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

Abstract

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).

Figure 1

Schematic representation of (A) the synthesis of pCBMA-coated MBs and anti-L1CAM Ab conjugation. Inset shows zeta potential of bare MBs before (marked as Fe₃O₄) and after polymerization (pCBMA@Fe₃O₄). (B) Neuronal exosome isolation using anti-L1CAM pCBMA-coated MBs. (C) Label-free impedimetric assays for two internal markers (Ab1 = anti-α-Syn antibody, Ab2 = anti-Synt-1 antibody).

Figure 2

(A) Histogram depicting the quantified adsorption of recombinant α-Syn on different Ab-modified pCBMA@Fe₃O₄ MBs surfaces. The commercial carboxylic acid-terminated MBs were used as the control. (B) SEM image of serum-captured exosomes on anti-L1CAM-modified MBs versus anti-HA (control)-modified MBs (insert). Scale bar 1 μm. (C) Immunoblotting of lysates of immunocaptured vesicles confirming the detection of both transmembrane proteins (L1CAM, CD81) and internal protein Synt-1 from exosomes. Specific electrochemiluminescence detection of α-Syn (D) in neuronal exosomes immunocaptured from serum with anti-L1CAM vs anti-HA (control)-modified pCBMA@Fe₃O₄ MBs.

Figure 3

Box plots for impedimetric exosomal quantifications of (A) α-Syn and (B) Synt-1 from 40 clinical samples (PD and HC, n = 20 per group). In the box plots, the lower and upper boundaries indicate the 25th and 75th percentiles, respectively. The line within the box marks the median, and the blue circle within the box marks the mean. Diamonds represent individual patient sample data points (the mean value of nine measurements across three electrodes for each clinical sample).