Laser Emission of Thioflavin T Uncovers Protein Aggregation in Amyloid Nucleation Phase

Abstract

There is currently no definitive test for early detection of neurodegeneration which is linked with protein aggregation. Finding methods capable of detecting intermediate states of protein aggregates, named oligomers, is critical for the early stage diagnosis of over 30 neurodegenerative diseases including Alzheimer’s or Parkinson’s. Currently, fluorescence-based imaging using Thioflavin T (ThT) dye is the gold standard for detecting protein aggregation. It is used to detect aggregation in vitro and in various tissues, including the cerebrospinal fluid (CSF), whereby the disease-related protein recombinant is seeded with the patient’s fluid. The major drawback of ThT is its lack of sensitivity to oligomeric forms of protein aggregates. Here, we overcome this limitation by transferring a ThT–oligomer mixture into solid state thin films and detecting fluorescence of ThT amplified in the process of stimulated emission. By monitoring the amplified spontaneous emission (ASE) we achieved a remarkable recognition sensitivity to prefibrillar oligomeric forms of insulin and lysozyme aggregates in vitro, to Aβ42 oligomers in the human protein recombinants seeded with CSF and to Aβ42 oligomers doped into brain tissue. Seeding with Alzheimer patient’s CSF containing Aβ42 and Tau aggregates revealed that only Aβ42 oligomers allowed generating ASE. Thus, we demonstrated that, in contrast to the current state-of-the-art, ASE of ThT, a commonly used histological dye, can be used to detect and differentiate amyloid oligomers and evaluate the risk levels of neurodegenerative diseases to potential patients before the clinical symptoms occur.

Influence of Certain D-Metals on Formation of Advanced Glycation End Products, Aggregation and Amyloid Transformation of Albumin in Glycation Reaction

“…Последнее нивелирует потенциальную полезность замедления амилоидной трансформации под действием гликирования. Согласно данным литературы [54,55], ThT может не выявлять образования олигомерных форм амилоида, в отличие от иных форм (мономеры, фибриллы). Можно выдвинуть предположение о том, что сравнительно меньшая интенсивность флуоресценции ThT в пробах, гликированных в присутствии всех ионов d-металлов за исключением меди (II), соотносится с возможностью замедления реакции на стадии образования олигомеров, что, однако, будет являться предметом дальнейших исследований.…”

Monitoring Amyloidogenesis with a 3D Deep-Learning-Guided Biolaser Imaging Array

“…26,27 For instance, Piotr et al demonstrated amplified stimulated emission with ThT oligomers in solidstate thin films, where different conformations would lead to changes in the lasing threshold. 16 Zhang et al developed a whispering gallery mode (WGM) biolaser by incorporating fluorescein sodium salt into silk fibroin microspheres, and the structural change of the silk fibroin is observed extrinsically from changes of lasing wavelength shifts. 28 Although lasing spectra can provide highly distinguishable and notable optical signatures, most studies require spectrometric systems and precise measurements at a single optical cavity level for readout.…”

“…15 The major challenge of fluorescence-based imaging is its low sensitivity to oligomeric forms of protein aggregates, which is considered to be the turning point in neurodegenerative diseases. 16,17 To circumvent these challenges, optical technologies have been developed to enhance the signals from protein interactions. One of the well-known emergent approaches is to take advantage of active photonic resonators, also known as biological lasers.…”

“…As amyloid fibrils bind with ThT, the internal nonradiative rotation of the benzothiazole and aminobenzene rings of ThT molecules will be restricted, thus giving rise to a significant increase in the fluorescence quantum yield . The major challenge of fluorescence-based imaging is its low sensitivity to oligomeric forms of protein aggregates, which is considered to be the turning point in neurodegenerative diseases. , …”

“…Unlike conventional fluorescence-based sensing systems, biological lasers utilize biological materials as part of their gain medium or cavity . Thanks to the strong optical feedback provided by the cavity, a small change in the gain material is significantly amplified, leading to a high signal-to-noise ratio, , high sensitivity toward minute changes in the bioprocesses, , threshold behavior which eradicates interfering background signals, and narrow line width that facilitates monitoring of biological processes. , Recent studies in biolasers have also demonstrated their capability to detect structural changes in biological gain materials or microcavities. , For instance, Piotr et al demonstrated amplified stimulated emission with ThT oligomers in solid-state thin films, where different conformations would lead to changes in the lasing threshold . Zhang et al developed a whispering gallery mode (WGM) biolaser by incorporating fluorescein sodium salt into silk fibroin microspheres, and the structural change of the silk fibroin is observed extrinsically from changes of lasing wavelength shifts .…”