Tumor cell-derived exosomes are considered a potential source of cancer biomarkers. Here, we developed an electrochemical sensing platform for the rapid and simple detection of exosomes, using the CCRF-CEM exosome as a model. The platform utilizes cyclic nicking enzyme cleavage and a hybridization chain reaction (HCR) for dual-signal amplification. A hairpin aptamer probe (HAP) containing an aptamer was designed for the assay. The specific binding between the aptamer and PTK7, present on the exosome surface, causes a conformational change in the HAP. This facilitates hybridization between the HAP and the linker DNA, which subsequently triggers cyclic cleavage of the nicking endonuclease towards the linker DNA. Therefore, exosome detection is transformed into DNA detection. By combining this approach with HCR signal amplification, we achieved high-sensitivity electrochemical detection of CCRF-CEM exosomes, down to 1.1 × 104particles/mL. Importantly, this assay effectively detected tumor exosomes in complex biological fluids, demonstrating the potential for clinical diagnosis.Keywords:exosomes;electrochemistry;cyclic enzyme scission;hybridization chain reaction
Xueke Wu,Chenyuan Yan,Lixuan Mu,Yuan Wang,Guangwei She,Wensheng Shi
By assembling fluorophore-marked DNA stem-loops on the surface of gold nanobipyramids (AuNBPs) with an efficient photothermal conversion, we fabricated a multifunctional photothermal reagent (AuNBPs-mPEG/AA-TR). The sgc8 sequences that were embedded inside the DNA stem-loops can specifically target the PTK7, an overexpressed protein located on the surface of CCRF-CEM cells (human acute lymphoblastic leukemia T lymphocyte) to realize an active targeting function. Before the AuNBPs-mPEG/AA-TR bind to the PTK7 proteins, the DNA stem-loops are folded, and the fluorophore linked on the DNA stem-loop will be close to the AuNBPs. In this configuration, the fluorescence from the fluorophore is quenched by the AuNBPs. However, when the AuNBPs-mPEG/AA-TR bound to the PTK7 proteins through the sgc8 sequences, the DNA stem-loops can be opened partially to enable the fluorophore to be far away from the AuNBPs. As a result, the fluorescent signal will be activated. Therefore, the fluorescence can be used as a visual signal to reveal the binding between the AuNBPs-mPEG/AA-TR and CCRF-CEM cells (usually called activatable fluorescence labelling). While under irradiation of an 808 nm laser, the CCRF-CEM cells could be ablated through the photothermal therapy of the AuNBPs-mPEG/AA-TR with a high photothermal efficiency. Applying the AuNBPs-mPEG/AA-TR to Human B lymphoma cells (Ramos cells) and CCRF-CEM cells, we found that only the CCRF-CEM cells can be effectively targeted, actively fluorescently labeled, and readily ablated by the AuNBPs-mPEG/AA-TR. This work provides an alternative photothermal agent for the targeted therapy of tumors.
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