Fluorescent Filter-Trap Assay for Amyloid Fibril Formation Kinetics in Complex Solutions

Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid β (Aβ) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aβ allows for their detection. The times of half completion of the process (<i>t</i>_1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aβ fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration

Screening raw data, generated by the plate reader.

<p>The interactions between 7 proteins and 2 particles was followed with ANS fluorescence monitored at 3 emission wavelength; blue = 460 nm, green = 475 and red = 520 nm, over time. The shown results are the average of three sample replicates.</p

Schematic illustration of the 6 scenarios leading to the observed fluorescence intensity.

<p>Left side shows the situation when the fluorophore <b>do not</b> adsorb to the nanoparticle (1a, 2a, and 3a) and the right side shows the situations when the fluorophore <b>do</b> adsorb to the nanoparticles (1b, 2b, and 3b).</p

Summary of the screening data.

<p>^a Scenario No. as presented in the text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136687#pone.0136687.g004" target="_blank">Fig 4</a>.</p><p>^b Combined Analysis taken in consideration also the DLS data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136687#pone.0136687.t001" target="_blank">Table 1</a>.</p><p>^c The protein adsorbs to the nanoparticle.</p><p>^d not applicable.</p><p>Summary of the screening data.</p

NP sizes before and after screening experiment.

<p>¹ Size measured by DLS, ~48 h after sample mixture.</p><p>² Size measured by DLS, ~12 h after sample mixture.</p><p>³ Visible precipitated aggregates</p><p>NP sizes before and after screening experiment.</p

Screening results for 7 proteins and PS-COOH, ANS data in panel A and NR data in panel B, with corresponding controls.

<p>Bars represent the mean intensity value from three individual samples, with standard deviation in corresponding error bars, at 6 different time points; 0 min (blue), 15 min (red), 30 min (gray), 2 h (yellow), 5 h (dark blue) and 8 h (green) for the ANS data and 10 h (green) for the NR data. Fluorescence intensity of fluorophore with 1: Buffer only, 2: Particle only, 3: Lysozyme + particle, 4: HSA + particle, 5: OVA + particle, 6: CA-I + particle, 7: trCA-II + particle, 8: β-lactoglobulin + particle, 9: Calbindin + particle. Sky blue lines show the protein controls (i.e. protein with fluorophore) I_F at each time point. In both panels a yellow dashed line is drawn across all samples representing the mean fluorescence from the particle control at time point 0 min. In panel A, two insets are also shown for trCA-II (number 7) and for β-lactoglobulin(number 8) data.</p

Method outline.

<p>Short description of the method.</p

Screening results for 7 proteins and PS-NH₂, ANS data in panel A and NR data in panel B, with corresponding controls.

<p>Bars represent the mean intensity value from three individual samples, with standard deviation in corresponding error bars, at 6 different time points; 0 min (blue), 15 min (red), 30 min (gray), 2 h (yellow), 5 h (dark blue) and 8 h (green) for the ANS data and 10 h (green) for the NR data. Fluorescence intensity of fluorophore with 1: Buffer only, 2: Particle only, 3: Lysozyme + particle, 4: HSA + particle, 5: OVA + particle, 6: CA-I + particle, 7: trCA-II + particle, 8: β-lactoglobulin + particle, 9: Calbindin + particle. Sky blue lines show the protein controls (i.e. protein with fluorophore) I_F at each time point. In both panels a yellow dashed line is drawn across all samples representing the mean fluorescence from the particle control at time point 0 min.</p