Electrified nanopipettes decrease diagnostic time for amyloid diseases from days to hours

When it comes to neurodegenerative diseases, patients want an inexpensive, reliable, and rapid diagnostic test. However, certain diseases like Parkinson’s or Alzheimer’s, categorized as amyloid diseases for their characteristic accumulation of pathogenic proteins with amyloid morphology, require a certain amount of these amyloids in the brain before the diagnosis becomes conclusive. Therefore, as soon as we reach that threshold, patients want to know our technology can identify their change in homeostatic state and give more time to undertake new therapeutic strategies.

That’s what motivated the authors of this study to harness solid-state nanopore technology and detect amyloid seeds at the single-cell level. Instead of the real-time quaking induced conversion assay currently used in the clinic, the authors Meyer et al. developed a new assay for real time fast amyloid seeding and translation, called RT-FAST for short. This assay takes advantage of protein adsorption to capture and amplify our proteins of interest and resistive pulse sensing to analyze the size, shape, and quantity of these early disease markers.

Figure 1.(A-E) Cartoon schematic and photographs of the RT-FAST amyloid capture, amplification and detection of α-synuclein (α-syn) seeds. (G) The “disruption” events of the resistive pulse sequencing, depicted as large spikes that denote the stoppage of current at the pore opening of the nanopipette. Image used with permission from Meyer et al. ACS Cent. Sci. 2022, 8 (4), 441-448.

In the RT-FAST method, a small clinical sample (around 35 μL or less than a drop of water) is introduced into a glass nanopipette (Fig 1A and 1B), containing the disease associated proteins – like α-synuclein in Parkinson’s disease. The proteins attach to the walls of the reservoir, accumulating on the walls as the sample sits in the nanopipette and amplifying the initial “seeding” event. This leads to many amyloid fibrils and the formation of pathogenic aggregates in the pipette. Every 30 minutes, a voltage is applied across the reservoir channel. As these pathogenic aggregates grow large enough, they are able to disrupt the voltage and minimize the current detected by the sensor, quantified by the number of “events” (Figs. 1C, 1D). A low number of events (Fig 1G, blue) indicate the absence of amyloid proteins, while a high number of disruptive events (Fig 1G, red and yellow) indicate a large amount of amyloid fibrils – and a higher likelihood of an amyloid-related disease diagnostic.

Importantly, some variations of α-synuclein show a stronger correlation with disease onset, i.e., they are more likely to form amyloid fibrils than the kind that is normally expressed in our brains. One of these variants, known as A53T (wherein the 53rd residue in the α-synuclein protein sequence is changed from an alanine to a threonine), is linked to familial and early-onset Parkinson’s disease.

The difference between their disease-causing ability is reflected in the difference between wildtype and A53T to generate amyloid fibrils in the glass nanopipette. The WT seed forms large oligomers (multi-protein structures), though these oligomers are susceptible to rapidly returning to their monomeric form as quickly as the oligomer forms (Fig 2). In contrast, the A53T sample seemed to form larger, more stable amyloid fibrils, causing a greater number of current-disrupting events over time. The authors concluded that not only did A53T variant have a greater propensity for aggregation (Fig 3A vs. 3B), but also they showed greater diversity in the types of aggregates formed, as indicated by the wide deviation of detected events (the range of dots in Fig 3C compared to dots in Fig 3D).

Figure 2. The various aggregation and fibril forming pathways that α-synuclein can undertake. On-pathway indicates amyloid fibril forming pathways while off-pathway and disordered aggregates indicate off-target pathways that will cause disruptive events but not indicate amyloid formation. Certain α-synuclein variants like A53T are more prone to form amyloid fibrils than the wild-type. Image adapted from Meyer et al. ACS Cent. Sci. 2022, 8 (4), 441-448.

The authors Meyer et al. also demonstrated two additional benefits for their RT-FAST assay. Not only was this test proven to be reliably qualitative, confidently identifying the presence or absence of amyloid-forming proteins, but showed quantitative results. The initial amount of α-synuclein strongly influenced the number of disruptive events seen in their resistive pulse sequencing output, whereby a larger initial quantity led to more events over time. Additionally, shown in Figure 3E, whether there was 2, 20, or 200 picomolar (pM) concentrations of wild-type α-synuclein, the same number of events after a 30 minute incubation was seen as after the 90 minute incubation. This indicated that their signal was stable over time and suggested that time of sample collection and analysis would not influence the diagnostic results. This is especially important for clinical samples as the clinicians would not have to worry about how long after collection their samples would need to be run to obtain accurate results.

Figure 3. (A-B) Comparison of the number of disruption events (f) to time in minutes for α-synuclein WT and A53T seeds to a control sample (no or random artifact events). (C) Comparison of the intensity of the disruption events for the control, WT, and A53T α-synuclein samples in three samples (n1, n2, n3). The dots represent the events seen over the one-hour period. (C-D) Comparison of the intensity of the disruptive events (ΔI/I0) for wild-type (WT) α-synuclein seeds and A53T α-synuclein seeds. (E) Comparison of the number of disruption events per second to the initial seed concentration (in picomolar or pM) after 30- and 90-minute incubation periods for WT α-synuclein compared to control. The similarity between the samples indicate that the sample can be processed for 30 minutes and give consistent results even hours later. Image adapted from Meyer et al. ACS Cent. Sci. 2022, 8 (4), 441-448.

As an inexpensive, rapid, and accurate diagnostic test, the RT-FAST meets all the marks, with strong potential for further optimization for quantitative analysis. Additionally, the RT-FAST could distinguish between wild-type and more pathogenic variants of α-synuclein, allowing an earlier diagnosis for those with strong risk of early onset or familial Parkinson’s. We may soon be able to diagnose individuals with Parkinson’s disease or other amyloid diseases with a common glass pipette and electric current output.

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