Electrochemical Sandwich Immunosensor for Determination of Exosomes Based on Surface Marker-Mediated Signal Amplification

Abstract

Extracellular vesicles (EVs), namely, exosomes and microvesicles, are important mediators of intercellular communication pathways. Since EVs can be detected in a variety of biofluids and contain a specific set of biomarkers which are reminiscent of their parental cells, they show great promise in clinical diagnostics as EV analysis can be performed in minimally invasive liquid biopsies. However, reliable, fast and cost-effective methods for their determination are still needed, especially if decentralized analysis is intended. In this study, we developed an electrochemical biosensor which works with 1.5 μL sample volume and can detect as low as 200 exosomes per microliter, with a linear range spanning almost 4 orders of magnitude. The sensor is specific and readily differentiates exosomes from microvesicles in samples containing 1000-fold excess of the latter. Capability of detecting exosomes in real samples (diluted serum) was shown. This was achieved by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for detection of captured exosomes. Signal amplification is presumably obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. Detection is performed based on electrochemical reduction of 3,3',5,5'-tetramethyl benzidine (TMB) after addition of horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for EV determination.

Biological Functions and Current Advances in Isolation and Detection Strategies for Exosome Nanovesicles

“…This method has shown good sensitivity, with a detection limit of 4.7 × 10 5 exosomes µL −1 . Doldan et al developed another electrochemical sandwich approach for exosomes determination . This approach uses gold electrodes prefunctionalized with α‐CD9 antibodies.…”

“…For instance, while conventional ELISA suffers from long assay times, laborious sample loading, reagent addition, washing and incubation steps, a modified ELISA integrated with a lab‐on‐a‐chip microfluidic platform offers a simple, fast, and automated analysis of exosomes . Much attention has also been given to developing new strategies based on microfluidics and electrochemical biosensors . Among these, electrochemical biosensor approaches have shown great promise due to their fast, simple, and cost‐effective procedures, as well as the requirement for less sample volume.…”

Progress in Microfluidics‐Based Exosome Separation and Detection Technologies for Diagnostic Applications

“…In this way, choosing a proper biomarker of exosomes is the crucial factor that impacts the separation purity and throughput of the immunoassays. Recently, some achievements in the immunological exosome separation field revealed that antibody‐coated magnetic beads, chromatography matrices, and plates can be effectively used to separate specific exosomes from body fluids . Unlike other exosome separation techniques, the immunological separation method is a time‐saving method and can directly separate exosomes from body fluids.…”

Exosomes: Isolation, Analysis, and Applications in Cancer Detection and Therapy

“…Many electrochemically based exosome biosensors have also been developed, because of their simplicity, rapid response, and high sensitivity . The Tan group, for example, developed a sensor for electrochemical detection of cancerous exosomes .…”