Multiplexed Ultrasensitive Sample-to-Answer RT-LAMP Chip for the Identification of SARS-CoV-2 and Influenza Viruses.

Prompt on-site diagnosis of SARS-CoV-2 with other respiratory infections will have minimized the global impact of the COVID-19 pandemic through rapid, effective management. However, no such multiplex point-of-care (POC) chip has satisfied a suitable sensitivity of gold-standard nucleic acid amplification tests (NAATs). Here, a rapid multiplexed ultrasensitive sample-to-answer loop-mediated isothermal amplification (MUSAL) chip operated by simple LED-driven photothermal amplification to detect six targets from single-swab sampling is presented. First, the MUSAL chip allows ultrafast on-chip sample preparation with ≈500-fold preconcentration at a rate of 1.2 mL min-1 . Second, the chip enables contamination-free amplification using autonomous target elution into on-chip reagents by photothermal activation. Finally, the chip accomplishes multiplexed on-chip diagnostics of SARS-CoV-2 and influenza viruses with a limit of detection (LoD) of 0.5 copies µL-1 . The rapid, ultrasensitive, cost-effective sample-to-answer chip with a multiplex capability will allow timely management of various pandemics situations that may be faced shortly.

KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney.

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro . KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

Asymmetric Cell Division of Fibroblasts is An Early Deterministic Step to Generate Elite Cells during Cell Reprogramming.

Cell reprogramming is considered a stochastic process, and it is not clear which cells are prone to be reprogrammed and whether a deterministic step exists. Here, asymmetric cell division (ACD) at the early stage of induced neuronal (iN) reprogramming is shown to play a deterministic role in generating elite cells for reprogramming. Within one day, fibroblasts underwent ACD, with one daughter cell being converted into an iN precursor and the other one remaining as a fibroblast. Inhibition of ACD significantly inhibited iN conversion. Moreover, the daughter cells showed asymmetric DNA segregation and histone marks during cytokinesis, and the cells inheriting newly replicated DNA strands during ACD became iN precursors. These results unravel a deterministic step at the early phase of cell reprogramming and demonstrate a novel role of ACD in cell phenotype change. This work also supports a novel hypothesis that daughter cells with newly replicated DNA strands are elite cells for reprogramming, which remains to be tested in various reprogramming processes.

Nanophotonic Cell Lysis and Polymerase Chain Reaction with Gravity-Driven Cell Enrichment for Rapid Detection of Pathogens.

Rapid and precise detection of pathogens is a critical step in the prevention and identification of emergencies related to health and biosafety as well as the clinical management of community-acquired urinary tract infections or sexually transmitted diseases. However, a conventional culture-based pathogen diagnostic method is time-consuming, permitting physicians to use antibiotics without ample clinical data. Here, we present a nanophotonic Light-driven Integrated cell lysis and polymerase chain reaction (PCR) on a chip with Gravity-driven cell enrichment Health Technology (LIGHT) for rapid precision detection of pathogens (<20 min). We created the LIGHT, which has the three functions of (1) selective enrichment of pathogens, (2) photothermal cell lysis, and (3) photonic PCR on a chip. We designed the gravity-driven cell enrichment via a nanoporous membrane on a chip that allows an effective bacterial enrichment of 40 000-fold from a 1 mL sample in 2 min. We established a light-driven photothermal lysis of preconcentrated bacteria within 1 min by designing the network of nanoplasmonic optical antenna on a chip for ultrafast light-to-heat conversion, created the nanoplasmonic optical antenna network-based ultrafast photonic PCR on a chip, and identified Escherichia coli. Finally, we demonstrated the end-point detection of up to 103 CFU/mL of E. coli in 10 min. We believe that our nanophotonic LIGHT will provide rapid and precise identification of pathogens in both developing and developed countries.

Bubble-free rapid microfluidic PCR.

Microfluidic polymerase chain reaction (PCR) has been of great interest owing to its ability to perform rapid and specific nucleic acid amplification and analysis on small volumes of samples. One of the major drawbacks of microfluidic PCR is bubble generation and reagent evaporation, which can cause malfunctions. Here, through theoretical modeling and characterization of bubble behavior, we propose a bubble-free microfluidic PCR device via controlled fluid transfer. Our approach exploits a thin impermeable polyethylene (PE) top layer that minimizes the generation of bubbles by inhibiting mass transport along a vertical direction. Simulation results demonstrate that a calculated mass flow difference of approximately 370% can be obtained by utilizing an impermeable membrane as the vertical barrier layer. To demonstrate proof-of-concept, two nanoporous polymeric materials, poly(dimethylsiloxane) (PDMS) and PE, were used for stand-alone self-powered sample loading (approximately 70 s) and for use as a vertical barrier layer, respectively. Consequently, we demonstrate successful amplification of the cMET gene, a nucleic acid (NA) biomarker for lung cancer, and complete an ultrafast PCR test in less than 3 min using a high powered Peltier-based thermal cycler under bubble-free conditions. This approach will result in a new paradigm for ultrafast molecular diagnosis and can facilitate NA-based nearly instantaneous diagnostics for point-of-care testing and for personalized and preventive medicine.

Preclinical Animal Models for Dravet Syndrome: Seizure Phenotypes, Comorbidities and Drug Screening.

Epilepsy is a common chronic neurological disease affecting almost 3 million people in the United States and 50 million people worldwide. Despite availability of more than two dozen FDA-approved anti-epileptic drugs (AEDs), one-third of patients fail to receive adequate seizure control. Specifically, pediatric genetic epilepsies are often the most severe, debilitating and pharmaco-resistant forms of epilepsy. Epileptic syndromes share a common symptom of unprovoked seizures. While some epilepsies/forms of epilepsy are the result of acquired insults such as head trauma, febrile seizure, or viral infection, others have a genetic basis. The discovery of epilepsy associated genes suggests varied underlying pathologies and opens the door for development of new "personalized" treatment options for each genetic epilepsy. Among these, Dravet syndrome (DS) has received substantial attention for both the pre-clinical and early clinical development of novel therapeutics. Despite these advances, there is no FDA-approved treatment for DS. Over 80% of patients diagnosed with DS carry a de novo mutation within the voltage-gated sodium channel gene SCN1A and these patients suffer with drug resistant and life-threatening seizures. Here we will review the preclinical animal models for DS featuring inactivation of SCN1A (including zebrafish and mice) with an emphasis on seizure phenotypes and behavioral comorbidities. Because many drugs fail somewhere between initial preclinical discovery and clinical trials, it is equally important that we understand how these models respond to known AEDs. As such, we will also review the available literature and recent drug screening efforts using these models with a focus on assay protocols and predictive pharmacological profiles. Validation of these preclinical models is a critical step in our efforts to efficiently discover new therapies for these patients. The behavioral and electrophysiological drug screening assays in zebrafish will be discussed in detail including specific examples from our laboratory using a zebrafish scn1 mutant and a summary of the nearly 3000 drugs screened to date. As the discovery and development phase rapidly moves from the lab-to-the-clinic for DS, it is hoped that this preclinical strategy offers a platform for how to approach any genetic epilepsy.

Microphysiological Analysis Platform of Pancreatic Islet ß-Cell Spheroids.

The hallmarks of diabetics are insufficient secretion of insulin and dysregulation of glucagon. It is critical to understand release mechanisms of insulin, glucagon, and other hormones from the islets of Langerhans. In spite of remarkable advancements in diabetes research and practice, robust and reproducible models that can measure pancreatic ß-cell function are lacking. Here, a microphysiological analysis platform (MAP) that allows the uniform 3D spheroid formation of pancreatic ß-cell islets, large-scale morphological phenotyping, and gene expression mapping of chronic glycemia and lipidemia development is reported. The MAP enables the scaffold-free formation of densely packed ß-cell spheroids (i.e., multiple array of 110 bioreactors) surrounded with a perfusion flow network inspired by physiologically relevant microenvironment. The MAP permits dynamic perturbations on the ß-cell spheroids and the precise controls of glycemia and lipidemia, which allow us to confirm that cellular apoptosis in the ß-cell spheroid under hyperglycemia and hyperlipidemia is mostly dependent to a reactive oxygen species-induced caspase-mediated pathway. The ß-cells' MAP might provide a potential new map in the pathophysiological mechanisms of ß cells.

Asymmetric Nanocrescent Antenna on Upconversion Nanocrystal.

Frequency upconversion activated with lanthanide has attracted attention in various real-world applications, because it is far simpler and more efficient than traditional nonlinear susceptibility-based frequency upconversion, such as second harmonic generation. However, the quantum yield of frequency upconversion of lanthanide-based upconversion nanocrystals remains inefficient for practical applications, and spatial control of upconverted emission is not yet developed. Here, we developed an asymmetric nanocrescent antenna on upconversion nanocrystal (ANAU) to deliver excitation light effectively to the core of upconversion nanocrystal by nanofocusing light and generating asymmetric frequency upconverted emission concentrated toward the tip region. ANAUs were fabricated by high-angle deposition (60°) of gold (Au) on the isolated upconversion nanoparticles supported by nanopillars then moved to refractive-index matched substrate for orientation-dependent upconversion luminescence analysis in the single-nanoparticle scale. We studied shape-dependent nanofocusing efficiency of nanocrescent antennae as a function of the tip-to-tip distance by modulating the deposition angle. The generation of asymmetric frequency upconverted emission toward the tip region was simulated by the asymmetric far-field radiation pattern of dipoles in the nanocrescent antenna and experimentally demonstrated by the orientation-dependent photon intensity of frequency upconverted emission of an ANAU. This finding provides a new way to improve frequency upconversion using an antenna, which locally increases the excitation light and generates the radiation power to certain directions for various applications.

Assessing Antibiotic Permeability of Gram-Negative Bacteria via Nanofluidics.

While antibiotic resistance is increasing rapidly, drug discovery has proven to be extremely difficult. Antibiotic resistance transforms some bacterial infections into deadly medical conditions. A significant challenge in antibiotic discovery is designing potent molecules that enter Gram-negative bacteria and also avoid active efflux mechanisms. Critical analysis in rational drug design has been hindered by the lack of effective analytical tools to analyze the bacterial membrane permeability of small molecules. We design, fabricate, and characterize the nanofluidic device that actively loads more than 200 single bacterial cells in a nanochannel array. We demonstrate a gigaohm seal between the nanochannel walls and the loaded bacteria, restricting small molecule transport to only occur through the bacterial cell envelope. Quantitation of clindamycin translocation through wild-type and efflux-deficient (ΔtolC) Escherichia coli strains via nanofluidic-interfaced liquid chromatography mass spectrometry shows higher levels of translocation for wild-type E. coli than for an efflux-deficient strain. We believe that the assessment of compound permeability in Gram-negative bacteria via the nanofluidic analysis platform will be an impactful tool for compound permeation and efflux studies in bacteria to assist rational antibiotic design.

Clemizole and modulators of serotonin signalling suppress seizures in Dravet syndrome.

Dravet syndrome is a catastrophic childhood epilepsy with early-onset seizures, delayed language and motor development, sleep disturbances, anxiety-like behaviour, severe cognitive deficit and an increased risk of fatality. It is primarily caused by de novo mutations of the SCN1A gene encoding a neuronal voltage-activated sodium channel. Zebrafish with a mutation in the SCN1A homologue recapitulate spontaneous seizure activity and mimic the convulsive behavioural movements observed in Dravet syndrome. Here, we show that phenotypic screening of drug libraries in zebrafish scn1 mutants rapidly and successfully identifies new therapeutics. We demonstrate that clemizole binds to serotonin receptors and its antiepileptic activity can be mimicked by drugs acting on serotonin signalling pathways e.g. trazodone and lorcaserin. Coincident with these zebrafish findings, we treated five medically intractable Dravet syndrome patients with a clinically-approved serotonin receptor agonist (lorcaserin, Belviq®) and observed some promising results in terms of reductions in seizure frequency and/or severity. Our findings demonstrate a rapid path from preclinical discovery in zebrafish, through target identification, to potential clinical treatments for Dravet syndrome.

Dual transcript and protein quantification in a massive single cell array.

Recently, single-cell molecular analysis has been leveraged to achieve unprecedented levels of biological investigation. However, a lack of simple, high-throughput single-cell methods has hindered in-depth population-wide studies with single-cell resolution. We report a microwell-based cytometric method for simultaneous measurements of gene and protein expression dynamics in thousands of single cells. We quantified the regulatory effects of transcriptional and translational inhibitors on cMET mRNA and cMET protein in cell populations. We studied the dynamic responses of individual cells to drug treatments, by measuring cMET overexpression levels in individual non-small cell lung cancer (NSCLC) cells with induced drug resistance. Across NSCLC cell lines with a given protein expression, distinct patterns of transcript-protein correlation emerged. We believe this platform is applicable for interrogating the dynamics of gene expression, protein expression, and translational kinetics at the single-cell level - a paradigm shift in life science and medicine toward discovering vital cell regulatory mechanisms.

A Novel Long-term, Multi-Channel and Non-invasive Electrophysiology Platform for Zebrafish.

Zebrafish are a popular vertebrate model for human neurological disorders and drug discovery. Although fecundity, breeding convenience, genetic homology and optical transparency have been key advantages, laborious and invasive procedures are required for electrophysiological studies. Using an electrode-integrated microfluidic system, here we demonstrate a novel multichannel electrophysiology unit to record multiple zebrafish. This platform allows spontaneous alignment of zebrafish and maintains, over days, close contact between head and multiple surface electrodes, enabling non-invasive long-term electroencephalographic recording. First, we demonstrate that electrographic seizure events, induced by pentylenetetrazole, can be reliably distinguished from eye or tail movement artifacts, and quantifiably identified with our unique algorithm. Second, we show long-term monitoring during epileptogenic progression in a scn1lab mutant recapitulating human Dravet syndrome. Third, we provide an example of cross-over pharmacology antiepileptic drug testing. Such promising features of this integrated microfluidic platform will greatly facilitate high-throughput drug screening and electrophysiological characterization of epileptic zebrafish.

Spontaneous Self-Formation of 3D Plasmonic Optical Structures.

Self-formation of colloidal oil droplets in water or water droplets in oil not only has been regarded as fascinating fundamental science but also has been utilized in an enormous number of applications in everyday life. However, the creation of three-dimensional (3D) architectures by a liquid droplet and an immiscible liquid interface has been less investigated than other applications. Here, we report interfacial energy-driven spontaneous self-formation of a 3D plasmonic optical structure at room temperature without an external force. Based on the densities and interfacial energies of two liquids, we simulated the spontaneous formation of a plasmonic optical structure when a water droplet containing metal ions meets an immiscible liquid polydimethylsiloxane (PDMS) interface. At the interface, the metal ions in the droplet are automatically reduced to form an interfacial plasmonic layer as the liquid PDMS cures. The self-formation of both an optical cavity and integrated plasmonic nanostructure significantly enhances the fluorescence by a magnitude of 1000. Our findings will have a huge impact on the development of various photonic and plasmonic materials as well as metamaterials and devices.

Integrated Microalgae Analysis Photobioreactor for Rapid Strain Selection.

Algal photosynthesis is considered to be a sustainable, alternative, and renewable solution to generating green energy. For high-productivity algaculture in diverse local environments, a high-throughput screening method is needed to select algal strains from naturally available or genetically engineered strains. Herein, we present an integrated plasmonic photobioreactor for rapid, high-throughput screening of microalgae. Our 3D nanoplasmonic optical cavity-based photobioreactor permits the amplification of a selective wavelength favorable to photosynthesis in the cavity. The hemispheric plasmonic cavity allows intercellular interaction to be promoted in the optically favorable milieu and also permits effective visual examination of algal growth. Using Chlamydomonas reinhardtii, we demonstrated a 2-fold enhanced growth rate and a 1.5-fold lipid production rate with no distinctive lag phase. By facilitating growth and biomass conversion rates, the integrated microalgae analysis platform will serve as rapid microalgae screening platforms for biofuel applications.

Rapid Optical Cavity PCR.

Recent outbreaks of deadly infectious diseases, such as Ebola and Middle East respiratory syndrome coronavirus, have motivated the research for accurate, rapid diagnostics that can be administered at the point of care. Nucleic acid biomarkers for these diseases can be amplified and quantified via polymerase chain reaction (PCR). In order to solve the problems of conventional PCR--speed, uniform heating and cooling, and massive metal heating blocks--an innovative optofluidic cavity PCR method using light-emitting diodes (LEDs) is accomplished. Using this device, 30 thermal cycles between 94 °C and 68 °C can be accomplished in 4 min for 1.3 µL (10 min for 10 µL). Simulation results show that temperature differences across the 750 µm thick cavity are less than 2 °C and 0.2 °C, respectively, at 94 °C and 68 °C. Nucleic acid concentrations as low as 10(-8) ng µL(-1) (2 DNA copies per µL) can be amplified with 40 PCR thermal cycles. This simple, ultrafast, precise, robust, and low-cost optofluidic cavity PCR is favorable for advanced molecular diagnostics and precision medicine. It is especially important for the development of lightweight, point-of-care devices for use in both developing and developed countries.

Human iPSC-based cardiac microphysiological system for drug screening applications.

Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non-human animal models cannot adequately represent human biology. Thus, there is an urgent need for high-content in vitro systems that can better predict drug-induced toxicity. Systems that predict cardiotoxicity are of uppermost significance, as approximately one third of safety-based pharmaceutical withdrawals are due to cardiotoxicty. Here, we present a cardiac microphysiological system (MPS) with the attributes required for an ideal in vitro system to predict cardiotoxicity: i) cells with a human genetic background; ii) physiologically relevant tissue structure (e.g. aligned cells); iii) computationally predictable perfusion mimicking human vasculature; and, iv) multiple modes of analysis (e.g. biological, electrophysiological, and physiological). Our MPS is able to keep human induced pluripotent stem cell derived cardiac tissue viable and functional over multiple weeks. Pharmacological studies using the cardiac MPS show half maximal inhibitory/effective concentration values (IC50/EC50) that are more consistent with the data on tissue scale references compared to cellular scale studies. We anticipate the widespread adoption of MPSs for drug screening and disease modeling.

Graphene nanopore with a self-integrated optical antenna.

We report graphene nanopores with integrated optical antennae. We demonstrate that a nanometer-sized heated spot created by photon-to-heat conversion of a gold nanorod resting on a graphene membrane forms a nanoscale pore with a self-integrated optical antenna in a single step. The distinct plasmonic traits of metal nanoparticles, which have a unique capability to concentrate light into nanoscale regions, yield the significant advantage of parallel nanopore fabrication compared to the conventional sequential process using an electron beam. Tunability of both the nanopore dimensions and the optical characteristics of plasmonic nanoantennae are further achieved. Finally, the key optical function of our self-integrated optical antenna on the vicinity of graphene nanopore is manifested by multifold fluorescent signal enhancement during DNA translocation.

Hemolysis-free blood plasma separation.

Hemolysis, involving the rupture of red blood cells (RBCs) and release of their contents into blood plasma, is a major issue of concern in clinical fields. Hemolysis in vitro can occur as a result of errors in clinical trials; in vivo, hemolysis can be caused by a variety of medical conditions. Blood plasma separation is often the first step in blood-based clinical diagnostic procedures. However, inhibitors released from RBCs due to hemolysis during plasma separation can lead to problems in diagnostic tests such as low sensitivity, selectivity and inaccurate results. In particular, a general lack of simple and reliable blood plasma separation methods has been a major obstacle for microfluidic-based point-of-care (POC) diagnostic devices. Here we present a hemolysis-free microfluidic blood plasma separation platform. A membrane filter was positioned on top of a vertical up-flow channel (filter-in-top configuration) to reduce clogging of RBCs by gravity-assisted cells sedimentation. With this device, separated plasma volume was increased approximately 4-fold (2.4 µL plasma after 20 min with 38% hematocrit human whole blood), and hemoglobin concentration in separated plasma was decreased approximately 90% due to the prevention of RBCs hemolysis, when compared to conventional filter-in-bottom configuration blood plasma separation platforms. On-chip plasma contained ~90% of protein and ~100% of nucleic acids found in off-chip centrifuged plasma, confirming comparable target molecule recovery efficiency. This simple and robust on-chip blood plasma separation device integrates with downstream detection modules to ultimately create sample-to-answer microfluidic POC diagnostics devices.

A micropatterning approach for imaging dynamic Cx43 trafficking to cell-cell borders.

The precise expression and timely delivery of connexin 43 (Cx43) proteins to form gap junctions are essential for electrical coupling of cardiomyocytes. Growing evidence supports a cytoskeletal-based trafficking paradigm for Cx43 delivery directly to adherens junctions at the intercalated disc. A limitation of Cx43 localization assays in cultured cells, in which cell-cell contacts are essential, is the inability to control for cell geometry or reproducibly generate contact points. Here we present a micropatterned cell pairing system well suited for live microscopy to examine how the microtubule and actin cytoskeleton confer specificity to Cx43 trafficking to precisely defined cell-cell junctions. This system can be adapted for other cell types and used to study dynamic intracellular movements of other proteins important for cell-cell communication.

Interfacial liquid-state surface-enhanced Raman spectroscopy.

Oriented assemblies of functional nanoparticles, with the aid of external physical and chemical driving forces, have been prepared on two-dimensional solid substrates. It is challengeable, however, to achieve three-dimensional assembly directly in solution, owing to thermal fluctuations and free diffusion. Here we describe the self-orientation of gold nanorods at an immiscible liquid interface (that is, oleic acid-water) and exploit this novel phenomenon to create a substrate-free interfacial liquid-state surface-enhanced Raman spectroscopy. Dark-field imaging and Raman scattering results reveal that gold nanorods spontaneously adopt a vertical orientation at an oleic acid-water interface in a stable trapping mode, which is in good agreement with simulation results. The spontaneous vertical alignment of gold nanorods at the interface allows one to accomplish significant additional amplification of the Raman signal, which is up to three to four orders of magnitude higher than that from a solution of randomly oriented gold nanorods.