Sensitive detection of biomarkers is highly desirable for disease diagnosis and postoperative examination. As a signal amplification technique, nonlinear hybridization chain reactions (NHCR) based on DNA self-assembly has been widely adopted to versatile biosensor platforms for signal output and sensitivity enhancement. Herein, we proposed a novel hairpin-free NHCR based flow cytometric immunoassay for prostate specific antigen (PSA) detection. In this study, Ab1-Ag-Ab2-streptavidin-trigger DNA complexes were formed on the magnetic beads (MBs), and each trigger DNA initiated a round of NHCR amplification to form dendritic DNA nanostructures with many fluorescent signal molecules. The robust flow cytometric fluorescent analysis was finally employed for the quantitation of target protein on the MBs. As far as we know, this is the first time to combine the hairpin-free NHCR strategy with fluorescent immunoassay on MBs to detect protein biomarkers. In addition to the high selectivity of immunoassay itself, the characteristics of isothermal, enzyme-free, and exponential amplification efficiency of hairpin-free NHCR endow this developed immunoassay with a detection limit that exceeds 100-folds that of commercially available PSA kits. Moreover, this MBs-based platform of this immunoassay is also amenable to target enrichment and removal of spontaneous NHCR signal through magnetic separation, greatly eliminating the background signal interference. With our efforts, this newly developed biosensor exhibits a detection limit of 1.92 pg/mL and shows high selectivity. It has also been successfully applied to the quantitative detection of PSA in serum samples. With these merits, this convenient biosensor platform has the potential for medical research and disease diagnosis.

生物标志物的灵敏检测对于疾病诊断和术后检查是非常可取的。作为一种信号放大技术，基于DNA自组装的非线性杂交链式反应（NHCR）已被广泛应用于多功能生物传感器平台，用于信号输出和灵敏度增强。在这里，我们提出了一种新的基于无发夹 NHCR 的流式细胞免疫测定法，用于前列腺特异性抗原 (PSA) 检测。在这项研究中，Ab1-Ag-Ab2-链霉亲和素-触发 DNA 复合物在磁珠 (MB) 上形成，每个触发 DNA 启动一轮 NHCR 扩增，形成具有许多荧光信号分子的树突状 DNA 纳米结构。强大的流式细胞荧光分析最终被用于对 MB 上的目标蛋白进行定量。据我们了解，这是第一次将无发夹的 NHCR 策略与 MB 上的荧光免疫分析相结合来检测蛋白质生物标志物。除了免疫分析本身的高选择性外，无发夹NHCR的等温、无酶和指数扩增效率的特点，使该开发的免疫分析的检测限超过市售PSA试剂盒的100倍。此外，该免疫分析基于MBs的平台还可以通过磁分离靶向富集和去除自发NHCR信号，大大消除了背景信号干扰。通过我们的努力，这种新开发的生物传感器的检测限为 1.92 pg/mL，并显示出高选择性。它还成功地应用于血清样品中PSA的定量检测。