敏感生物标志物检测技术对于疾病诊断和术后检查都有益处。非线性杂交链反应（NHCR）广泛用作生物传感器平台的输出信号放大技术。本研究开发了一种无发夹的NHCR，作为流式细胞仪免疫测定法，用于检测甲胎蛋白（AFP）和前列腺特异性抗原（PSA）。首先，将目标AFP捕获在捕获抗体修饰的磁珠（MBs）上。然后添加准备好的生物素-链霉亲和素-生物素（B-S-B）系统，将生物素化的检测抗体和生物素化的触发DNA通过生物素-链霉亲和素相互作用的高亲和力连接在一起，标记目标AFP，形成三条触发DNA链的夹心复合物。每个触发DNA链都会在非线性杂交链反应后生长出树枝状DNA纳米结构。由于基质流链标记有荧光染料，所以树状DNA的自组装过程伴随着荧光染料的持续释放。强荧光的树枝最终形成在MBs的表面。最后，通过分析流式细胞仪中的荧光MBs来定量检测目标AFP。所提出的免疫测定法具有高选择性以及等温、无酶和指数放大效率。它显示了1.74 pg mL-1的检测限制（LOD）。该生物传感器还成功地用于定量检测血清样品中的AFP。通过改变MBs和抗体-抗原对的大小，可以同时检测多个肿瘤标记物。大多数肿瘤标志物只与肿瘤诊断相关，但缺乏特异性，因此通过联合检测多个肿瘤标志物可以提高早期肿瘤诊断的准确性。

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. The nonlinear hybridization chain reaction (NHCR) is widely used as an output signal amplification technique for biosensor platforms. A novel hairpin-free NHCR was developed in this study as a flow cytometric immunoassay to detect alpha-fetoprotein (AFP) and prostate specific antigen (PSA). First, the target AFP is captured on magnetic beads (MBs) that are modified with capture antibodies. Then, the prepared biotin-streptavidin-biotin (B-S-B) system, which links biotinylated detection antibodies and biotinylated trigger DNA together through the high affinity between biotin-streptavidin interaction, is added to label the target AFP, forming a sandwich complex with three trigger DNA chains. Each trigger DNA chain grows a dendritic DNA nanostructure following a nonlinear hybridization chain reaction. As the substrate flue chains are labeled with fluorophores, the self-assembly process of dendritic DNA is accompanied by the continuous release of fluorophores. Dendrites with strong fluorescence then form on the surface of MBs. Finally, the target AFP is quantified by analyzing the fluorescent MBs using flow cytometry. The proposed immunoassay has a high selectivity along with isothermal, enzyme-free, and exponential amplification efficiency. It shows a limit of detection (LOD) of 1.74 pg mL-1. The proposed biosensor was also successfully used to quantitatively detect AFP in serum samples. It may be utilized to detect multiple tumor markers simultaneously by changing the size of MBs and antibody-antigen pairs. Most tumor markers are only related to tumor diagnosis but without specificity, so the combined detection of multiple tumor markers can improve the accuracy of early tumor diagnoses.