Label-free detection of hypoxia-induced extracellular vesicle secretion from MCF-7 cells

Abstract

Nanoscale extracellular vesicles (EVs) including exosomes (50-150 nm membrane particles) have emerged as promising cancer biomarkers due to the carried genetic information about the parental cells. However the sensitive detection of these vesicles remains a challenge. Here we present a label-free electrochemical sensor to measure the EVs secretion levels of hypoxic and normoxic MCF-7 cells. The sensor design includes two consecutive steps; i) Au electrode surface functionalization for anti-CD81 Antibody and ii) EVs capture. The label-free detection of EVs was done via Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS). The working linear range for the sensor was 10²-10⁹ EVs/ml with an LOD 77 EVs/mL and 379 EVs/ml for EIS and DPV based detection. A blood-abundant protein, RhD was used for the selectivity test. In order to assess the performance of the biosensor, the level of EVs secretion by the human breast cancer MCF-7 cell line was compared with enzyme-linked immunosorbent assays (ELISA) and Nanoparticle Tracking Analysis (NTA). Designed label-free electrochemical sensors utilized for quantification of EVs secretion enhancement due to CoCl₂-induced hypoxia and 1.23 fold increase with respect to normoxic conditions was found.

Figure 1

Experimental steps followed throughout the work. MCF-7 cells were exposed to either CoCl₂- induced hypoxic or normoxic conditions. Isolation of EVs were done via ultracentrifugation. Characterization and quantification of EVs were done via NTA and EVs biosensors that are designed to capture CD-81 EVs biomarker via biotinylatd anti-CD81 antibody immobilized through streptavidin-biotin interaction on SAM modified Au SPE surface.

Figure 2

Characterization of EVs via NTA. Size distrubution analysis of EVs as a function of particle concentration. (A) Representative image used for size distribution analysis of extracellular vesicles (scale bar represents 200 nm).

Figure 3

Electrochemical characterization of the surface functionalization based on EIS (A) and DPV (B) according to the steps detailed in section 2.3.

Figure 4

Assessment of biosensor sensitivity via concentration studies. Differential pulse voltammograms recorded for various EVs concentrations (10–10¹⁰ EVs/ml) (A), bar graphs represent the average Ip_A of redox probe at each concentration, (B) semi-log calibration curve drawn for DPV based detection of EVs in the concentration range of 10²–10⁷ EVs/ml (C). EIS measurements in the concentration range of 10²–5 × 10⁶ EVs/ml (D), bar graphs with represent the average R_ct of redox probe at each concentration (E) Semi-log calibration curve drawn for EIS based detection of EVs (F). Data are expressed as mean ± SD of three measurement. Experimental steps are detailed in methods section.

Figure 5

Calibration curves drawn by ELISA standards based on DPV (A) EIS (B) and ELISA (C). Data are expressed as mean ± SD of three measurements. Experimental steps are detailed in methods section.

Figure 6

Results of the biosensor selectivity study showing the effect of RhD immobilization on Anti-CD81 Ab immobilized electrodes. Nyquist plots (A) and bar graphs representing R_ct values calculated from simulation results (B). Differential pulse voltammogram (B), and bar graphs representing Ip_A values. Data are expressed as mean ± standard deviation of three measurements. Experimental steps are detailed in methods section.

Figure 7

Effect of CoCl₂-induced hypoxia on MCF-7 cell viability, HIF-1 alpha expression and EVs secretion. Effect of CoCl₂ on cell viability; microscope images shows the cell morphology after (A-a) and before (A-b) CoCl₂ exposure for incubation period of 3 days. Statistical analysis of total and viable cell numbers before and after CoCl₂ exposure (A-c). Assessment of HIF1-α expression (in green) after exposure to CoCl₂. DAPI was used as a counter stain (D), DPV results presented via voltammograms (B) and bar graphs representing normalized IpA (C), EIS results presented via Nyquist plots (E) and bar graphs representing normalized R_ct (F). Data are expressed as mean ± SD of three repeats.Experimental steps are detailed in methods section. Annotations * correspond to differences with P values < 0.05.