On-exosome-membrane DNA polymerization-powered magneto-electrochemical aptasensing for osteosarcoma

The development of exosomes-based non-invasive liquid biopsy method is essential for an early diagnosis of cancers such as osteosarcoma (OS). The emerging isothermal nucleic acid amplification (NAA)-integrated electrochemical aptasensor (E-aptasensor) holds a great promise due to its affordability, rapid response, high sensitivity, and multiplexing capability. However, these aptasensors often require a challenging and complicated integration of signal trigger sequences into the parent aptamers that regulates the NAA. Thus, it is highly desirable to develop a template-free and universal isothermal NAA-intergrated E-aptasensor for OS-derived exosome detection.

Herein, via terminal deoxynucleotidyl transferase (TdTase)-mediated DNA polymerization on exosome membrane, we firstly proposed a membrane-initiated enzymatic polymerization (MIEP)-based magneto-driven E-aptasensor for an ultrasensitive detection of OS cell-derived exosomes. Specifically, the LC09 aptamers-modified magnetic microbeads (MMBs) efficiently captured the specific exosomes. Afterwards, the direct on-exosome membrane DNA polymerization was performed by TdTase and then large amounts of horseradish peroxidases were bound via immunoreaction, leading to a strong catalytic current in the substrate of 3,3',5,5'-tetramethylbenzidine/hydrogen peroxide on the surface of a magnetic glassy carbon electrode. This MIEP-based E-aptasensor reported a satisfactory detection result, displaying a broad linear range (6 × 10⁷∼6 × 10¹⁰ particles/mL), a low detection limit (60 particles/μL), a good specificity toward other tumor cell-derived exosomes, and a good recovery (87.9 %-98.3 % in human plasma). Finally, this method was applied to different clinical samples of micro-volume plasma and distinguished them successfully.

The proposed aptasensor can serve as a powerful and potential tool for liquid biopsy of OS, owing to its high generality, low sample demand, and high practicability. Furthermore, the innovative design strategy established in this work provides a new and versatile avenue for the construction of advanced exosome biosensors, thereby broadening their application in clinical research and precision medicine.