Zheng Zongmei, Wang Mingyang, Yuan Jinlong, Liu Junchong, Yu Haipeng, Yang Zhonghuang, Liu Wanjian, Liu Aihua
Institute for Chemical Biology & Biosensing, College of Life Sciences, Qingdao University, 308 Ningxia Road, Qingdao, 266071, China.
Qingdao Richgen Biological Technology Co., Ltd, 369 Hedong Road, Qingdao National Hi-tech Industrial Development Zone, Qingdao, 266112, China.
Anal Chim Acta. 2025 Jun 15;1355:344015. doi: 10.1016/j.aca.2025.344015. Epub 2025 Apr 4.
It is crucial to timely and accurately identify the causative virus for early treatment and urgent prevention. Viral antigen detection can identify those people who are most likely at risk of spreading the disease, but most based on antibodies with limited stability and sensitivity. Peptides offer several advantages over antibodies, such as low cost, smaller size and good stability. The development of electrochemical immunoassay using specific peptide probes have the merits of good sensitivity and selectivity as well as good stability. Herein we report an amperometric immunosensor using peptides as capture probe and recognition probe. The molecular docking suggests that the two peptides of Pi (sequence: NFWISPKLAFALGGGKKKSC) and FK11 (sequence: WFLNDSELISML), bioscreened from phage display, bind to N-terminal domain of SARS-CoV-2 spike protein (SP). The peptide Pi is assembled on MoS@AuNPs modified electrode to capture SARS-CoV-2 SP, which is recognized by peptide FK11-displayed phage to form Pi/SARS-CoV-2 SP/FK11-phage sandwich. Then anti-M13 phage conjugated horseradish peroxidase (HRP) (anti M13-HRP) was introduced to recognize the phage capsid protein pVIII to form M13 phage/anti M13-HRP to enrich thousands of HRP, which can further electrochemically catalyze HO reduction at highly conductive MoS@AuNPs at - 0.35 V. Then amperometric immunosensor was constructed with linear range of 0.1-5000 pg/mL SARS-CoV-2 SP and detection limit of 0.074 pg/mL. The sensor also has good selectivity, batch reproducibility and stability, capable of detecting down to 10 transducing units/mL SARS-CoV-2 pseudoviruses. This work represents the first example of dual-peptide probes based sandwich-type electrochemical immunosensor integrated with dual signal amplification, which may provide a cost-effective assay platform in detecting real SARS-CoV-2 viruses for early diagnosis. The flexible and modular strategy can be extended to develop other type biosensors for a wide range of applications.
及时准确地识别致病病毒对于早期治疗和紧急预防至关重要。病毒抗原检测可以识别那些最有可能传播疾病的人，但大多数基于稳定性和灵敏度有限的抗体。与抗体相比，肽具有几个优点，如成本低、尺寸小和稳定性好。使用特定肽探针开发的电化学免疫分析法具有良好的灵敏度、选择性和稳定性。在此，我们报道了一种以肽为捕获探针和识别探针的安培免疫传感器。分子对接表明，从噬菌体展示中筛选出的两种肽Pi（序列：NFWISPKLAFALGGGKKKSC）和FK11（序列：WFLNDSELISML）与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白（SP）的N端结构域结合。肽Pi组装在二硫化钼@金纳米颗粒修饰电极上以捕获SARS-CoV-2 SP，其被展示肽FK11的噬菌体识别以形成Pi/SARS-CoV-2 SP/FK11-噬菌体夹心结构。然后引入抗M13噬菌体缀合辣根过氧化物酶（HRP）（抗M13-HRP）以识别噬菌体衣壳蛋白pVIII，形成M13噬菌体/抗M13-HRP以富集数千个HRP，其可以在-0.35 V下在高导电性二硫化钼@金纳米颗粒上进一步电化学催化过氧化氢还原。然后构建安培免疫传感器，其线性范围为0.1-至5000 pg/mL的SARS-CoV-2 SP，检测限为0.074 pg/mL。该传感器还具有良好的选择性、批次重现性和稳定性，能够检测低至10个转导单位/mL的SARS-CoV-2假病毒。这项工作代表了基于双肽探针的夹心型电化学免疫传感器与双信号放大相结合的首个实例，这可能为早期诊断检测真实的SARS-CoV-2病毒提供一个具有成本效益的检测平台。这种灵活且模块化的策略可以扩展到开发用于广泛应用的其他类型生物传感器。
