Point-of-care (POC) detection technologies that enable decentralized, rapid, sensitive, low-cost diagnostics of COVID-19 infection are urgently needed around the world. With many technologies approved for commercialization in the past 10 months, the field of COVID-19 POC diagnostics is rapidly evolving. In this Perspective, we analyze the current state of POC technologies for the diagnosis and monitoring of COVID-19 infection and discuss future challenges in COVID-19 diagnostics. As the COVID-19 pandemic becomes endemic, the advances gained during this past year will likely also be utilized for future prediction of emerging outbreaks and pandemics.
The rapid and unexpected spread of SARS-CoV-2 worldwide has caused unprecedented disruption to daily life and has brought forward critical challenges for public health. The disease was the largest cause of death in the United States in early 2021. Likewise, the COVID-19 pandemic has highlighted the need for rapid and accurate diagnoses at scales larger than ever before. To improve the availability of current gold standard diagnostic testing methods, the development of point-of-care devices that can maintain gold standard sensitivity while reducing the cost and providing portability is much needed. In this work, we combine the amplification capabilities of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) techniques with high-sensitivity end-point detection of crumpled graphene field-effect transistors (cgFETs) to develop a portable detection cell. This electrical detection method takes advantage of the ability of graphene to adsorb single-stranded DNA due to noncovalent π–π bonds but not double-stranded DNA. These devices have demonstrated the ability to detect the presence of the SARS-CoV-2 virus in a range from 10 to 10 4 copies/μL in 20 viral transport medium (VTM) clinical samples. As a result, we achieved 100% PPV, NPV, sensitivity, and specificity with 10 positive and 10 negative VTM clinical samples. Further, the cgFET devices can differentiate between positive and negative VTM clinical samples in 35 min based on the Dirac point shift. Likewise, the improved sensing capabilities of the crumpled gFET were compared with those of the traditional flat gFET devices.
Investigation of the dielectric properties of cell membranes plays an important role in understanding the biological activities that sustain cellular life and realize cellular functionalities. Herein, the variable dielectric polarization characteristics of cell membranes are reported. In controlling the dielectric polarization of a cell using dielectrophoresis force spectroscopy, different cellular crossover frequencies were observed by modulating both the direction and sweep rate of the frequency. The crossover frequencies were used for the extraction of the variable capacitance, which is involved in the dielectric polarization across the cell membranes. In addition, this variable phenomenon was investigated by examining cells whose membranes were cholesterol-depleted with methyl-β-cyclodextrin, which verified a strong correlation between the variable dielectric polarization characteristics and membrane composition changes. This study presented the dielectric polarization properties in live cells’ membranes that can be modified by the regulation of external stimuli and provided a powerful platform to explore cellular membrane dielectric polarization.
The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.
Characterization of cellular dielectrophoretic (DEP) behaviors, when cells are exposed to an alternating current (AC) electric field of varying frequency, is fundamentally important to many applications using dielectrophoresis. However, to date, that characterization has been performed with monotonically increasing or decreasing frequency, not with successive increases and decreases, even though cells might behave differently with those frequency modulations due to the nonlinear cellular electrodynamic responses reported in previous works. In this report, we present a method to trace the behaviors of numerous cells simultaneously at the single-cell level in a simple, robust manner using dielectrophoretic tweezers-based force spectroscopy. Using this method, the behaviors of more than 150 cells were traced in a single environment at the same time, while a modulated DEP force acted upon them, resulting in characterization of nonlinear DEP cellular behaviors and generation of different cross-over frequencies in living cells by modulating the DEP force. This study demonstrated that living cells can have non-linear di-polarized responses depending on the modulation direction of the applied frequency as well as providing a simple and reliable platform from which to measure a cellular cross-over frequency and characterize its nonlinear property.
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