Dark Field Microscopic Sensitive Detection of Amyloid Fibrils Using Gold Nanoparticles Modified with Antibody

Abstract

Dark field microscopy (DFM) was employed to detect amyloid β (Aβ) fibrils-induced gold nanoparticle (AuNP) aggregation at the single-particle level, with a detection limit of 40 pM fibrils. The sensitivity of this method is higher than that of the current fibril-specific detection method using probe dye, such as thioflavin T, for which sub-μM level of fibrils are necessary. This study further proved the potential application of DFM in the analytical methods based on AuNP aggregation.

Single-molecule detection on a portable 3D-printed microscope

“…2c, direct counting of individual aggregates achieves an increase in sensitivity of at least five orders of magnitude compared with conventional bulk methods. The sensitivity of our simple benchtop instrument even compares favourably to more complex amyloid detection methods (10 p m by single-molecule TIRF imaging 26 and another confocal setup 27 and 40 p m using immunogold labelling and dark field microscopy 28 ). A key advantage of the direct detection of individual diffusing particles is also the deconvolution of the number and size of aggregates, which is crucial in the context of disease, as a number of studies have linked smaller aggregated species to an increase in cellular toxicity 29–31 .…”

Protein-Functionalized Gold Nanoparticles for Antibody Detection Using the Darkfield Microscopic Observation of Nanoparticle Aggregation

“…The formation of the anti-BSA-induced rBSA-AuNP aggregates was analyzed by darkfield microscopy (DFM), which detects scattered light from individual nanostructures 17,18 . We have previously demonstrated the application of DFM for the facile and sensitive detection of target molecules such as DNA, thrombin, and protein amyloids by analysing the AuNP aggregates formed by each of the target molecules [19][20][21] . In this study, DFM results revealed the formation of the rBSA-AuNP aggregates upon the addition of 20 nM anti-BSA.…”

Detection of Gold Nanoparticles Aggregation Using Light Scattering for Molecular Sensing

“…DFM images of the AuNP samples with various concentrations of thrombin were obtained using a BX53 microscope (Olympus, Tokyo, Japan) equipped with a UP73 CCD camera, UPlanFLN 60× objective lens, and U-DCW dark field condenser as described. 18,19 The APTES treated glass (APTES-glass) is used for darkfield imaging in this study. 25 To prepare the APTESglass, a glass slide was soaked for 30 min in 1% APTES solution.…”

“…For the analysis of AuNP aggregates, we then utilized darkfield microscopy (DFM), which can detect the scattering light from nanoparticles. [16][17][18][19] It was previously shown that the digital color of the AuNP spots in the darkfield images can be used to determine the nanoparticle size. 20,21 We demonstrate that digital color analysis of darkfieldimages of AuNP aggregates can also be used for detecting targets without spectroscopic analysis.…”