【摘要】 基于逆转录定量聚合酶链式反应（RT-qPCR）的分子诊断依赖于温度变化驱动的酶促反应过程，因此需要体系与热循环条件具有良好的兼容性。本研究采用水包油乳液策略结合原位琼脂糖凝胶化方法，制备了热响应性单室多糖微反应器。研究了油相组成及琼脂糖浓度对微球形成、粒径分布及蛋白包封效率的影响，并确定异辛醇可作为一种有效油相，在实现稳定乳化的同时降低界面泄漏。随后，将逆转录酶和DNA聚合酶共封装于微反应器中构建单腔室酶促反应体系，并通过一步法RT-qPCR评估其生化兼容性。结果表明，包封酶体系的扩增曲线及循环阈值（Ct值）与游离酶对照组相当，未观察到明显的反应抑制或动力学干扰。此外，多重RT-qPCR实验进一步验证了体系在检测特异性方面的完整性。上述结果表明，热响应性琼脂糖微反应器具备结构稳定性与功能惰性，有望成为一种兼容RT-qPCR流程的分子诊断平台。更多还原

【Abstract】 Molecular diagnostics based on reverse transcription quantitative polymerase chain reaction(RTqPCR) relies on temperature-dependent enzymatic reactions that require compatibility with thermal cycling processes. In this study, thermally responsive single-compartment polysaccharide microreactors were fabricated using a water-in-oil emulsion strategy combined with in situ agarose gelation. The effects of oil phase composition and agarose concentration on microsphere formation, size distribution, and protein encapsulation were systematically investigated, and isooctanol was identified as an effective oil phase for achieving stable emulsification with reduced interfacial leakage. Reverse transcriptase and DNA polymerase were subsequently encapsulated to construct single-compartment microreactors, and the biochemical compatibility of these microreactors was evaluated using a one-step RT-qPCR assay. The encapsulated enzyme systems exhibited amplification curves and cycle threshold values comparable to those of free enzyme controls, without detectable inhibition or kinetic interference.Moreover, multiplex RT-qPCR assays confirmed preserved detection specificity without apparent cross-reactivity.These results demonstrate that thermally responsive agarose microreactors provide a structurally stable and functionally neutral platform that is compatible with RT-qPCR workflows for molecular diagnostic applications.更多还原