Intracellular Amyloid-β Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS

Abstract

Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-β peptide (Aβ) oligomers that appear as intermediates along the Aβ aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aβ from in vitro and animal models, there is little known about intracellular Aβ in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aβ species in specific brain cell subpopulations can provide insight into the role of Aβ in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aβ species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aβ species from as few as 20 human brain cells.

Figure 1

MIMAS schematics. (a) Microfluidic device top view. Fluidic layer is filled in solid gray. The control layer is marked with dotted red lines. Inset, top: cross-sections of a normally closed valve. Inset, bottom: a valve opened by vacuum action, upon which two adjacent wells become connected. (b) Cross-section of a collection-layer funnel-shaped well, aligned over an upward-facing MIMAS device well. (c) Cross-section of a MIMAS device assembled on an ITO-coated glass slide. (d) Analyte-matrix co-crystals over an ITO-coated glass slide after MIMAS manifold removal, being ionized during MALDI-MS.

Figure 2

Collection of microdissected brain cells. Dissection schematics and representative microscopy images of collected cells into (a) a 500 μm × 500 μm MIMAS well (indicated by dashed lines), some cells are found outside the well; (b) a 2 mm-diameter milli-well and (c) a 1 mm-diameter collection layer funnel on a MIMAS well. (d) Cell capture efficiency for (a–c); error bars represent the standard deviation.

Figure 3

Aβ-M IgG 6E10 immunocapture using the MIMAS platform. (a) Aβ_1–40-M immunocapture exhibits [Aβ + H]⁺ at m/z 4330 and [Aβ + 2H]²⁺ at m/z 2165. (b) Aβ_1–42-M immunocapture exhibits [Aβ + H]⁺ at m/z 4514 and [Aβ + 2H]²⁺ at m/z 2257. Yellow arrows indicate the m/z ∼ 3880 peak associated with the BSA blocking step. Aβ species concentration was 200 nM in each case.

Figure 4

Aβ-O IgG 6E10 immunocapture in the MIMAS platform. (a) Aβ_1–40-O immunocaptured monomers, dimers, and tetramers. Peaks with m/z ∼ 4720 and m/z ∼ 8570 are labeled U₁ and U₂. (b) Aβ_1–42-O immunocaptured monomers, dimers, and trimers. Peaks with m/z ∼ 4720 and m/z ∼ 8570 are labeled U₃ and U₄. Green arrows indicate peaks related to the blocking step. Aβ concentration (prior oligomerization) = 1 μM.

Figure 5

Immunoassay performed in milli-wells using brain cells. (a) Representative MS spectrum resulting from the immunoassay in milliwells using 100 cell bodies dissected from a brain section. (b) Zoom-in spectrum of section A showing the peaks with m/z 4270, m/z 4374, and m/z 4490. (c) Zoom-in of section B showing peaks with m/z 8570, m/z 8760, and m/z 8970.

Figure 6

Schematics of the LCM-MIMAS workflow: (1) brain cell microdissection and loading into the microfluidic wells with the help of the collection layer, (2) removal of the collection layer, (3) assembly of the MIMAS manifold with the collected cells in the wells of fluidic line I onto an ITO glass slide, (4) immobilization of antibodies and blocking step in fluidic line II, (5) mixing wells I and II contents and incubation for immunocapture, (6) wash to remove noncaptured material, (7) matrix solution loading and mixing with the sample, and (8) removal of the MIMAS elastomeric manifold to expose the sample-matrix crystals for MS analysis.

Figure 7

Analysis of Aβ in cells from archived brain tissue with the LCM-MIMAS approach. Representative MS spectra from the MIMAS immunoassay, using a collection layer to load 20 brain tissue cells directly into MIMAS wells. Peaks m/z 4270.2 and m/z 8557.9 match with peaks observed in the milli-wells assay.