Early accurate profiling of pathogenic bacteria in patients with nosocomial bloodstream infections (nBSIs) is crucial to optimize antibiotic therapy and patient survival. We present a novel autocatalytic Multi-component DNAzyme (MNAzyme)-integrated surface plasmon resonance (SPR) biosensor assay that rapidly and sensitively detects the DNA of three nBSI pathogenic bacteria. This strategy combines autocatalytic MNAzyme cleavage on magnetic beads, as the molecular recognition and signal amplification element, with hybridization chain reaction (HCR)-mediated nanowire assembly on SPR sensing film, as the signal readout and further amplification. Once the MNAzyme complex was activated as a biocatalyst by co-recognizing the bacterial DNA and initial probes, the MNAzyme substrate was catalytically cleaved into two DNA segments. One hybridized with immobilized probes triggering folded hairpin probes to self-assemble into massive nanowire structures via HCR on the sensing interface, and the other co-recognized the initial probes trigging another cycle of multi-turnover cleavage. As a result, the amount of cleaved DNA segments was increasing and greatly enhancing the SPR signal from the analyte. This assay system can provide the fast and quantitative analysis at low concentration (67 cfu/mL of S. aureus, 57 cfu/mL of K. pneumonia and 61 cfu/mL of E. coli, respectively) in a nonenzymatic manner. More importantly, the biosensor was successfully applied to DNA extracted from clinical blood specimens of patients with nBSIs, showing good consistence and practicability. The developed autocatalytic MNAzyme-integrated SPR biosensor provides a sensitive, rapid, reliable and culture-independent approach to efficiently determine multiple pathogenic bacteria for the early diagnosis of nBSIs.

医院血流感染（nBSI）患者中病原细菌的早期准确分析对于优化抗生素治疗和患者生存至关重要。我们提出了一种新型的自动催化多组分DNA酶（MNAzyme）集成的表面等离振子共振（SPR）生物传感器测定法，可以快速，灵敏地检测三种nBSI致病菌的DNA。该策略将磁珠上的自催化MNA酶裂解（作为分子识别和信号放大元件）与SPR传感膜上的杂交链反应（HCR）介导的纳米线组装相结合，作为信号读出和进一步放大。一旦通过共同识别细菌DNA和初始探针将MNAzyme复合物激活为生物催化剂，就将MNAzyme底物催化切割成两个DNA片段。一个与固定探针杂交，触发折叠的发夹状探针，通过传感界面上的HCR自组装成块状纳米线结构，另一个与他人共同认识到，初始探针触发了另一次多翻转切割循环。结果，切割的DNA片段的数量增加并且极大地增强了来自分析物的SPR信号。该测定系统可以以非酶促方式对低浓度（分别为67 cfu / mL的金黄色葡萄球菌，57 cfu / mL的肺炎克雷伯菌和61 cfu / mL的大肠杆菌）进行快速定量分析。更重要的是，该生物传感器已成功应用于从nBSIs患者的临床血液样本中提取的DNA，具有良好的一致性和实用性。集成了自动催化MNAzyme的SPR生物传感器可提供灵敏，快速，