Nanoparticle-Enhanced RT-QuIC (Nano-QuIC) Diagnostic Assay for Misfolded Proteins

Abstract

Misfolded proteins associated with various neurodegenerative diseases often accumulate in tissues or circulate in biological fluids years before the clinical onset, thus representing ideal diagnostic targets. Real-time quaking-induced conversion (RT-QuIC), a protein-based seeded-amplification assay, holds great potential for early disease detection, yet challenges remain for routine diagnostic application. Chronic Wasting Disease (CWD), associated with misfolded prion proteins of cervids, serves as an ideal model for evaluating new RT-QuIC methodologies. In this study, we investigate the previously untested hypothesis that incorporating nanoparticles into RT-QuIC assays can enhance their speed and sensitivity when applied to biological samples. We show that adding 50 nm silica nanoparticles to RT-QuIC experiments (termed Nano-QuIC) for CWD diagnostics greatly improves the performance by reducing detection times 2.5-fold and increasing sensitivity 10-fold by overcoming the effect of inhibitors in complex tissue samples. Crucially, no false positives were observed with these 50 nm silica nanoparticles, demonstrating the enhanced reliability and potential for diagnostic application of Nano-QuIC in detecting misfolded proteins.

Seeding activity of human superoxide dismutase 1 aggregates in familial and sporadic amyotrophic lateral sclerosis postmortem neural tissues by real-time quaking-induced conversion

“…From the fits from Eq. 1 , we used these four parameters to calculate 50% ThT fluorescence and lag phase (time to a designated threshold of positive fluorescence), as previously described [ 16 ]. For each ALS patient spinal cord and motor cortex dilution, we calculated mea n ± standard deviation for lag phase and 50% ThT fluorescence, and graphed 50% ThT fluorescence versus lag phase for each ALS neural tissue dilution.…”

“…where y 0 is initial ThT fluorescence, A is ThT amplitude (final – initial fluorescence), x 0 is time to reach 50% ThT fluorescence, and τ is a fibril time constant. The time to fluorescence positivity threshold or lag phase is equal to x 0 – 2 τ [ 16 ]. We fit our raw data to this model for each kinetic curve using constraints > 0, to estimate x 0, y 0 , τ , and A .…”