RAPID: Dual COVID-19 and Influenza Virus Detection via Target Antibody-Functionalized Graphene Field-Effect Sensing

新冠肺炎和流感都是传染性呼吸道疾病，早期症状很难区分。流感，俗称流感，每年在世界各地爆发，主要在秋冬季爆发，每年造成多达65万人死亡，严重病例为300-500万人。新近出现的新冠肺炎已经在全球近400万人中检测到阳性。由于缺乏有效、低成本的护理点检测方法，估计受影响的人数可能高出10倍。尽管科学家、医生和整个国家都在努力解决这场大流行，但迫切需要能够区分新冠肺炎和流感的诊断方法。目前的情况是，冠状病毒传播的第二波可能会在2020年秋冬季出现，这也恰逢流感的季节性爆发。因此，迅速开发一种特异和选择性的双重生物传感器，直接确认患者体液(如唾液)中是否存在流感和/或新冠肺炎病毒是非常重要的。这项工作的作者建议使用超灵敏的石墨烯纳米材料晶体管，这种晶体管与抗体一起功能化，作为病毒特异性生物传感器。研究人员的目标是建造一种专门用于直接检测病毒体的传感器。该项目的成功完成将带来一种区分流感和新冠肺炎的早期诊断工具，但它将被集成到单一设备中，这对于控制即将到来的疫情以促进公众健康至关重要。研究人员打算通过构建一种特殊的双重生物电子传感器来解决病毒检测问题，该传感器使用电解液门控石墨烯基场效应晶体管。在其他电势检测方法和传感器中，电解液门控石墨烯场效应管以其对分析物的极高灵敏度而闻名。此外，石墨烯具有生物相容性，在环境中稳定，在水环境中具有惰性。石墨烯通道将通过1-戊二酸丁二酰亚胺酯(Pase)连接物分子与特定抗体进行功能化。PASE将促进与新冠肺炎特异性抗体和流感特异性抗体的特定结合。在Pase-抗体直接结合的基础上，研究人员计划探索另一种更健壮和模块化的途径，即使用石墨烯和单链DNA的结合，并使用抗体：CompDNA杂交物来改善抗体在石墨烯上的附着。石墨烯-SARS-CoV-2和石墨烯-H1N1特异性抗体的特殊结合将在工作范围内进行研究。为了测试新冠肺炎和流感生物传感器，将分别使用尖峰蛋白(S1)和H1血凝素蛋白(H1 HA)作为分析物。在蛋白质生物传感的基础上，了解生物传感器的检测极限、灵敏度、动态范围、准确度和响应时间。在两个石墨烯通道与特定抗体双重结合后，另一个通道将被化学钝化，以提供参考信号并允许去除非特定信号。灭活的SARS-CoV-2和H1N1病毒将被用来探索所建立的生物传感器的真实应用特性，探索这种传感器的特异性和选择性、交叉反应性和吞吐量。这种特殊的钝化将在下一阶段的病毒检测中实现可靠的信号记录，使用真实的体液样本来评估生物传感器。将SARS-CoV-2和H1N1病毒的特定和可靠的生物分子集成与石墨烯的电子读数相结合，将能够对这两种致命的呼吸道疾病进行准确、特定和快速的测试。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Both COVID-19 and Influenza are infectious respiratory diseases, and their symptoms are hard to distinguish at the early stage. Influenza, commonly known as the flu, spreads through the world in yearly outbreaks, mainly in the fall-winter season, and causes up to 650,000 death per year with 3-5 million severe cases. COVID-19, emerged recently, has already been positively detected in almost 4 million individuals worldwide. Due to the absence of effective, low-cost, point-of-care detection methods, the number of affected individuals is estimated to be, perhaps, 10- fold higher. While scientists, doctors, and whole nations are hard at work to resolve the pandemic, there exists a pressing and urgent need for diagnostics that can differentiate between COVID-19 and influenza. The situation is such that a second wave of the coronavirus spread will likely appear in the fall/winter of 2020, which also coincides with the seasonal outbreak of influenza. Therefore, it is of rapid importance to develop a dual specific and selective biosensor for direct confirmation of the presence of influenza and/or COVID-19 virus within the patient’s body fluids, such as saliva. Authors of the work propose to utilize ultrasensitive graphene nanomaterial transistors, that are functionalized with the antibodies as the virus-specific biosensors. The researchers aim to build a sensor explicitly designed towards the detection of virus bodies directly. Successful completion of this project will lead to an early-stage diagnosis tool that differentiates between influenza and COVID-19, yet embodied into a single device, which is crucial for containing the forthcoming outbreak in order to promote public health.The investigators intend to approach the problem of virus detection by building a specific dual bioelectronic sensor that employs electrolyte-gated graphene-based field-effect transistors. Among other potential detection methods and transducers, electrolyte-gated graphene field-effect transistors are known for their extremely high sensitivity to analytes. Besides, graphene is biocompatible, stable in ambient, and inert in the aqueous environments. The graphene channel will be functionalized with specific antibodies through a 1-pyrenebutanoic acid, succinimidyl ester (PASE) linker molecule. PASE will facilitate the particular conjugation with both COVID-19 specific, and influenza-specific antibodies. Building upon the direct PASE-antibody conjugation, the researchers plan to explore another, more robust and modular path, which employs conjugation of graphene with ssDNA and use the antibody:compDNA hybrids to improve the antibody attachment onto graphene. The particular conjugation of graphene-to-SARS-CoV-2 and graphene-to-H1N1 specific antibodies will be studied within the scope of the work. In order to test the COVID-19 and Influenza biosensors, the spike protein (S1) and H1 hemagglutinin protein (H1 HA) will be used as analytes, respectively. Based on the protein biosensing, knowledge on the biosensor’s detection limit, their sensitivity, dynamic range, accuracy, and response time will be obtained. Following the dual conjugation of two graphene channels with specific antibodies, one additional channel will be chemically passivated in order to provide a reference signal and allow for the removal of non-specific signals. The inactivated SARS-CoV-2 and H1N1 viruses will then be used to explore the real-world application properties of the built biosensor, exploring specificity and selectivity of such a sensor, cross-reactivity, and throughput. The specific passivation will enable reliable signal recording at the next stage of virus detection, using real body fluid samples to assess the biosensor. Combining a specific and dependable biomolecular integration of both SARS-CoV-2 and H1N1 viruses with graphene’s electrical readout will enable an accurate, specific, and rapid test for the two deadly respiratory diseases.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.