胃癌（GC）是全球最常见的肿瘤之一，也是肿瘤相关死亡的主要原因。传统的生物标志物和筛查方法无法满足临床需求。对于早期胃癌（EGC）筛查，迫切需要高灵敏度的诊断标志物以及准确的定量方法。在这里，利用创新应用的两个靶标响应链迁移系统与单扩增循环元素协同响应的荧光纳米机器被开发出来，用于同时检测GC生物标志物miR-5585-5p和PLS3 mRNA，这些靶标是通过下一代测序和RT-qPCR选定的。这也是一种免RNA提取、无PCR、非酶生物传感器，可实现肿瘤细胞成像和血清诊断。只需要20μL血清样本和20分钟的孵育时间，该纳米机器可以实现fM级别的超灵敏检测限，具有从fM到nM的广泛线性范围。更重要的是，与临床常用生物标志物CA 72-4相比，纳米机器获得了更高的AUC值（0.884），成功区分了GC患者。值得注意的是，对于EGC患者的关键诊断问题，纳米机器也实现了令人满意的AUC值（0.859）。总之，本研究筛选获得了多个生物标志物，并开发了一种荧光纳米机器，用于结合诊断GC，为功能化DNA纳米机器的巧妙设计和血清生物标志物向临床诊断的可行策略提供了方法。

Gastric cancer (GC) is one of the most common tumors worldwide and is the leading cause of tumor-related mortality. Traditional biomarkers and screening methods cannot meet the clinical demands. There is an urgent need for highly sensitive diagnostic markers as well as accurate quantification methods for early gastric cancer (EGC) screening. Here a dual-target cooperatively responsive fluorescent nanomachine by the innovative application of two targets─responsive strand migration system with a single-amplification-cycle element was developed for the simultaneous detection of GC biomarkers miR-5585-5p and PLS3 mRNA, which were selected by next-generation sequencing and RT-qPCR. It was also an RNA extraction-free, PCR-free, and nonenzymatic biosensor to achieve tumor cell imaging and serum diagnosis. Requiring only a 20 μL serum sample and 20 min incubation time, the nanomachine achieved an ultrasensitive detection limit of fM level with a broad linear range from fM to nM. More importantly, a higher AUC value (0.884) compared to the clinically used biomarker CA 72-4 was obtained by the nanomachine to distinguish GC patients successfully. Notably, for the key concerns of diagnosis of EGC patients, the nanomachine also achieved a satisfactory AUC value of 0.859. Taken together, this work has screened and obtained multiple biomarkers and developed a fluorescent nanomachine for combination diagnosis of GC, providing an ingenious design of a functionalized DNA nanomachine and a feasible strategy for the transformation of serum biomarkers into clinical diagnosis.