Roberts Akanksha, Dhanze Himani, Sharma G Taru, Gandhi Sonu
DBT-National Institute of Animal Biotechnology (NIAB) Hyderabad Telangana India.
DBT-Regional Centre for Biotechnology (RCB) Faridabad Haryana India.
Bioeng Transl Med. 2023 Apr 13;8(3):e10506. doi: 10.1002/btm2.10506. eCollection 2023 May.
Japanese encephalitis (JE), a neglected tropical zoonotic disease prevalent in south-east Asian and western pacific countries, caused by the flavivirus JE virus (JEV), has a dearth of electrochemical point-of-care (PoC) diagnostic tools available to manage endemic breakouts. To overcome this, we have developed a screen-printed carbon electrode (SPCE) immunosensor for rapid PoC detection of JEV nonstructural 1 (NS1) antigen (Ag), found circulating in serum of infected individuals using a smartphone based portable "Sensit" device. The modification of SPCE surface with JEV NS1 antibody (Ab) was confirmed via observation of globular protein structures via scanning electron microscopy (SEM), increase in electrode surface hydrophilicity via contact angle measurement and decrease in current via differential pulse voltammetry (DPV). The fabrication and testing parameters were optimized based on highest current output obtained using DPV. The SPCE was tested for detection limit of target JEV NS1 Ag ranging from 1 fM to 1 μM, which was determined as 0.45 fM in spiked serum. The disposable immunosensor was also found to be highly specific in detecting JEV NS1 Ag over other flaviviral NS1 Ag. Finally, the modified SPCE was clinically validated by testing 62 clinical JEV samples using both a portable miniaturized electrochemical "Sensit" device coupled with a smartphone and a laboratory-based potentiostat. The results were corroborated with gold-standard RT-PCR and showed 96.77% accuracy, 96.15% sensitivity, and 97.22% specificity. Hence, this technique may further be developed into a one-step rapid diagnostic tool for JEV, especially in rural areas.
日本脑炎（JE）是一种被忽视的热带人畜共患病，在东南亚和西太平洋国家流行，由黄病毒科日本脑炎病毒（JEV）引起，目前缺乏用于管理地方性疫情爆发的电化学即时检测（PoC）诊断工具。为了克服这一问题，我们开发了一种丝网印刷碳电极（SPCE）免疫传感器，用于使用基于智能手机的便携式“Sensit”设备快速即时检测感染个体血清中循环的JEV非结构1（NS1）抗原（Ag）。通过扫描电子显微镜（SEM）观察球状蛋白质结构、通过接触角测量增加电极表面亲水性以及通过差分脉冲伏安法（DPV）降低电流，证实了SPCE表面用JEV NS1抗体（Ab）进行了修饰。基于使用DPV获得的最高电流输出，对制造和测试参数进行了优化。对SPCE进行了测试，以确定目标JEV NS1 Ag的检测限，范围为1 fM至1 μM，在加标血清中测定为0.45 fM。还发现这种一次性免疫传感器在检测JEV NS1 Ag方面比其他黄病毒NS1 Ag具有高度特异性。最后，通过使用与智能手机耦合的便携式小型化电化学“Sensit”设备和基于实验室的恒电位仪对62份临床JEV样本进行测试，对修饰后的SPCE进行了临床验证。结果与金标准逆转录聚合酶链反应（RT-PCR）得到了证实，显示出96.77%的准确率、96.15%的灵敏度和97.22%的特异性。因此，这项技术可能会进一步发展成为一种用于JEV的一步式快速诊断工具，特别是在农村地区。
