Ion-Exchange Membrane Bio-Sensing in Early Alzheimer's Disease Screening

Alzheimer’s Disease (AD) is the most common form of severe dementia in the world and continues to grow in prevalence as global populations age. AD often goes undiagnosed or underdiagnosed due to current limitations in the gold standards for AD detection. AD has no cure, and current treatments have low efficacy at even mild stages of AD and become even less effective at later stages. Early detection is imperative for favorable patient outcomes. Plasma based diagnostics offer potential for a minimally invasive and easily administered method of early AD screening, even at pre-clinical stages. Unfortunately, the concentration of neuron-derived biomarkers in plasma is below the limit of detection for many commercial tests. This work details the development of two microfluidic platforms. The first platform is for irreversible dissociation and sensing of stable ribonucleoprotein (RNP) constituents, which have been found to be strong early AD biomarkers. The second platform is for colocalized detection of co-expressed AD and extracellular vesicle (EV) biomarkers on carrier neuron derived extra-cellular vesicles (NDEVs). The RNP dissociation platform employs cation exchange membranes to pre-concentrate RNP complexes in a microfluidic channel. The concentrated RNP is found to irreversibly dissociate and isolate under the large electric field in pre-concentration. Integration of a ssDNA functionalized sensor close to the pre-concentrated region in the channel allows for the selective measurement of dissociated RNA and direct quantification of dissociation. However, the lack of established RNA markers for AD prevents AD sample testing with this new technology. The colocalized NDEV platform was used to profile patient plasma at various stages of AD. Strong colocalized NDEV markers were identified that correlated with AD progression. ATP1A3 and SNAP25 expression with CD81 was found to be highly over-expressed in mild cognitive impairment (MCI) patients compared to healthy individuals. ATP1A3 was found to drop in expression with CD63 in these same patients. The total expression of all SNAP25 and ATP1A3 NDEVs was found to slightly drop from healthy to MCI and AD patients. This new result suggests that these overexpressed AD biomarkers are preferentially secreted as ectosomes by the membrane “budding” mechanism via CD81-associated pathway, instead of the usual CD63-associated “endosomal” pathway that secrets exosomes. With selective colocalization probing we not only see the change in ATP1A3 expression on specific extracellular vesicle, but also correlate that change to disease stages. The IEM was successfully benchmarked against two orthogonal assays, SPR and ELISA, that require extensive pretreatment or lack sensitivity for early-stage detection. This platform shows the potential to detect pre-clinical AD but also differentiate between stages of neurodegeneration. Blood based biosensors offer a promising method of pre-clinical AD screening and diagnostics to the aging world population. Both platforms developed in this work have shown pilot scale success and open further doors for easily and rapidly administered diagnostic sensing of AD.