Facile Impedimetric Analysis of Neuronal Exosome Markers in Parkinson's Disease Diagnostics.

The egress of α-synuclein in neuronally derived exosomes predates the clinical presentation of Parkinson's disease (PD), offering a means of developing a predictive or prognostic test. Here, we report the reagentless impedimetric assay of two internal exosome markers (α-synuclein and syntenin-1) from neuronal exosomes. Exosomes were efficiently extracted from patient sera using anti-L1CAM conjugated zwitterionic polymer-modified magnetic beads prior to lysis and analyzed by electrochemical impedance spectroscopy. The quantification of α-synuclein level across 40 clinical samples resolved statistically significant differences between PD patients and healthy controls (HC).

A Paradigm in Immunochemistry, Revealed by Monoclonal Antibodies to Spatially Distinct Epitopes on Syntenin-1.

Syntenin-1 is an essential multi-functional adaptor protein, which has multiple roles in membrane trafficking and exosome biogenesis, as well as scaffolding interactions with either the actin cytoskeleton or focal adhesions. However, how this functional multiplicity relates to syntenin-1 distribution in different endosome compartments or other intracellular locations and its underlying involvement in cancer pathogenesis have yet to be fully defined. To help facilitate the investigation of syntenin-1 biology, we developed two specific monoclonal antibodies (Synt-2C6 and Synt-3A11) to spatially distinct linear sequence epitopes on syntenin-1, which were each designed to be unique at the six-amino acid level. These antibodies produced very different intracellular staining patterns, with Synt-2C6 detecting endosomes and Synt-3A11 producing a fibrillar staining pattern suggesting a cytoskeletal localisation. Treatment of cells with Nocodazole altered the intracellular localisation of Synt-3A11, which was consistent with the syntenin-1 protein interacting with microtubules. In prostate tissue biopsies, Synt-3A11 defined atrophy and early-stage prostate cancer, whereas Synt-2C6 only showed minimal interaction with atrophic tissue. This highlights a critical need for site-specific antibodies and a knowledge of their reactivity to define differential protein distributions, interactions and functions, which may differ between normal and malignant cells.

Concentration-Normalized Electroanalytical Assaying of Exosomal Markers.

Exosomes are both active in mediating intracellular communication and potentially present a potent cargo of disease biomarkers to an assay. The robust evaluation of exosomal markers could lead to a paradigm shift in clinical analysis and associated care. To date, much of this has been hindered by issues of sample preparation and assay signal-to-noise. We introduce here the use of ultrasensitive electrochemical impedance spectroscopy to quantify both external (tetraspanin) and internal (syntenin) exosome-specific markers. Associated exosome detection limits are 1.9 × 105 particles mL-1 (equivalent to 320 aM or 9500 exosomes in 50 µL) for intact exosomes and 3-5 picomolar for internal exosomal syntenin levels with almost 5 decades of linear dynamic range. Sample preparation can be carried out by simple fine filtering of cell-conditioned medium prior to a non-NTA-determined (i.e., nanoparticle tracking analysis) exosome concentration analysis, lysing, and subsequent internal syntenin quantification. Such concentration-normalized dual-marker analysis can be used to define "analytical zones" in a manner which is then independent of absolute exosome concentration and sample preparation.