Magneto-immunocapture with on-bead fluorescent labeling of amyloid-ß peptides: towards a microfluidized-bed-based operation.

A new sample treatment approach for sensitive determination of three amyloid-ß peptides (Aß 1-42, Aß 1-40 and Aß 1-38) with capillary electrophoresis coupled with laser induced fluorescent detection is reported herein. These Aß peptides are considered an important family of biomarkers in the cerebrospinal fluid (CSF) for early diagnosis of Alzheimer's disease (AD). Due to their extremely low abundance in CSF (down to sub nM ranges), batch-wise preconcentration via magneto-immunocapture with enrichment factors up to 100 was implemented. The Aß peptides were first captured onto magnetic micro-beads. Then, on-beads fluorescent labeling of the captured Aß peptides were carried out to avoid the unwanted presence of extra fluorescent dye in the eluent as in the case of in-solution labeling. Finally thermal elution was performed and eluted labeled peptides were analyzed off line with CE-LIF. The Aß-capturing efficiencies of different commercially available antibodies grafted onto magnetic beads were tested. Aß peptides in CSF samples collected from AD's patients and healthy persons (used as controls) were measured and evaluated. As a proof of concept, the developed strategy was adapted into a miniaturized fluidized bed configuration that has the potential for coupling with a microchip separation system.

Monodisperse carboxyl-functionalized poly(ethylene glycol)-coated magnetic poly(glycidyl methacrylate) microspheres: application to the immunocapture of ß-amyloid peptides.

Identification and evaluation of small changes in ß-amyloid peptide (Aß) levels in cerebrospinal fluid is of crucial importance for early detection of Alzheimer's disease. Microfluidic detection methods enable effective preconcentration of Aß using magnetic microparticles coated with Aß antibodies. Poly(glycidyl methacrylate) microspheres are coated with α-amino-ω-methoxy-PEG5000 /α-amino-ω-Boc-NH-PEG5000 Boc groups deprotected and NH2 succinylated to introduce carboxyl groups. Capillary electrophoresis with laser-induced fluorescence detection confirms the efficient capture of Aß 1-40 peptides on the microspheres with immobilized monoclonal anti-Aß 6E10. The capture specificity is confirmed by comparing Aß 1-40 levels on the anti-IgG-immobilized particles used as a control.