RAPID: Determination of the cross-over frequency of SARS-CoV-2 for rapid identification concentration

威斯康星大学密尔沃基分校（University of Wisconsin-Milwaukee）获得了一项奖项，研究一种电捕获技术，该技术可以快速浓缩SARS-CoV-2病毒，以生产用于开发疫苗和治疗方法的高纯度样品。目前，通过过滤和离心的多重串联应用进行纯化需要多达两天的时间来制备病毒样本。不幸的是，用常规方法收集后，分离的病毒的纯度和活力仍然很低。在本研究中，研究了病毒、纳米颗粒大小和电介电捕获条件之间的相关性，以确定浓缩80 - 160 nm大小的SARS-CoV-2的最佳操作条件。所开发的方法将能够从细胞培养和患者样本中快速浓缩纯病毒。浓缩样本将通过快速提供高度浓缩的纯病毒样本，促进传染病疫苗、治疗和快速诊断方法的开发。因此，该研究将有助于改善与传染病有关的公共安全以及诊断和治疗领域的经济。所开发的方法也适用于生物分子的浓度，包括DNA、蛋白质、细胞裂解片段和颗粒，除了病毒。因此，这项研究将大大提高分子在各个学科中的快速表征和应用，如病毒学、基因组学、蛋白质组学、诊断、法医学，以及疫苗和治疗学的开发。本研究开发的新型介电泳纳米颗粒操作方法可以从病毒感染的细胞培养基和患者的样本中快速浓缩病毒到高纯度的样品中。当介电粒子暴露在不均匀分布的电场中时，就会发生电荷极化。根据给定的条件，如粒子和介质的电势、频率和介电常数，粒子将移动到密集或稀疏的电场区域。粒子的运动方向随频率而反转；这被称为交叉频率。交叉频率取决于粒子的特性，如成分、结构、电荷等。在本研究中，将使用热处理的SARS-CoV-2和纳米颗粒来研究60至500纳米尺寸的不同生物纳米颗粒的交叉频率。最初，不同大小的纳米粒子的交叉频率将被确定使用介电泳陷阱，通过修改应用电位，频率和粒度。然后，将相同的操作条件筛选策略应用于热处理的SARS-CoV-2。利用开发的条件确定分离的纳米颗粒和SARS-CoV-2的纯度和效率。所开发的方法将克服目前用于分离病毒的串行处理方法存在的缺点，即费时、费力、复杂和效果较差。因此，该研究将通过快速提供更高纯度的样品，大大提高对生物纳米颗粒（如活病毒）结构和特性的理解。获得的结果将迅速应用于其他病毒和颗粒，以进一步表征和检测，以及开发应用。威斯康星大学密尔沃基分校的这项RAPID奖是由生物基础设施部利用《冠状病毒援助、救济和经济安全（关怀）法案》的资金颁发的。该奖项反映了美国国家科学基金会的法定使命，并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，被认为值得支持。

The University of Wisconsin-Milwaukee received an award to investigate an electrical trapping technique to rapidly concentrate SARS-CoV-2 virus to produce highly pure samples for the development of vaccines and therapeutics. Currently, purification through multiple in-series applications of filtration and centrifugation require up to two days to prepare virus samples. Unfortunately, the purity and viability of the separated viruses are still low after being collected by conventional methods. In this research, a correlation between the virus, nanoparticle size, and electrical dielectrophoretic trapping conditions is investigated to determine the best operating conditions to concentrate 80 – 160 nm size SARS-CoV-2. The developed method will enable the rapid concentration of pure viruses from cell culture and patient samples. The concentrated samples will enhance the development of vaccines, cures, and rapid diagnostic methods for infectious diseases by rapidly supplying highly concentrated pure virus samples. Thus, the research will contribute to improve the public safety related to infectious diseases and economy in the diagnostics and therapeutics area. The developed method is also applicable to the concentration of biomolecules, including DNA, protein, cell lysed fragments and particles, in addition to viruses. Thus, this research will give a substantial improvement on the rapid characterization and applications of molecules in various disciplines, such as virology, genomics, proteomics, diagnosis, forensic science, in addition to vaccine and therapeutics development. The novel dielectrophoretic nanoparticle manipulating method developed in this research can rapidly concentrate viruses into highly pure samples from virus-infected cell media and samples from patients. A charge polarization happens to a dielectric particle when the particle is exposed to an unevenly distributed electric field. The particle will move to either a dense or sparse electric field area depending on the given condition, such as electrical potential, frequency, and permittivities of the particle and medium. The direction of particle movement is inverted depending on the frequency; this is called cross-over frequency. The cross-over frequency varies depending on the characteristics of the particles, such as components, structure, charge, etc. In this research, the cross-over frequency of different bio-nano-particles, ranged from 60 to 500 nm size, will be investigated using heat-treated SARS-CoV-2 and nanoparticles. Initially, the cross-over frequency of different size nano-particles will be determined using dielectrophoretic traps, by modifying the applied potential, frequency, and particle size. Then, the same operating condition screening strategy will be applied to heat-treated SARS-CoV-2. Purity and efficiency of the isolated nano-particles and SARS-CoV-2 will be determined using the developed conditions. The developed method will circumvent the drawbacks that exist with the current serial treatment method used to separate virus, which are: time-intensive, laborious, complicated, and less effective. Consequently, this research will significantly enhance the understandings of the structures and characteristics of bio-nano-particles, such as live viruses, by supplying higher purity samples rapidly. Obtained results will be rapidly applied to other viruses and particles for further characterization and detection, as well as development of applications. This RAPID award to the University of Wisconsin-Milwaukee is made by the Division of Biological Infrastructure using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.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.