College of Pharmaceutical Sciences, Key Laboratory of Public Health Safety of Hebei Province, Hebei University, Baoding 071002, China.
Department of Medical Oncology, Hebei Key Laboratory of Cancer Radiotherapy and Chemotherapy, Affiliated Hospital of Hebei University, Baoding 071000, China.
Anal Chem. 2021 Apr 27;93(16):6534-6543. doi: 10.1021/acs.analchem.1c00528. Epub 2021 Apr 14.
Exosomes have become the most ideal analysis target for liquid biopsy since they carry a large amount of genetic materials. The study on exosomes has great significance for cancer diagnosis and prognosis. However, the extremely low concentration renders the development of a robust exosomes enrichment technique, with the merits of low nonspecific cell adhesion, high-capture efficiency, and easy nondestructive release of captured exosomes, of vital significance. We successfully designed and developed a novel Tim4@ILI-01 immunoaffinity flake material. First, a strongly hydrophilic ILI-01 MOFs matrix material was fabricated with cationic ionic liquid 1,3-bis(4-carboxybutyl)imidazolium bromide as the organic ligand. The nonspecific adsorption of the ILI-01 MOFs material was only 0.7% after two washings with a neutral buffer. Moreover, based on the inherent abundant carboxyl groups on the ILI-01 MOFs flake, they can be facilely functionalized with an anti-Tim4 antibody with the bonding efficiency of 82.4%. The capture efficiency of the developed Tim4@ILI-01 immunoaffinity material for exosomes reached 85.2%, which is 5.2 times higher than that via the gold standard ultracentrifugation method. Furthermore, based on the Ca-dependent characteristic of the binding between the Tim4@ILI-01 immunoaffinity material and phosphatidylserine (PS) on the surfaces of exosomes, the captured exosomes can be easily released with the addition of a chelating agent under neutral eluent conditions. Thus, the captured exosomes maintained good biological activity. The developed Tim4@ILI-01 immunoaffinity flake was successfully applied for enrichment of exosomes from serums of healthy persons and lung adenocarcinoma patients. The levels of the expressed CD44 gene significantly changed under different stages of lung adenocarcinoma cancer. All these results demonstrate that the Tim4@ILI-01 immunoaffinity flake is a robust enrichment material and has a good potential in practical clinical applications.
外泌体因其携带大量遗传物质而成为液体活检中最理想的分析靶标。对外泌体的研究对癌症的诊断和预后具有重要意义。然而，极低的浓度使得开发一种强大的外泌体富集技术变得至关重要，该技术具有非特异性细胞黏附低、捕获效率高、易于无损释放捕获的外泌体等优点。我们成功设计并开发了一种新型 Tim4@ILI-01 免疫亲和薄片材料。首先，采用阳离子离子液体 1,3-双（4-羧基丁基）咪唑溴盐作为有机配体制备了强亲水性的 ILI-01 MOFs 基质材料。经过两次中性缓冲液洗涤后，ILI-01 MOFs 材料的非特异性吸附仅为 0.7%。此外，基于 ILI-01 MOFs 薄片上固有丰富的羧基，它们可以很容易地与 Tim4 抗体结合，结合效率为 82.4%。所开发的 Tim4@ILI-01 免疫亲和材料对外泌体的捕获效率达到 85.2%，比金标准超速离心法高 5.2 倍。此外，基于 Tim4@ILI-01 免疫亲和材料与外泌体表面磷脂酰丝氨酸（PS）之间 Ca 依赖性结合的特性，在中性洗脱条件下加入螯合剂可轻松释放捕获的外泌体。因此，捕获的外泌体保持良好的生物学活性。所开发的 Tim4@ILI-01 免疫亲和薄片成功应用于健康人和肺腺癌患者血清中外泌体的富集。在不同阶段的肺腺癌癌症中，表达的 CD44 基因水平明显变化。所有这些结果表明，Tim4@ILI-01 免疫亲和薄片是一种强大的富集材料，在实际临床应用中具有良好的潜力。
