Introduction

Neurodegenerative diseases affect tens of millions of people worldwide, significantly increasing disability-adjusted life years, devastating family units and ultimately costing global economies hundreds of billions of dollars annually1,2. The prevalence of such diseases, including Alzheimer’s, Parkinson’s (PD), and Creutzfeldt Jakob Disease (CJD), is predicted to increase in the coming decades2,3,4. These conditions, collectively known as proteinopathies, are associated with misfolding and aggregation of proteins3,5,6,7. Parkinson’s disease, in particular, is a global concern affecting over six million individuals worldwide, with its prevalence predicted to double within the next three decades4,8. PD symptoms can include tremors, walking imbalance, cognitive impairment, and more9.

PD is the most prevalent of a subclass of proteinopathies known as synucleinopathies10. All humans express cellular alpha-synuclein (α-syn) protein. α-syn is found in many tissues, but it is particularly abundant in neural tissue, specifically in presynaptic terminals9,11. In PD, misfolded α-syn fibrils recruit cellular α-syn monomers thus contributing to the build-up of large abnormal aggregates known as Lewy bodies, a common feature found in PD patients12,13,14. Abnormal alpha synuclein aggregates are found throughout the body including in the brain11, skin15,16,17, CSF18, olfactory mucosa19, and submandibular glands20. Currently, clinical diagnosis of symptoms remains the standard for classification due to the lack of readily available antemortem biomarker testing4,21,22,23,24,25. Relying on clinical diagnosis of symptoms greatly limits the effectiveness of potential therapeutics to treat PD. Therefore, to aid the early detection of disease-associated misfolded proteins, it is desirable to create cheap, sensitive assays that can be utilized across a host of pathologies.

Chronic wasting disease (CWD) is a proteinopathy, found in cervids (deer, elk, moose), that negatively impacts both animal health and society. In CWD, native prion proteins misfold and accumulate in lymphatic and nervous tissues. Currently, CWD has been found in 32 US states and 5 other countries (Canada, South Korea, Norway, Sweden, Finland) and is rapidly spreading26,27,28, leading to great concerns for the health and stability of cervid populations and surrounding economies. Moreover, concerns regarding the potential for zoonotic spillover of CWD to wildlife, livestock and humans are increasing29,30. In CWD, post-mortem testing is largely performed using antibody-based enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) tests, as they are the only USDA approved methods for regulatory decisions associated with the disease31. However, these technologies are often costly, time-consuming, and require substantial training and expertise to operate23. In addition, a major limitation of ELISA and IHC assays is that the antibodies routinely used cannot distinguish between native protein and the disease-associated forms such as PrPCWD thus requiring protein digestion steps to enrich for misfolded prion proteins32,33, a methodology that likely reduces diagnostic sensitivity through the destruction of particular prion protein strains32,34,35,36. Collectively, these antibody-based assays have limited performance in the identification of early-stage prion infections; therefore, they are insufficient to facilitate effective CWD management and halt the spread of the disease. In addition, current USDA-approved methods are primarily used on tissues collected post-mortem and, subsequently, unable to aid live-animal testing and surveillance for CWD.

The detection of both human (PD) and animal (CWD) proteinopathies was recently enhanced by various assays involving the amplification of protein misfolding in vitro, including protein misfolding cyclic amplification (PMCA)18,23,37,38,39, end-point quaking-induced conversion (EP-QuIC)40,41 and real-time quaking-induced conversion (RT-QuIC)4,23,42,43,44,45,46,47,48,49. These assays are also known as seeded amplification assays (SAAs). The recent versions of these amplification assays utilize a recombinant mammalian protein substrate (such as prion protein or α-syn) that is incubated and shaken with the diagnostic samples. When the infectious form of misfolded protein is present within a given amplification reaction (such as RT-QuIC), it induces a conformational change of the substrate protein, forming a beta-sheet enriched mixture that is detected by fluorescence signal of Thioflavin T (ThT). Despite the advantages of amplification assays, these methods have limitations, including the need for expensive and large laboratory equipment and complex strategies for visualizing and analyzing results, thus limiting access to diagnostic tests.

To design a diagnostic platform addressing these challenges that can be utilized across multiple neurodegenerative diseases, we first observed that post-QuIC amplified solutions have visible differences in their liquid air interface depending on the presence of misfolded proteins (Fig. 1a). To characterize this in a more quantitative manner, we examine the differences in liquid height for capillary tubes contacted with post-QuIC-amplified misfolded versus native human α-syn associated with Parkinson’s disease. As a secondary model system for validating this novel capillary method (i.e., Cap-QuIC), we also study the prions associated with CWD in wild white-tailed deer (WTD, Odocoileus virginianus).

Fig. 1: Introduction and overview of Cap-QuIC method.
figure 1

a Cuvettes containing post-amplified solutions of misfolded vs. native proteins. Meniscus of air-water interface reveals pattern between misfolded and native samples. b Overview of the Cap-QuIC assay method. Samples are incubated with solutions with concentrated recombinant protein substrate. Contrary to RT-QuIC, which utilizes a plate reader, our QuIC protocol employs a simple thermomixer. This shift not only simplifies the process, but also significantly reduces the overall cost and enhances the portability compared with the conventional RT-QuIC. After the quaking amplification, capillary tubes are placed into the wells and then measured (see methods). A representative image of the capillaries is shown on the right.

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Our experiments showed a clear difference in the capillary action of solutions containing misfolded and native conformations of both α-syn and prions (Figs. 1b–3). We propose that these variations in capillary action are due to the effects of protein surface modification on the hydrophobicity of a glass capillary as supported by the experiments in Supplementary Fig. 1 and main Figs. 4 and 5. Utilizing this phenomenon, we present the capillary quaking-induced conversion (Cap-QuIC) method for misfolded protein detection. With the proof-of-concept experiments illustrating its potential for α-syn and CWD diagnostics, we posit that with further development Cap-QuIC could have utility not only in PD and CWD diagnostics but also in a variety of protein misfolding diseases such as Alzheimer’s, ALS, and CJD.

Fig. 2: Comparison of capillary action for α-syn and PrP.
figure 2

a Differences in capillary height for native (neg) (n = 12, average: 44.2 mm, stdev: 1.3 mm), spontaneous misfolded solutions (pos) (n = 12, average: 49.1 mm, stdev: 2.0 mm), and buffer only control (n = 12, average: 50.3 mm, stdev: 1.3 mm) for α-syn. b Differences in capillary height for CWD negative lymph node (neg) (n = 8, average: 42.0 mm, stdev: 1.1 mm), misfolded solutions from CWD positive lymph node (pos) (n = 8, average: 51.1 mm, stdev: 1.4 mm), and buffer only control (n = 8, average: 52.6 mm, stdev: 2.3 mm). ɑ-syn = recombinant human ɑ-syn substrate, PrP = recombinant hamster prion protein substrate. p = 1.2 × 10−6 and 2.1 × 10−9 for ɑ-syn and PrP, respectively (two sample T-test).

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Fig. 3: Cap-QuIC for CWD detecion in WTD.
figure 3

a Comparison of the detection of misfolded proteins in QuIC reactions seeded with CWD-positive and CWD-negative lymph node tissue samples, using both capillary action and ThT fluorescence. Each sample was replicated eight times. A sample was classified as positive if at least four out of eight replicates showed misfolded protein detection. b A correlation between average capillary height and average ThT fluorescence from the eight replicates of each tissue sample. The horizontal axis shows the sample ID number. These results illustrate the striking correspondence between the capillary-based method and ThT fluorescence detection in identifying misfolded proteins in tissue samples. (for a and b; horizontal axis shows sample ID; meta data in Supplementary Table 1 error bars show stdev).

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Fig. 4: Mechanism overview.
figure 4

a Schematic of the proposed mechanism of action. Native and substrate proteins coat a larger section of the interior surface of the capillary causing macroscale changes to its hydrophobicity compared to misfolded protein. b Capillary distances traveled by buffer solution in the capillaries pre-treated with negative (native), positive (misfolded), or buffer (no protein) samples. Eight and six technical replicates for PrP and alpha syn respectively (error bars show stdev). c Example of contact angle measurements of glass slides pre-treated with misfolded PrP vs native PrP. The dashed line denotes the glass surface, distinguishing the droplet from the reflection of the droplet. d Contact angle measurements for glass slides pre-treated with PrP and α-syn.

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Fig. 5: Concentration experiments.
figure 5

The effect of diluting post-amplified misfolded and native solutions on capillary distance for (a) α-syn (n = 4 per condition) and (b) prion (n = 6 per condition). Error bars show stdev.

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Results

Capillary action comparison between misfolded and native protein

Misfolded/native α-syn and CWD positive/negative tissue samples were seeded into QuIC reactions containing recombinant α-syn or PrP substrates, respectively. After a 24-h amplification period on a thermomixer, glass capillaries (0.4 mm inner diameter) were immersed in the amplified solutions (Fig. 1b). Both α-syn and PrP showed a significant difference between samples seeded with misfolded protein and samples with native protein (Fig. 2a, b, p = 1.2 × 10−6 and 2.1 × 10−9 respectively), demonstrating the utility of capillaries for classifying post-amplified solutions of misfolded and native proteins. Capillary distances of 46 mm and 45 mm for α-syn and PrP respectively, were determined as the cutoff values distinguishing misfolded vs. native samples. Where below the threshold indicates the presence of native protein and above the threshold indicates the presence of misfolded protein.

Performance comparison between capillary-based detection and Thioflavin T (ThT) fluorescence detection

To compare how capillary-based detection performs compared to the current gold standard, Thioflavin T (ThT) fluorescence detection, we selected a set of 40 WTD retropharyngeal lymph nodes (20 CWD positive and 20 CWD negative samples, Supplementary Table 1). These tissues were seeded with 8 replicates each into QuIC reactions with ThT on a 96-well plate that were then shaken and incubated for 24 h on a thermomixer. After amplification, ThT fluorescence from the plate was measured and a capillary was immersed into each well. We found that for a threshold of 50% of technical replicates crossing the 45 mm threshold, capillaries classified the tissues with 90% sensitivity and 95% specificity. The comparison revealed a high degree of agreement between capillary- and ThT fluorescence-based tests. When comparing each specific technical replicate with capillary and ThT, capillary classification matched the state-of-the-art ThT classification for 306 of the total 320 technical replicates (95.6%). (Fig. 3a, b). Additionally, the trends in the average height of the capillaries for each tissue sample correlated well with the average fluorescence of each tissue sample, indicating a robust performance of the Cap-QuIC method (Fig. 3b).

Understanding the mechanism underlying capillary detection

Capillary action is the affinity of liquids to travel through small tubes and porous material without, and even in opposition to, external forces such as pumps, gravity, etc50. It is a result of the balance of intermolecular forces of cohesion (a liquid’s affinity to itself) and adhesion (a liquid’s affinity to surfaces it is in contact with). Liquids are always trying to minimize their free surface energy. For example, it is less energetically favorable for a water solution to be in contact with a hydrophobic surface thus it will minimize its surface area by beading up. For hydrophilic surfaces the opposite is true, it is more energetically favorable for the water solution to interact with the solid surface, thus the liquid will maximize its surface area by spreading out on a solid it is in contact with. In a vertical capillary tube, the hydrophobicity of the inner capillary surface and the force of gravity combine to determine the rise of a solution in a capillary tube.

We propose that a large portion of the observed difference in capillary height arises from the differential binding behavior of native proteins and their misfolded counterparts to the glass capillary surface (Fig. 4a). Monomeric native proteins are posited to bind to the capillary surface, whereas misfolded proteins prone to forming large aggregates, result in a diminished amount of available protein for surface binding. This reduces the amount of protein available to coat the surface, rendering solutions containing misfolded proteins nearly indistinguishable from buffer controls. Once bound, the native proteins increase the hydrophobicity of the capillary tube wall, making capillary action less energetically favorable as reflected by the native protein solutions giving a lower capillary height.

To scrutinize this mechanism, we incubated capillaries in solutions containing either native, misfolded, or no proteins (buffer-only control) for both PrP and α-syn. After subsequent rinsing and drying steps, we immersed the treated capillaries into a buffer control solution. For both α-syn and PrP, there was a clear difference between capillaries treated with native protein versus the other solutions (Fig. 4b), indicating that native proteins persistently bind to the inner surface of the capillary, even after rinse and drying steps.

To further support the capillary effects seen are due to protein binding to the inner wall, we incubated capillaries in poly(diallyldimethylammonium chloride) (PDDA) solution. PDDA combined with the Sodium Dodecyl Sulfate (SDS) in QuIC solutions creates a layer that passivates the capillary walls and blocks the protein from binding to bare glass. When capillaries incubated in PDDA were immersed in solutions of native, misfolded, and buffer-only controls, the difference between the capillary heights disappeared (Supplementary Fig. 1) because an insufficient quantity of protein was allowed to bind to the walls. This supports the hypothesis that the difference in capillary distance is due to protein binding to the inner capillary wall.

Characterizing surface hydrophobicity and protein concentration effects

The hydrophobicity of a surface can be gauged by how much a droplet of water spreads out or beads up on the surface. When liquid spreads out, the angle the edge of the drop makes with the surface is small and the surface is said to be hydrophilic. Conversely, when the liquid beads up the angle is large and the surface is deemed more hydrophobic (Fig. 4c). Droplet contact angle measurements were performed to examine the effect of native, misfolded, and buffer-only controls on the hydrophobicity of glass surfaces. Glass slides were incubated in solutions and then put through rinse and wash cycles. Droplets of DI water were then applied to the glass surface and the profile of the droplets was analyzed (Fig. 4c). It was found that for both PrP and α-syn the native protein caused the glass to become more hydrophobic than solutions incubated with misfolded protein or buffer-only controls (Fig. 4d). This supports our hypothesis that native proteins bind to the glass to a larger extent, increasing its hydrophobicity thus decreasing capillary action.

To examine the effects protein concentration has on capillary height, post-amplified solutions were diluted in buffer (containing no proteins) from concentrations of 0% (buffer only) to 100% (undiluted post-amplified sample). These solutions were then contacted with capillary tubes. For both α-syn and PrP, we found that diluting the misfolded samples did not affect the capillary height significantly (Fig. 5a, b). Since diluting the solution does not affect the capillary height, we propose that the misfolded protein is simply not present in large enough free-floating quantities to effectively coat the inner surface area of the capillary. Since monomeric protein has aggregated into large fibrils it cannot interact with enough glass surface area to make a macroscopic difference in the capillary height. On the other hand, native proteins showed a strong trend with dilution and capillary height (Fig. 5a, b). As the native solutions become more diluted, there is less free-floating protein to interact with the inner walls of the capillary and thus the stunted capillary height disappears.

Discussion

In this work, we demonstrated that using glass capillaries to interact with post-QuIC amplified solutions of both PrP and α-syn (Cap-QuIC) results in statistically significant differences in capillary action. Our Cap-QuIC method showed diagnostic utility in detecting protein misfolding disease in real tissue samples from 40 WTD, with capillary classification matching the state-of-the-art ThT classification in 95.6% of replicates. Using protein and PDDA coating experiments as support, we suggest that the native proteins coat the inner walls of the capillaries, leading to the observed differences in capillary action between native and misfolded protein solutions. Contact angle measurements show that native proteins render glass surfaces more hydrophobic than misfolded protein solutions. Furthermore, dilution experiments of post-amplified solutions revealed that only the capillary action of native protein solutions has a concentration dependence. This indicates that misfolded protein solutions likely do not have sufficient free-floating proteins to cause a macroscopic difference in capillary height.

Given the growing prevalence of protein misfolding diseases in both human and animal populations (e.g., PD, Alzheimer’s, CWD, etc.), the development of effective point-of-care diagnostic assays are imperative. Ideally, diagnostic solutions would not require costly equipment or extensive training and would be readily applicable to a host of diseases. This would allow testing not to be exclusively limited to large, well-equipped urban diagnostic labs in developed countries. To help solve this issue, assays such as RT-QuIC are being developed for a host of misfolding diseases4,23,44,45,46,47,51. While the RT-QuIC method is sensitive and relatively simple to perform, it requires tens of thousands of USD worth of capital equipment to run a single plate. This may be acceptable for a large well-funded research laboratory performing a limited number of tests, but for laboratories with high demand for machine time or small laboratories in developing nations, the expense of extra equipment quickly becomes a bottleneck. Additionally for applications in wildlife disease surveillance such as CWD, having diagnostic testing outside of a traditional laboratory is highly desirable52.

While it is almost certain RT-QuIC will play an important role in mitigating the impact of proteinopathies in the future, there is still a need for robust inexpensive testing. Cap-QuIC leverages the power of sensitive QuIC amplification while only requiring one-fifth of the specialized equipment cost of RT-QuIC. The thermomixer used in Cap-QuIC is robust and portable and has been shown to perform well in non-laboratory settings52. Since the Cap-QuIC method leverages simple capillary action, it can be potentially integrated into microfluidic systems to enhance point-of-care technologies for a host of proteinopathies. One such microfluidic system utilizes acoustic agitation to amplify and detect misfolded proteins53. The current form of the Cap-QuIC protocol should be done in a BSL-2 setting. There is evidence supporting that amplification assays performed with truncated recombinant PrP (as used in Cap-QuIC for CWD) do not amplify the infectivity of the original sample significantly, likely due to a lack of cofactors54,55,56. However, the infectivity of QuIC products of full-length human α-syn is not known and thus advanced safety precautions such as a biosafety cabinet should be employed until future research on sample infectivity is performed.

By integrating QuIC amplification and glass capillaries, we have demonstrated a novel method for distinguishing misfolded proteins from their native counterparts. Because Cap-QuIC leverages the universal characteristic of aggregation and clumping associated with protein misfolding diseases, we hypothesize, that with further development, capillary action-based assays would be useful for diagnostics in a whole host of diseases such as PD, Alzheimer’s, BSE, and CWD. To support this notion, we successfully distinguished native from misfolded protein for α-syn and PrP, two relevant proteins associated with CWD and PD, respectively.

For the CWD study, we performed experiments on a set of medial retropharyngeal lymph node tissues from 40 wild WTD (20 CWD positive and 20 negative based on regulatory ELISA and IHC methods). Our findings revealed that the capillary classification for each technical replicate (8 per sample) across all 40 samples matched the state-of-the-art ThT classification for 306 of the total 320 technical replicates (95.6%). Utilizing the 50% replicate threshold, capillaries classified WTD samples with 90% sensitivity and 95% specificity. Using the ThT classification gave 90% sensitivity and 95% specificity. It should be noted that one of the samples Cap-QuIC missed was picked up by ThT, and vice versa. Thus, the two positive samples the capillaries mis-classified were likely not due to a flaw with capillary readout but rather a suboptimal 24 h QuIC protocol. Often QuIC assays are performed on plate reader systems and routinely get sensitivity >90%23 with lymph node samples thus it is suspected that future work needs to be done to optimize the thermomixer QuIC amplification protocol. However, these experiments still give strong evidence that capillaries can be used to classify misfolded vs native proteins.

A key advantage of QuIC-based amplification is that advances in methods such as RT-QuIC could likely be adapted for Cap-QuIC. For instance, RT-QuIC protocols have been developed for various antemortem diagnostics15,43,57,58,59. Moreover, our recent work on nanoparticle-enhanced QuIC (Nano-QuIC)60 demonstrates the potential to enhance the speed, sensitivity, and specificity of the protein amplification assay in complex tissue samples. Thus, we posit that Cap-QuIC offers great promise for the visual detection of a host of neurodegenerative diseases. By eliminating the need for delicate or expensive equipment and being readily compatible with microfluidic point-of-care technologies, our proof-of-concept hybrid assay has the potential to greatly increase access to neurodegenerative testing. With further development, we envision that methods like our Cap-QuIC assay could be deployed in clinics around the world for early detection of neurodegenerative disease, ultimately aiding to early application of diagnostics.

Methods

Sample collection

This study used medial retropharyngeal lymph node samples obtained opportunistically for CWD testing purposes from white-tailed deer that were hunter-harvested or culled by the Minnesota Department of Natural Resources (MN DNR). None of the animals were specifically sacrificed for this study. No animal use protocol approval was required. WTD RPLNs were homogenized in PBS (10% w:v) in 2mL tubes containing 1.5 mm zirconium beads with a BeadBug Homogenizer (Beanchmark Scientific, Sayreville New Jersey USA) on max speed for 90 s.

QuIC amplification of misfolded prions

For QuIC analysis (both Cap-QuIC and RT-QuIC), a PrP master mix was made to the following specifications: 1X PBS, 1 mM Ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl, 10 μM thioflavin T (ThT), and 0.1 mg/mL rPrP (~6.25 µM). Recombinant hamster PrP (HaPrP90-231) production and purification followed methods in Schwabenlander et al.46 In instances where the end reaction would be analyzed using capillaries, ThT could be excluded. The 10% tissue homogenates (prepared as described above) were further diluted 100-fold in 0.1% Sodium Dodecyl Sulfate (SDS) using methods from Schwabenlander et al.46 (final tissue dilution: 0.1%). 1 μl of N-2 Supplement [Life Technologies Corporation, Carlsbad, California, USA]) was added to 99 μl dilution of SDS (for some Cap-QuIC reactions N-2 could be excluded from SDS). 2 μL of the diluent was added to each well containing 98 μL of master mix. Plates for Cap-QuIC were amplified on a ThermoMixer® C equipped with SmartBlock plate and Thermotop (Eppendorf, Enfield, Connecticut, USA) for 24 h at 48 °C at 600 rpm.

Plates for RT-QuIC were amplified on a FLUOstar® Omega plate reader (BMG Labtech, Cary, North Carolina, USA; 42 °C, 700 rpm, double orbital, shake for 60 s, rest for 60 s). Fluorescent readings were taken at ~45 min increments.

PrP capillary experiments

After amplification as described above, plates were removed from the ThermoMixer® C. Each well was then contacted with a 0.4 mm × 75 mm (ID × L) borosilicate capillary tube (Drummond Scientific Company, Broomall, PA USA) for 1 min. Capillaries were then removed and imaged.

Spontaneous α-synuclein seeds

To make a spontaneously misfolded α-synuclein seed, a master mix was made to the following specifications: 1X PBS, 1 mM Ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl, 10 μM thioflavin T (ThT), and 7.14 μM α-synuclein (from R&D systems). These plates were shaken on a FLUOstar® Omega plate reader (BMG Labtech, Cary, North Carolina, USA; 42 °C, 700 rpm, double orbital, shake for 60 s, rest for 60 s) for 3–4 days until spontaneous misfolding occurred. These solutions were then frozen and used to seed future α-synuclein reactions.

QuIC amplification of α-synuclein

For Cap-QuIC, to create positive seeds 10 µl of the spontaneously misfolded seed (described above) was added to 90 μl of 0.1% SDS in 1X PBS (no N2). To create misfolded α-synuclein samples, 2 μl of this seed solution was added to a 96-well plate with an α-synuclein master mix made to the following specifications: pH 5.8 PBS buffer (120 mM phosphate, 137 mM NaCl, 2.7 mM KCl), 1 mM Ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl, 10 μM thioflavin T (ThT), and 6.43 μM α-synuclein (R&D systems). To create negative α-synuclein samples, 2 ul of 0.1% SDS was added to the α-synuclein master mix solution. All samples were shaken for 24 h on a ThermoMixer® C equipped with SmartBlock plate and Thermotop (Eppendorf, Enfield, Connecticut, USA).

α-synuclein capillary experiments

After amplification as described above, plates were removed from the ThermoMixer® C. Each well was then contacted with a 0.4 mm × 75 mm (ID × L) borosilicate capillary tube (Drummond Scientific Company, Broomall, PA USA) for 1 min. Capillaries were then removed and imaged.

α-synuclein and PrP dilution experiments

For the dilution experiments in Fig. 5a, Post-amplified α-synuclein solutions with and without spontaneously misfolded seeds were diluted in α-synuclein dilution buffer (pH 5.8 phosphate buffer[see above], 1 mM Ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl, 10 μM thioflavin T (ThT), and 1/5X PBS.) to six concentrations ranging from 0% (all buffer) to 100% (straight off the amplified plate). Each dilution had four replicates. Capillaries were inserted into each replicate well for 1 min. After this, the capillaries were imaged. The process for PrP concentration experiments in Fig. 5b was much the same.

Post-amplified PrP solutions with seeds from CWD positive and negative tissue were diluted in PrP dilution buffer (1X PBS, 1 mM Ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl, 10 μM thioflavin T (ThT), and 1.4 mM pH 6.1 phosphate buffer) to six PrP concentrations ranging from 0% (all buffer) to 100% (straight off the amplified plate). Each dilution had six replicates. Capillaries were inserted into each replicate well for 1 min. After this, the capillaries were imaged.

PrP and α-synuclein coating experiments

For PrP coating capillary experiments (Fig. 4b), capillaries were filled with and incubated for 10 min in post-amplified PrP solutions with seeds from CWD positive tissue or with seeds from negative tissue or with dilution buffer (see above) with no PrP. To dry, liquid was removed from the capillary using the wicking properties of a KimwipeTM and then dried in a stream of air. The capillaries were then filled with and incubated in deionized water for 1 min and dried. This water rinse was repeated once for a total of two water rinses. After preparation, capillaries were inserted into wells containing PrP dilution buffer for 1 min. The capillaries treated with misfolded PrP, native PrP and PrP dilution buffer all had eight replicates. The capillaries were then removed and imaged. α-synuclein coating capillary experiments were performed in much the same way. Capillaries were filled with and incubated for 10 min in post-amplified misfolded α-synuclein solutions or nonmisfolded α-synuclein solutions or with α-synuclein dilution buffer (see above). To dry, liquid was removed from the capillary using the wicking properties of a KimwipeTM and then dried in a stream of air. The capillaries were then filled with and incubated in deionized water for 1 min and dried. This water rinse was repeated once for a total of two water rinses. After preparation, capillaries were inserted into wells containing α-synuclein dilution buffer for 1 min. The capillaries treated with misfolded α-synuclein, native α-synuclein, and α-synuclein dilution buffer all had four replicates.

PrP and α-synuclein PDDA experiments

For coating experiments in Supplementary Fig 1 capillaries were incubated and filled with a solution of 1 mg/ml poly(diallyldimethylammonium chloride) (PDDA) MW 200–350 kDa (MilliporeSigma) in deionized water. Capillaries were left to incubate for 1 h. To dry, liquid was removed from the capillary using the wicking properties of a KimwipeTM and then dried in a stream of air. Capillaries were then filled and incubated with deionized water for 10 min and then dried. Capillaries were then filled and incubated with deionized water for 1 min and then dried. After preparation, PDDA-coated capillary tubes were inserted into wells of post-amplified CWD positive, post-amplified CWD negative or PrP dilution buffer solutions. There were eight replicates for each solution. After 1 min of incubation, the capillaries were removed and imaged. For α-synuclein PDDA coating experiments in Supplementary Fig 1 PDDA coated capillaries were prepared identically to the PrP PDDA experiment. After preparation, PDDA-coated capillary tubes were inserted into wells of post-amplified misfolded α-synuclein, post-amplified native α-synuclein or α-synuclein dilution buffer solutions. There were six replicates for each solution. After 1 min of incubation, the capillaries were removed and imaged.

Statistical methods

Unless otherwise noted, significance was determined using a two-tailed, two-sample t-test in Origin 2022b software (significance level = 0.05). Equal variance was not assumed and compensated for using Welch correction.

Supplementary information

The Supplementary Information is available free of charge via the NPJ Biosensing website. Supplementary information includes Supplementary Fig. 1 and the metadata for the samples used in Fig. 3.