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Multispectral Nanoparticle Tracking Analysis for the Real-Time and Label-Free Characterization of Amyloid-β Self-Assembly In Vitro.

ABSTRACT: The deposition of amyloid β (Aβ) plaques and fibrils in the brain parenchyma is a hallmark of Alzheimer's disease (AD), but a mechanistic understanding of the role Aβ plays in AD has remained unclear. One important reason could be the limitations of current tools to size and count Aβ fibrils in real time. Conventional techniques from molecular biology largely use ensemble averaging; some microscopy analyses have been reported but suffer from low throughput. Nanoparticle tracking analysis is an alternative approach developed in the past decade for sizing and counting particles according to their Brownian motion; however, it is limited in sensitivity to polydisperse solutions because it uses only one laser. More recently, multispectral nanoparticle tracking analysis (MNTA) was introduced to address this limitation; it uses three visible wavelengths to quantitate heterogeneous particle distributions. Here, we used MNTA as a label-free technique to characterize the in vitro kinetics of Aβ_1-42 aggregation by measuring the size distributions of aggregates during self-assembly. Our results show that this technology can monitor the aggregation of 10⁶-10⁸ particles/mL with a temporal resolution between 15 and 30 min. We corroborated this method with the fluorescent Thioflavin-T assay and transmission electron microscopy (TEM), showing good agreement between the techniques (Pearson's r = 0.821, P < 0.0001). We also used fluorescent gating to examine the effect of ThT on the aggregate size distribution. Finally, the biological relevance was demonstrated via fibril modulation in the presence of a polyphenolic Aβ disruptor. In summary, this approach measures Aβ assembly similar to ensemble-type measurements but with per-fibril resolution.

SUBMITTER: Moore C

PROVIDER: S-EPMC8411845 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Micromotor (MM) technology offers a valuable and smart on-the-move biosensing microscale approach in clinical settings where sample availability is scarce in the case of Alzheimer's disease (AD). Soluble amyloid-β protein oligomers (AβO) (mainly AβO42) that circulate in biological fluids have been recognized as a molecular biomarker and therapeutic target of AD due to their high toxicity, and they are correlated much more strongly with AD compared to the insoluble Aβ monomers. A graphene oxide (GO)-gold nanoparticles (AuNPs)/nickel (Ni)/platinum nanoparticles (PtNPs) micromotors (MMGO-AuNPs)-based electrochemical label-free aptassay is proposed for sensitive, accurate, and rapid determination of AβO42 in complex clinical samples such as brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. An approach that implies the in situ formation of AuNPs on the GO external layer of tubular MM in only one step during MM electrosynthesis was performed (MMGO-AuNPs). The AβO42 specific thiolated-aptamer (AptAβO42) was immobilized in the MMGO-AuNPs via Au-S interaction, allowing for the selective recognition of the AβO42 (MMGO-AuNPs-AptAβO42-AβO42). AuNPs were smartly used not only to covalently bind a specific thiolated-aptamer for the design of a label-free electrochemical aptassay but also to improve the final MM propulsion performance due to their catalytic activity (approximately 2.0× speed). This on-the-move bioplatform provided a fast (5 min), selective, precise (RSD < 8%), and accurate quantification of AβO42 (recoveries 94-102%) with excellent sensitivity (LOD = 0.10 pg mL-1) and wide linear range (0.5-500 pg mL-1) in ultralow volumes of the clinical sample of AD patients (5 μL), without any dilution. Remarkably, our MM-based bioplatform demonstrated the competitiveness for the determination of AβO42 in the target samples against the dot blot analysis, which requires more than 14 h to provide qualitative results only. It is also important to highlight its applicability to the potential analysis of liquid biopsies as plasma and CSF samples, improving the reliability of the diagnosis given the heterogeneity and temporal complexity of neurodegenerative diseases. The excellent results obtained demonstrate the analytical potency of our approach as a future tool for clinical/POCT (Point-of-care testing) routine scenarios.

Dataset Information

Multispectral Nanoparticle Tracking Analysis for the Real-Time and Label-Free Characterization of Amyloid-β Self-Assembly In Vitro.

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