The isolation and detection of exosomes as tumor markers are of vital importance for the early diagnosis, therapeutic monitoring, and mechanistic studies of tumors. Here, exosomes derived from breast cancer cells were chosen as model targets, and a wash-free, enzyme-free, handheld mini centrifugation method based on hydrogels was developed to effectively isolate and detect breast cancer exosomes. Dual aptamers (CD63-T1 and EpCAM-T2) were employed to specifically recognize and capture breast cancer exosomes. This specific recognition triggered the formation of hybridization chain reaction (HCR) nanostructures on the captured exosomes through the interaction of hairpin 1 and the alginate complex (H1-Alg) and hairpin 2 (H2-Cy3). The interaction of Ca²⁺ and alginate enabled the in situ formation of a hydrogel on the exosome surface. Subsequent low-speed centrifugation using a handheld mini centrifuge facilitated the efficient isolation of the exosomes, thereby eliminating the need for tedious washing steps. Utilizing the classical chelation reaction of ethylene diamine tetraacetic acid (EDTA) with Ca²⁺, the hydrogel can be rapidly cleaved for enzyme-free release of exosomes. The method demonstrated excellent capture and release efficiencies of approximately 85% and 98%, respectively, for specific cancerous exosomes. Notably, the exosomes isolated by the hydrogel system retained excellent biological activity, making them suitable for further analysis and potential applications. Meanwhile, the highly sensitive detection of breast cancer exosomes based on this strategy could also be achieved with a lower limit of detection as low as 3.2 × 10³ particles/mL. This work provides a novel and cost-effective strategy for the effective isolation and detection of tumor-derived exosomes, which can help to promote the subsequent application of exosomes in research.

中文翻译：

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通过原位形成的水凝胶对肿瘤来源的外泌体进行免清洗分离和定量

作为肿瘤标志物的外泌体的分离和检测对于肿瘤的早期诊断、治疗监测和机制研究至关重要。在这里，选择来自乳腺癌细胞的外泌体作为模型靶标，并开发了一种基于水凝胶的免洗涤、无酶、手持式小离心法，以有效分离和检测乳腺癌外泌体。双适配体 （CD63-T1 和 EpCAM-T2） 用于特异性识别和捕获乳腺癌外泌体。这种特异性识别通过发夹 1 与藻酸盐复合物 （H1-Alg） 和发夹 2 （H2-Cy3） 的相互作用，触发了在捕获的外泌体上形成杂交链式反应 （HCR） 纳米结构。Ca²⁺ 和藻酸盐的相互作用能够在外泌体表面原位形成水凝胶。随后使用手持式微型离心机进行低速离心，有助于高效分离外泌体，从而消除了繁琐的洗涤步骤。利用乙二胺四乙酸 （EDTA） 与 Ca²⁺ 的经典螯合反应，水凝胶可以快速裂解，从而无酶释放外泌体。该方法对特定癌性外泌体表现出优异的捕获和释放效率，分别约为 85% 和 98%。值得注意的是，通过水凝胶系统分离的外泌体保留了优异的生物活性，使其适合进一步分析和潜在应用。同时，基于该策略的乳腺癌外泌体检测也可实现高灵敏度检测，检测下限低至 3.2 × 10³ 粒/mL。 这项工作为有效分离和检测肿瘤来源的外泌体提供了一种新颖且具有成本效益的策略，有助于促进外泌体在研究中的后续应用。