Ultrasensitive detection of exosomes by microchip electrophoresis combining with triple amplification strategies

Abstract

The analysis of exosomes is significant as they can be used for various pathophysiological processes, especially cancer related intercellular communication. Therefore, a convenient, reliable, and sensitive detection method is urgently needed. Strand displacement amplification (SDA) and catalytic hairpin assembly (CHA) are two kinds of effective isothermal nucleic acid amplification methods. In this article, an efficient quantitative MCE method for detecting human breast cancer cell (MCF-7) exosomes assisted by triple amplification strategies combining cholesterol probe (Chol-probe) with SDA-CHA was first developed. CD63 aptamer was immobilized on the avidin magnetic beads to specifically capture exosomes and then Chol-probe with high affinity was spontaneously inserted into the exosome membrane, which was the first step of amplification strategy to improve detection sensitivity. After magnetic separation, Chol-probe could complement ssDNA and trigger SDA, producing a large number of DNA sequences (Ta) to trigger CHA, achieving SDA-CHA amplification. Under optimal conditions, the detection limit (LOD) for MCF-7 exosomes was as low as 26 particle/μL (S/N = 3). This method provides an effective approach for sensitive and accurate quantification of tumor exosomes, and can be expected to detect exosomes in clinical samples.

Keywords: Catalytic hairpin assembly; Cholesterol probe; Exosomes; Microchip electrophoresis; Strand displacement amplification.