基于立足点介导的熵驱动双足DNA步行器及树枝状杂交链式反应的一步式循环肿瘤DNA微流控传感研究  

One-step Circulating Tumor DNA Microfluidic Sensing Based on Toehold-mediated Entropy-driven Bipedal DNA Walker and Dendritic Hybridization Chain Reaction

作　　者：杨梅[1] 傅裕 张何[1] 马文杰 豆玉豪 傅昕[1] 李满霞 曾超然 YANG Mei;FU Yu;ZHANG He;MA Wen-Jie;DOU Yu-Hao;FU Xin;LI Man-Xia;ZENG Chao-Ran(Hunan Provincial Key Laboratory of Environmental Catalysis and Waste Recycling,College of Materials and Chemical Engineering,Hunan Institute of Engineering,Xiangtan 411104,China)

机构地区：[1]湖南工程学院材料与化工学院,湖南省环境催化与废弃物再生化重点实验室,湘潭411104

出　　处：《分析化学》2024年第11期1755-1765,共11页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.21005067);湖南省重点研发计划项目(No.2020SK3019);湖南省研究生科研创新项目(No.CX20221292);湖南省教育厅科研项目(No.23A0529)资助。

摘　　要：血液中低丰度的循环肿瘤DNA(ctDNA)是一类关键癌症标志物,实现ctDNA的精准检测对于疾病的早期诊断、监测及预后评估均具有重要意义。本研究设计了一种基于微流控芯片的熵驱动双足DNA步行器与树枝状杂交链式反应相结合的级联信号放大策略,用于检测血液中的ctDNA。当靶标存在时,立足点A与靶标相互作用,借助燃料链F触发双向链置换反应,从而释放靶标及8-17 DNAzyme活性中心。其中,释放的靶标进入新一轮熵驱动链置换反应;同时,Pb^(2+)激活的8-17 DNAzyme活性中心将作用于微珠表面的切割位点,暴露出微珠表面的捕获探针,进而诱导树枝状杂交链式反应,使荧光信号富集于微珠表面。以乳腺癌突变基因PIK3CA^(E545K)为靶标模型,在最优实验条件下,此传感器的线性范围为50~10000 fmol/L,检出限(LOD,3σ)为0.94 fmol/L,回归方程为y=31.6539 lgC_(T)+474.0801(C_(T)为突变基因PIK3CA^(E545K)浓度,fmol/L)。将此方法应用于人血清中突变基因PIK3CA^(E545K)含量的检测,加标回收率在98.8%~106.5%之间。本方法灵敏度高、特异性强、抗干扰能力强,并具有高通量和一步式操作的特点,在复杂样品的快速分析中具有良好的应用潜力。Circulating tumor DNA(ctDNA)in blood,present at low abundance,serves as a critical biomarker for cancer.Precise detection of ctDNA is of great significance for early diagnosis,disease monitoring,and prognosis evaluation.In this study,a microfluidic chip-based entropy-driven bipedal DNA walker combined with dendritic hybridisation chain reaction was designed as a cascade signal amplification strategy for detection of ctDNA in blood in a microfluidic chip.In the presence of the target,toehold A interacted with target and triggerd a bidirectional strand displacement reaction with the aid of fuel strand F,thereby releasing the target and the 8-17 DNAzyme active centre.Among them,the released target was recycled in a new round of entropy-driven chain replacement reaction.Meanwhile,8-17 DNAzyme active center activated by Pb^(2+)would act on the cleavage site of the substrate hairpin at the microbead surface,exposing the capture probes on the surface of the microbead.The numerous capture probes induced a dendritic hybridization chain reaction,which resulted in fluorescent signals being concentrated o thesurface of the microbeads With the breastcancer mutant gene PIK3CAs4K as the target model,under the optimal experimental conditions,the linear range of sensor was 50-10000 fmol/L,the detection limit was 0.94 fmol/L(L0D,3o),and the regression equation was y=31.65 lgC_(T)+474.08(C_(T) is the concentration mutant gene PIK3CA^(E545K) sequence,fmol/L).This method showed spiked recoveries between 98.8%and 106.5%when applied to detection of mutant ene PIK3CA^(E545K) nhuman serum.Characterized by its sensitivity,specificity anti-interference capability,high throughput,and one-step operation,this method was ideally suited for the rapid analysis of complex samples.

关 键 词：DNA步行器 熵驱动链置换反应 微流控芯片 8-17 DNAzyme 树枝状杂交链式反应 

分 类 号：O657[理学—分析化学] R730.4[理学—化学]