Surface Assay for Specific Detection of Soluble Amyloid Oligomers Utilizing Pronucleon Peptides Instead of Antibodies.

Immunoassays such as enzyme-linked immunosorbent assays (ELISAs) are widely used for diagnostics; however, antibodies as detection reagents may be insufficiently selective and have other shortcomings. We present a novel non-antibody-based detection method based on binding target molecules to peptides (used as recognition molecules): a surface assay for A-ß oligomers employing a peptide comprising amino acid residues of the human ß-amyloid protein (Pronucleon peptide) as the capture agent. For the sake of convenience, we term this the "Pronucleon peptide-linked immunosorbent assay", or PLISA. Pronucleon peptides are amino acid sequences matched to target amyloids of interest, in particular soluble Aß-1-42 amyloid protein oligomers, which are widely considered as an early biomarker for Alzheimer's disease in body fluids. The Pronucleon peptide in a PLISA is immobilized on the surface and substitutes for the capture antibody used in an ELISA for binding the Aß-1-42 oligomers present in the sample. We present data comparing synthetic oligomer PLISAs in spiked buffer and body fluids (such as cerebrospinal fluid, brain extracts, or whole blood) to those from an ELISA and demonstrate better selectivity of the PLISA for amyloid ß-42 oligomers versus monomers and fibrils. The detection limit, calculated as the mean (blank) plus three standard deviations, was in the range of 0.35-1.5 pM (32-135 ng/L) (oligomers contained approximately 20 monomers on average).

Amyloid beta modulation of neuronal network activity in vitro.

In vitro assays offer a means of screening potential therapeutics and accelerating the drug development process. Here, we utilized neuronal cultures on planar microelectrode arrays (MEA) as a functional assay to assess the neurotoxicity of amyloid-ß 1-42 (Aß42), a biomolecule implicated in the Alzheimer׳s disease (AD). In this approach, neurons harvested from embryonic mice were seeded on the substrate-integrated microelectrode arrays. The cultured neurons form a spontaneously active network, and the spiking activity as a functional endpoint could be detected via the MEA. Aß42 oligomer, but not monomer, significantly reduced network spike rate. In addition, we demonstrated that the ionotropic glutamate receptors, NMDA and AMPA/kainate, play a role in the effects of Aß42 on neuronal activity in vitro. To examine the utility of the MEA-based assay for AD drug discovery, we tested two model therapeutics for AD, methylene blue (MB) and memantine. Our results show an almost full recovery in the activity within 24h after administration of Aß42 in the cultures pre-treated with either MB or memantine. Our findings suggest that cultured neuronal networks may be a useful platform in screening potential therapeutics for Aß induced changes in neurological function.

Freeze Drying Improves the Shelf-Life of Conductive Polymer Modified Neural Electrodes.

Coating microelectrodes with conductive polymer is widely recognized to decrease impedance and improve performance of implantable neural devices during recording and stimulation. A concern for wide-spread use of this approach is shelf-life, i.e., the electrochemical stability of the coated microelectrodes prior to use. In this work, we investigated the possibility of using the freeze-drying process in order to retain the native low impedance state and, thereby, improve the shelf-life of conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT)-PSS modified neural electrodes. Control PEDOT-PSS coated microelectrodes demonstrated a significant increase in impedance at 1 kHz after 41-50 days of room temperature storage. Based on equivalent circuit modeling derived from electrochemical impedance spectroscopy, this increase in impedance could be largely attributed to a decrease in the interfacial capacitance consistent with a collapse and closing of the porous structure of the polymeric coating. Time-dependent electrochemical impedance measurements revealed higher stability of the freeze-dried coated microelectrodes compared to the controls, such that impedance values after 41-50 days appeared to be indistinguishable from the initial levels. This suggests that freeze drying PEDOT-PSS coated microelectrodes correlates with enhanced electrochemical stability during shelf storage.