Shan Yuting, Zhang Dongmei, Liu Yao, Wang Yijie, Zhang Jiufa, Ma Cuiping, Li Yang, Shi Chao
Qingdao Nucleic Acid Rapid Testing International Science and Technology Cooperation Base, College of Life Sciences, Qingdao University, Qingdao 266071, People's Republic of China.
Qingdao Special Servicemen Recuperation Center of PLA Navy, Qingdao 266071, People's Republic of China.
Anal Methods. 2025 Jul 3;17(26):5498-5507. doi: 10.1039/d5ay00418g.
Respiratory viral infections continue to pose a significant challenge to global public health. Electrochemical nucleic acid sensors, with their high sensitivity and ease of miniaturization, demonstrate great diagnostic potential. However, the development of high-performance sensors requires comprehensive consideration of factors such as capture probe density, steric hindrance effects, and electrostatic repulsion, which pose significant challenges for the direct detection of long-sequence nucleic acids and limit the application of electrochemical sensors in pathogen diagnosis. This work reports a novel sensor platform based on a concatenated DNA circuit and modified electrodes to achieve efficient and rapid detection of long-sequence nucleic acids. First, the pathogen genomic target sequence replaces the Trigger strand (Ts) through a toehold displacement (TD) reaction. After this, Ts initiates the HCR and the two biotin-modified hairpins hybridize to generate a long double-stranded product. Crowding agents were introduced into the system to enhance the hybridization efficiency of the toehold displacement-mediated hybridization chain reaction (TDHCR) by increasing the local nucleic acid concentration and reducing the free water content in the system, resulting in a significant reduction in reaction time from 120 min to 30 min. Subsequently, the biotin labeled on the TDHCR products rapidly binds to streptavidin immobilized on the electrode surface, enabling efficient and rapid product immobilization. This strategy overcomes the common limitation of low nucleic acid hybridization efficiency at the solid-liquid interface. Finally, the HRP-catalyzed redox reaction between 3,3',5,5'-tetramethylbenzidine (TMB) and HO converts the DNA hybridization event into a measurable electrochemical signal, generating a significant current response. The entire detection process can be completed in less than 50 min, with a detection limit as low as 4.876 fM. This platform demonstrates great potential for clinical pathogen detection and is well-suited for integration into microfluidic devices.
呼吸道病毒感染继续对全球公共卫生构成重大挑战。电化学核酸传感器具有高灵敏度和易于小型化的特点，显示出巨大的诊断潜力。然而，高性能传感器的开发需要综合考虑捕获探针密度、空间位阻效应和静电排斥等因素，这对长序列核酸的直接检测提出了重大挑战，并限制了电化学传感器在病原体诊断中的应用。这项工作报道了一种基于串联DNA电路和修饰电极的新型传感器平台，以实现对长序列核酸的高效快速检测。首先，病原体基因组靶序列通过链置换反应取代触发链（Ts）。此后，Ts启动杂交链式反应（HCR），两个生物素修饰的发夹杂交生成一个长双链产物。将拥挤试剂引入系统，通过增加局部核酸浓度和降低系统中的自由水含量来提高链置换介导的杂交链式反应（TDHCR）的杂交效率，使反应时间从120分钟显著缩短至30分钟。随后，TDHCR产物上标记的生物素迅速与固定在电极表面的链霉亲和素结合，实现产物的高效快速固定。该策略克服了固液界面核酸杂交效率低的常见限制。最后，辣根过氧化物酶（HRP）催化的3,3',5,5'-四甲基联苯胺（TMB）与过氧化氢（HO）之间的氧化还原反应将DNA杂交事件转化为可测量的电化学信号，产生显著的电流响应。整个检测过程可在不到50分钟内完成，检测限低至4.876 fM。该平台在临床病原体检测方面显示出巨大潜力，非常适合集成到微流控设备中。
