Electrochemical Detection of Tumor-Derived Extracellular Vesicles on Nanointerdigitated Electrodes

Abstract

Tumor-derived extracellular vesicles (tdEVs) are attracting much attention due to their essential function in intercellular communication and their potential as cancer biomarkers. Although tdEVs are significantly more abundant in blood than other cancer biomarkers, their concentration compared to other blood components remains relatively low. Moreover, the presence of particles in blood with a similar size as that of tdEVs makes their selective and sensitive detection further challenging. Therefore, highly sensitive and specific biosensors are required for unambiguous tdEV detection in complex biological environments, especially for decentralized point-of-care analysis. Here, we report an electrochemical sensing scheme for tdEV detection, with two-level selectivity provided by a sandwich immunoassay and two-level amplification through the combination of an enzymatic assay and redox cycling on nanointerdigitated electrodes to respectively enhance the specificity and sensitivity of the assay. Analysis of prostate cancer cell line tdEV samples at various concentrations revealed an estimated limit of detection for our assay as low as 5 tdEVs/μL, as well as an excellent linear sensor response spreading over 6 orders of magnitude (10-10⁶ tdEVs/μL), which importantly covers the clinically relevant range for tdEV detection in blood. This novel nanosensor and associated sensing scheme opens new opportunities to detect tdEVs at clinically relevant concentrations from a single blood finger prick.

Keywords: Nanoelectrodes; enzymatic amplification; microfluidics; redox cycling; tumor-derived extracellular vesicles.

Figure 1

Schematic illustration of tdEV sensing using a sandwich immunoassay and redox cycling on nIDEs resulting in a two-level selectivity and a two-level amplification. tdEVs are captured using C-AE tethered to electrodes (first level of selectivity). The binding of R-AE to the tdEVs completes the antibody–antigen-antibody sandwich (second-level selectivity), after which the enzyme ALP is introduced using a biotin–SAV interaction. ALP provides an enzymatic amplification of pAPP to pAP by substrate cleavage (first-level amplification), which is followed by an electrochemical signal amplification via the oxidation of pAP to pQI and subsequent redox cycling thereof between the nIDE electrodes (second-level amplification).

Figure 2

Atomic force microscope height images: (a) bare electrodes before chemical modification and (b) after modification and capture of EpCAM-positive tdEVs derived from LnCAP cell lines on nIDEs. The captured objects are 30–150 nm in diameter, which is in good agreement with small EV dimensions (scale bar: 200 nm).

Figure 3

Cyclic voltammograms of tdEVs on nIDEs: evaluation of the device specificity. (a) Schematic representation of the electrochemical measurement setup. Cyclic voltammograms (CVs) recorded (b) after various steps of functionalization of the nIDEs, after C-AE surface modification (green), after tdEV capture (brown), and after formation of a sandwich with R-AE (blue), and (c) in the presence of tdEVs on nIDEs (brown) and on μIDEs (green), or in the presence of pdEVs on nIDEs (magenta). The background signal (blue) corresponds to a device after the antibody sandwich formation. The currents I_anode (solid lines) and I_cathode (dashed lines) were measured at the anode and the cathode set of electrodes of the nIDEs/μIDEs, respectively. CVs were acquired for a 1 mM pAPP solution in PBS (pH 7.4) between −0.1 V and +0.6 V vs Ag/AgCl at a scan rate of 50 mV/s.

Figure 4

Sensitivity and dynamic range of the assay for the detection of tdEVs. (a) Anodic cyclic voltammograms recorded at the anode for tdEV samples with concentrations ranging from 10 to 10⁶ tdEVs per μL. (b) Associated calibration curve (based on the limiting currents recorded at 0.6 V vs Ag/AgCl) revealing a dynamic range spanning at least 6 orders of magnitude (number of devices, n = 3). The horizontal dotted line depicts the background level plus three times the standard deviation (SD) of the redox current; from this horizontal line and the calibration curve, a theoretical LOD as low as 5 tdEVs/μL is found. (Conditions: 1 mM pAPP solution in PBS (pH 7.4); scan rate of 50 mV/s.)