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

COVID-19和流感都是传染性呼吸道疾病，其症状在早期很难区分。流行性感冒，通常被称为流感，每年爆发，主要在秋冬季节在世界各地传播，每年导致多达65万人死亡，其中300万至500万严重病例。最近出现的COVID-19已经在全球近400万人中检测出阳性。由于缺乏有效、低成本的即时检测方法，受影响的人数估计可能要高出10倍。尽管科学家、医生和整个国家都在努力解决这一大流行病，但仍然迫切需要能够区分COVID-19和流感的诊断方法。这种情况下，第二波冠状病毒传播可能会在2020年秋季/冬季出现，这也恰逢流感的季节性爆发。因此，开发双重特异性和选择性生物传感器以直接确认患者体液（例如唾液）内流感和/或COVID-19病毒的存在是非常重要的。这项工作的作者建议利用超灵敏的石墨烯纳米材料晶体管，这些晶体管被抗体功能化作为病毒特异性生物传感器。研究人员的目标是建立一个传感器，明确设计用于直接检测病毒体。该项目的成功完成将导致一个早期诊断工具，区分流感和COVID-19，但体现在一个单一的设备，这是至关重要的遏制即将到来的疫情，以促进公共卫生。研究人员打算通过建立一个特定的双生物电子传感器，采用电解质门控石墨烯基场效应晶体管来解决病毒检测的问题。在其他潜在的检测方法和换能器中，电解质门控石墨烯场效应晶体管因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，并且在水性环境中呈惰性。石墨烯通道将通过1-芘丁酸琥珀酰亚胺酯（PASE）接头分子用特异性抗体官能化。PASE将促进与COVID-19特异性抗体和流感特异性抗体的特定缀合。在直接的PASE-抗体缀合的基础上，研究人员计划探索另一种更强大和模块化的途径，该途径采用石墨烯与ssDNA的缀合，并使用抗体：compDNA杂交体来改善抗体与石墨烯的附着。 石墨烯与SARS-CoV-2和石墨烯与H1N1特异性抗体的特定结合将在工作范围内进行研究。为了测试COVID-19和流感生物传感器，将分别使用刺突蛋白（S1）和H1血凝素蛋白（H1 HA）作为分析物。基于蛋白质的生物传感，知识的生物传感器的检测限，他们的灵敏度，动态范围，准确度和响应时间将获得。在两个石墨烯通道与特异性抗体的双重缀合之后，一个额外的通道将被化学钝化，以提供参考信号并允许去除非特异性信号。然后，灭活的SARS-CoV-2和H1N1病毒将用于探索构建的生物传感器的现实应用特性，探索此类传感器的特异性和选择性、交叉反应性和通量。特异性钝化将使得能够在病毒检测的下一阶段进行可靠的信号记录，使用真实的体液样品来评估生物传感器。将SARS-CoV-2和H1N1病毒的特异性和可靠的生物分子整合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，特异和快速的测试。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

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.