Amyloid Fibril Formation using the UIP400MTP Microplate Sonicator

Amyloid fibrils, much like crystals, form through a process of nucleation and subsequent growth. However, due to the high free-energy barrier of nucleation, spontaneous amyloid fibril formation occurs only after a prolonged lag phase. Ultrasonication has emerged as a powerful tool for inducing amyloid nucleation, thereby significantly accelerating fibril formation. When combined with a microplate reader using thioflavin T (ThT) fluorescence, ultrasonication enables high-throughput detection of amyloid fibrils in multiple samples simultaneously.

Ultrasonically Induced Amyloid Fibril Formation with the UIP400MTP Microplate Sonicator

With the UIP400MTP multi-well plate sonicator, amyloid fibrils of same quality at large quantities can be rapidly synthesized for research purposes. This efficient approach allows studying protein amyloidogenicity. This technique facilitates rapid and reproducible amyloid fibrillation, as demonstrated with β2-microglobulin (β2-m), an amyloidogenic protein associated with dialysis-related amyloidosis.

Simple Experimental Approach: Ultrasonically Induced Amyloid Fibrillation

To induce fibril formation, a 96-well microplate was placed at the center of the UIP400MTP multi-well plate sonicator, which ensures uniform ultrasonic exposure across all wells. The experimental conditions were as follows:

1. Each well contained 0.2 ml of β2-microglobulin solution (0.3 mg/ml, pH 2.5) supplemented with 5 μM ThT.
2. The plate was subjected to ultrasonication cycles, such as 1-minute ultrasonication followed by 9 minutes of pause.
3. Post-sonication, ThT fluorescence was measured using a microplate reader.

(cf. So et al., 2011)

Comparison with Conventional Agitation

Compared to traditional agitation methods, ultrasonication drastically reduced the lag phase of fibril formation. Under conventional microplate shaking conditions, only 1 out of 10 wells exhibited increased ThT fluorescence after 20 hours. In contrast, using cycled ultrasonication (15 minutes sonication followed by 5 minutes quiescence), a significant ThT fluorescence increase was detected immediately after the first sonication treatment.

Rapid Acceleration of Fibrillation Kinetics

The results obtained from So et al. (2011) demonstrated that the spontaneous fibril formation of β2-microglobulin at pH 2.5 can be accelerated from several hours to just 10–15 minutes with ultrasonication.
Atomic force microscopy (AFM) images confirmed that fibrils generated via 10-minute ultrasonication every 15 minutes were morphologically indistinguishable from those formed using 1-minute ultrasonication every 10 minutes. This highlights the reproducibility and robustness of ultrasonically induced amyloid fibrillation.

AFM images of amyloid fibrils produced by 1-min ultrasonication every 10 min (i), by 10-min sonication every 15 min (ii), and by the seeding reaction without ultrasonication (iii). The white scale bar represents 1 μm.
Study and images: ©So et al., 2011

Fibrillation at Neutral pH Conditions

Even under neutral pH conditions, fibril formation was achieved after a lag time of 1.5 hours, demonstrating that ultrasonication significantly lowers the energetic barrier to nucleation and growth. This further supports the hypothesis that amyloid fibrillation is primarily a physical reaction, largely constrained by the nucleation energy barrier, which ultrasonication effectively reduces.

Impact on Amyloid-related Disease Research

The facile and reliable formation of amyloid fibrils using the UIP400MTP microplate sonicator holds significant implications for Alzheimer’s disease (AD) research and other amyloid-related disorders, such as Parkinson’s disease, type II diabetes, and systemic amyloidoses. In AD, amyloid-β (Aβ) aggregation is a key pathological hallmark, yet studying its fibrillation kinetics remains challenging due to long lag phases and variability in conventional methods. Ultrasonication-driven fibril formation accelerates nucleation, ensuring high reproducibility and reduced variability, which is crucial for screening potential inhibitors and understanding amyloidogenic mechanisms. Furthermore, the high-throughput capability of the UIP400MTP enables large-scale investigations into protein misfolding and aggregation, facilitating the discovery of therapeutic agents that can modulate fibril formation and potentially mitigate neurodegenerative progression.

This study establishes ultrasonication using the UIP400MTP multi-well plate sonicator as a highly efficient method for accelerating amyloid fibril formation. The key advantages of this approach include:

• Dramatic reduction in lag time for fibrillation.
• Uniform ultrasound exposure across all wells, enabling reproducible fibril formation.
• High-throughput screening capability, making it suitable for genome-wide searches of protein amyloidogenicity.

By integrating ultrasonication with ThT fluorescence detection, this method provides a rapid, scalable, and reliable platform for studying amyloid fibrillation. Given its efficiency and high-throughput potential, this approach may facilitate the facile synthesis of amyloid fibrils for biophysical and pharmaceutical research, offering a promising tool for amyloid-related studies and drug screening.