Polydopamine-assisted aptamer-carrying tetrahedral DNA microelectrode sensor for ultrasensitive electrochemical detection of exosomes

Abstract

Background: Exosomes are nanoscale extracellular vesicles (30-160 nm) with endosome origin secreted by almost all types of cells, which are considered to be messengers of intercellular communication. Cancerous exosomes serve as a rich source of biomarkers for monitoring changes in cancer-related physiological status, because they carry a large number of biological macromolecules derived from parental tumors. The ultrasensitive quantification of trace amounts of cancerous exosomes is highly valuable for non-invasive early cancer diagnosis, yet it remains challenging. Herein, we developed an aptamer-carrying tetrahedral DNA (Apt-TDNA) microelectrode sensor, assisted by a polydopamine (PDA) coating with semiconducting properties, for the ultrasensitive electrochemical detection of cancer-derived exosomes.

Results: The stable rigid structure and orientation of Apt-TDNA ensured efficient capture of suspended exosomes. Without PDA coating signal amplification strategy, the sensor has a linear working range of 10²-10⁷ particles mL^-1, with LOD of ~ 69 exosomes and ~ 42 exosomes for EIS and DPV, respectively. With PDA coating, the electrochemical signal of the microelectrode is further amplified, achieving single particle level sensitivity (~ 14 exosomes by EIS and ~ 6 exosomes by DPV).

Conclusions: The proposed PDA-assisted Apt-TDNA microelectrode sensor, which integrates efficient exosome capture, sensitive electrochemical signal feedback with PDA coating signal amplification, provides a new avenue for the development of simple and sensitive electrochemical sensing techniques in non-invasive cancer diagnosis and monitoring treatment response.

Keywords: Aptamer; Electrochemical sensing; Exosomes; Microelectrode; Polydopamine; Tetrahedra DNA.

Fig. 1

Schematic illustration of the PDA coating assisted Apt-TDNA microelectrode sensor

Fig. 2

Characterization of exosomes and Apt-TDNA. a Characterization of size and number of exosomes using nanoparticle tracking analysis. b The widespread distribution of CD63 protein on the membrane of A549-derived exosomes was characterized by flow cytometry. c Purified bare exosomes (left) and PDA coated exosomes (right) observed using TEM. d Four Apt-TDNA monomers before (1–4) and after (5–8) TCEP pretreatment were characterized by 12.5% PAGE. e Characterization of Apt-TDNA by AFM. f In 2.5% agarose, the migration rates of thiolated monomers, assembly intermediate and Apt-TDNA are from fast to slow

Fig. 3

Electrochemical characterization of the construction process of a PDA-assisted Apt-TDNA microelectrode sensor for exosome detection. a CV. b DPV. c EIS. d Anchoring time optimization of Apt-TDNA. e, f Feasibility of Apt-TDNA microelectrode sensor and evaluation of non-specific binding. Data represent mean ± SD, n = 3, three technical replicates. Two-tailed Student’s t tests were used for comparisons, ***P < 0.001, ****P < 0.0001

Fig. 4

The detection of purified cancerous exosomes with Apt-TDNA microelectrode sensor. a The typical impedance intensity curves in the presence of varying exosome levels. b Calibration curve of exosome concentration gradient versus Z. c Detection by DPV. d Calibration curve between peak current and exosome concentration. Data represent mean ± SD, n = 3, three technical replicates. Two-tailed Student’s t tests were used for comparisons, *P < 0.05

Fig. 5

Detection of exosomes based on a signal amplification strategy assisted by PDA coating. Additional CV electrochemical deposition helps the true signal of PDA coating appear: (a) EIS, (b) DPV. (c) Continuous CV promotes the electrochemical deposition of PDA. d Typical impedance intensity curves in the presence of varying exosome levels. e Calibration curve of EIS. f Detection by DPV. g Calibration curve of DPV. Data represent mean ± SD, n = 3, three technical replicates. Two-tailed Student’s t tests were used for comparisons, *P < 0.05