pTau concentrations were significantly increased on the surface of plasma NDEs from a small cohort of Alzheimer patients and healthy individuals we tested. This sensing platform allows for a rapid and precise measurement of multiple NDE surface biomarkers and the potential for colocalization studies of exosomal surface markers for the screening and progression monitoring of Alzheheimer and other neurological diseases.

[1] McCarthy, K., Go, D. B., Senapati, S. and Chang, H.-C., “An Integrated Ion-Exchange Membrane based microfluidic chip for irreversible dissociation and quantification of miRNA from ribonucleoproteins”, LabChip, 23:285-294(2022).

[2] Kumar, S., Maniya, N., Wang, C., Senapati, S. and Chang, H.-C., “Quantifying PON1 on HDL with Nanoparticle-Gated Electrokinetic Membrane Sensor for accurate cardiovascular risk assessment”, Nature Comm, 14:557(2023).