Accurate virus identification with interpretable Raman signatures by machine learning.

Rapid identification of newly emerging or circulating viruses is an important first step toward managing the public health response to potential outbreaks. A portable virus capture device, coupled with label-free Raman spectroscopy, holds the promise of fast detection by rapidly obtaining the Raman signature of a virus followed by a machine learning (ML) approach applied to recognize the virus based on its Raman spectrum, which is used as a fingerprint. We present such an ML approach for analyzing Raman spectra of human and avian viruses. A convolutional neural network (CNN) classifier specifically designed for spectral data achieves very high accuracy for a variety of virus type or subtype identification tasks. In particular, it achieves 99% accuracy for classifying influenza virus type A versus type B, 96% accuracy for classifying four subtypes of influenza A, 95% accuracy for differentiating enveloped and nonenveloped viruses, and 99% accuracy for differentiating avian coronavirus (infectious bronchitis virus [IBV]) from other avian viruses. Furthermore, interpretation of neural net responses in the trained CNN model using a full-gradient algorithm highlights Raman spectral ranges that are most important to virus identification. By correlating ML-selected salient Raman ranges with the signature ranges of known biomolecules and chemical functional groups­for example, amide, amino acid, and carboxylic acid­we verify that our ML model effectively recognizes the Raman signatures of proteins, lipids, and other vital functional groups present in different viruses and uses a weighted combination of these signatures to identify viruses.

Spontaneous chemical functionalization via coordination of Au single atoms on monolayer MoS2.

Surface functionalization of metallic and semiconducting 2D transition metal dichalcogenides (TMDs) have mostly relied on physi- and chemi-sorption at defect sites, which can diminish the potential applications of the decorated 2D materials, as structural defects can have substantial drawbacks on the electronic and optoelectronic characteristics. Here, we demonstrate a spontaneous defect-free functionalization method consisting of attaching Au single atoms to monolayers of semiconducting MoS2(1H) via S-Au-Cl coordination complexes. This strategy offers an effective and controllable approach for tuning the Fermi level and excitation spectra of MoS2 via p-type doping and enhancing the thermal boundary conductance of monolayer MoS2, thus promoting heat dissipation. The coordination-based method offers an effective and damage-free route of functionalizing TMDs and can be applied to other metals and used in single-atom catalysis, quantum information devices, optoelectronics, and enhanced sensing.

Selective Synthesis of Bi2Te3/WS2 Heterostructures with Strong Interlayer Coupling.

The vertical integration of atomically thin-layered materials to create van der Waals heterostructures (vdWHs) has been proposed as a method to design nanostructures with emergent properties. In this work, epitaxial Bi2Te3/WS2 vdWHs are synthesized via a two-step vapor deposition process. It is calculated that the vdWH has an indirect band gap with a valence band edge that bridges the vdW gap, resulting in a quenched photoluminescence (PL) from the WS2 monolayer, reduced intensity of its resonance Raman vibrational peaks, improved vertical charge transport, and a decrease in the intensity of second harmonic generation (SHG). Furthermore, it is observed that induced defects strongly influence the nucleation and growth of vdWHs. By creating point defects in WS2 monolayers, it is shown that the growth of Bi2Te3 platelets can be patterned. This work offers important insights into the synthesis, defect engineering, and moiré engineering of an emerging class of two-dimensional (2D) heterostructures.

Rapid Size-Based Isolation of Extracellular Vesicles by Three-Dimensional Carbon Nanotube Arrays.

Recent discoveries reveal that extracellular vesicles (EVs) play an important role in transmitting signals. Although this emerging transcellular pathway enables a better understanding of neural communication, the lack of techniques for effectively isolating EVs impedes their studies. Herein, we report an emergent high-throughput platform consisting of three-dimensional carbon nanotube arrays that rapidly capture different EVs based on their sizes, without any labels. More importantly, this label-free capture maintains the integrity of the EVs when they are excreted from a host cell, thus allowing comprehensive downstream analyses using conventional approaches. To study neural communication, we developed a stamping technique to construct a gradient of nanotube herringbone arrays and integrated them into a microdevice that allowed us processing of a wide range of sample volumes, microliters to milliliters, in several minutes through a syringe via manual hand pushing and without any sample preparation. This microdevice successfully captured and separated EVs excreted from glial cells into subgroups according to their sizes. During capture, this technology preserved the structural integrity and originality of the EVs that enabled us to monitor and follow internalization of EVs of different sizes by neurons and cells. As a proof of concept, our results showed that smaller EVs (∼80 nm in diameter) have a higher uptake efficiency compared to larger EVs (∼300 nm in diameter). In addition, after being internalized, small EVs could enter endoplasmic reticulum and Golgi but not the largest ones. Our platform significantly shortens sample preparation, allows the profiling of the different EVs based on their size, and facilitates the understanding of extracellular communication. Thus, it leads to early diagnostics and the development of novel therapeutics for neurological diseases.

A rapid and label-free platform for virus capture and identification from clinical samples.

Emerging and reemerging viruses are responsible for a number of recent epidemic outbreaks. A crucial step in predicting and controlling outbreaks is the timely and accurate characterization of emerging virus strains. We present a portable microfluidic platform containing carbon nanotube arrays with differential filtration porosity for the rapid enrichment and optical identification of viruses. Different emerging strains (or unknown viruses) can be enriched and identified in real time through a multivirus capture component in conjunction with surface-enhanced Raman spectroscopy. More importantly, after viral capture and detection on a chip, viruses remain viable and get purified in a microdevice that permits subsequent in-depth characterizations by various conventional methods. We validated this platform using different subtypes of avian influenza A viruses and human samples with respiratory infections. This technology successfully enriched rhinovirus, influenza virus, and parainfluenza viruses, and maintained the stoichiometric viral proportions when the samples contained more than one type of virus, thus emulating coinfection. Viral capture and detection took only a few minutes with a 70-fold enrichment enhancement; detection could be achieved with as little as 102 EID50/mL (50% egg infective dose per microliter), with a virus specificity of 90%. After enrichment using the device, we demonstrated by sequencing that the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for influenza virus. This enrichment method coupled to Raman virus identification constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real time.

Controlling Nitrogen Doping in Graphene with Atomic Precision: Synthesis and Characterization.

Graphene provides a unique platform for the detailed study of its dopants at the atomic level. Previously, doped materials including Si, and 0D-1D carbon nanomaterials presented difficulties in the characterization of their dopants due to gradients in their dopant concentration and agglomeration of the material itself. Graphene's two-dimensional nature allows for the detailed characterization of these dopants via spectroscopic and atomic resolution imaging techniques. Nitrogen doping of graphene has been well studied, providing insights into the dopant bonding structure, dopant-dopant interaction, and spatial segregation within a single crystal. Different configurations of nitrogen within the carbon lattice have different electronic and chemical properties, and by controlling these dopants it is possible to either n- or p-type dope graphene, grant half-metallicity, and alter nitrogen doped graphene's (NG) catalytic and sensing properties. Thus, an understanding and the ability to control different types of nitrogen doping configurations allows for the fine tuning of NG's properties. Here we review the synthesis, characterization, and properties of nitrogen dopants in NG beyond atomic dopant concentration.

A Portable Device Integrated with Aligned Carbon Nanotubes for Sensitive Virus Capture and Detection.

Point-of-care virus diagnosis is highly desirable in worldwide infectious disease control. Here we report a hand-held device for capturing viruses by applying physical size based exclusion inside a point-of-care device integrated with vertically aligned carbon nanotube (VACNT) nanostructures to achieve label-free and high throughput virus capture. The microfluidic device is constructed from a VACNT channel wall synthesized bottom-up via chemical vapor deposition (CVD). The VACNT has ~117 nm average gap size and ~97& porosity. By bonding with a polydimethylsiloxane (PDMS) cover sealing the top, the aqueous sample containing virus particles filter through the VACNT channel wall under negative pressure applied at the outlet end. We have demonstrated that the device is capable of filtering 50 µL of PBS containing ~6.3 × 104 counts of lentivirus particles in 10 minutes with 97& of capture efficiency, quantified by the cell infectious titration technique.

A carbon nanotube integrated microfluidic device for blood plasma extraction.

Blood is a complex fluid consisting of cells and plasma. Plasma contains key biomarkers essential for disease diagnosis and therapeutic monitoring. Thus, by separating plasma from the blood, it is possible to analyze these biomarkers. Conventional methods for plasma extraction involve bulky equipment, and miniaturization constitutes a key step to develop portable devices for plasma extraction. Here, we integrated nanomaterial synthesis with microfabrication, and built a microfluidic device. In particular, we designed a double-spiral channel able to perform cross-flow filtration. This channel was constructed by growing aligned carbon nanotubes (CNTs) with average inter-tubular distances of ~80 nm, which resulted in porosity values of ~93%. During blood extraction, these aligned CNTs allow smaller molecules (e.g., proteins) to pass through the channel wall, while larger molecules (e.g., cells) get blocked. Our results show that our device effectively separates plasma from blood, by trapping blood cells. We successfully recovered albumin -the most abundant protein inside plasma- with an efficiency of ~80%. This work constitutes the first report on integrating biocompatible nitrogen-doped CNT (CNxCNT) arrays to extract plasma from human blood, thus widening the bio-applications of CNTs.

Light-Emitting Transition Metal Dichalcogenide Monolayers under Cellular Digestion.

2D materials cover a wide spectrum of electronic properties. Their applications are extended from electronic, optical, and chemical to biological. In terms of biomedical uses of 2D materials, the interactions between living cells and 2D materials are of paramount importance. However, biointerfacial studies are still in their infancy. This work studies how living organisms interact with transition metal dichalcogenide monolayers. For the first time, cellular digestion of tungsten disulfide (WS2 ) monolayers is observed. After digestion, cells intake WS2 and become fluorescent. In addition, these light-emitting cells are not only viable, but also able to pass fluorescent signals to their progeny cells after cell division. By combining synthesis of 2D materials and a cell culturing technique, a procedure for monitoring the interactions between WS2 monolayers and cells is developed. These observations open up new avenues for developing novel cellular labeling and imaging approaches, thus triggering further studies on interactions between 2D materials and living organisms.

A Nanostructured Microfluidic Immunoassay Platform for Highly Sensitive Infectious Pathogen Detection.

Rapid and simultaneous detection of multiple potential pathogens by portable devices can facilitate early diagnosis of infectious diseases, and allow for rapid and effective implementation of disease prevention and treatment measures. The development of a ZnO nanorod integrated microdevice as a multiplex immunofluorescence platform for highly sensitive and selective detection of avian influenza virus (AIV) is described. The 3D morphology and unique optical property of the ZnO nanorods boost the detection limit of the H5N2 AIV to as low as 3.6 × 103 EID50 mL-1 (EID50 : 50% embryo infectious dose), which is ≈22 times more sensitive than conventional enzyme-linked immunosorbent assay. The entire virus capture and detection process could be completed within 1.5 h with excellent selectivity. Moreover, this microfluidic biosensor is capable of detecting multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 AIV can be released by simply dissolving ZnO nanorods under slightly acidic environment for subsequent off-chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low-cost and convenient biomarker detection.

Avian and human influenza virus compatible sialic acid receptors in little brown bats.

Influenza A viruses (IAVs) continue to threaten animal and human health globally. Bats are asymptomatic reservoirs for many zoonotic viruses. Recent reports of two novel IAVs in fruit bats and serological evidence of avian influenza virus (AIV) H9 infection in frugivorous bats raise questions about the role of bats in IAV epidemiology. IAVs bind to sialic acid (SA) receptors on host cells, and it is widely believed that hosts expressing both SA α2,3-Gal and SA α2,6-Gal receptors could facilitate genetic reassortment of avian and human IAVs. We found abundant co-expression of both avian (SA α2,3-Gal) and human (SA α2,6-Gal) type SA receptors in little brown bats (LBBs) that were compatible with avian and human IAV binding. This first ever study of IAV receptors in a bat species suggest that LBBs, a widely-distributed bat species in North America, could potentially be co-infected with avian and human IAVs, facilitating the emergence of zoonotic strains.

Evaluating a novel dimensional reduction approach for mechanical fractionation of cells using a tandem flexible micro spring array (tFMSA).

We present a novel methodology to establish experimental models for the rational design of cell fractionation based on physical properties of cells. Label-free microfluidic separation of cells based on size is a widely employed technique. However, close observation reveals that cell capture results cannot be explained by cell sizes alone. This is particularly apparent with viable cell fractionation, where cells retain their native deformability. We have developed a principal size cutoff (PSC) model based on the analysis of size distribution and size-based filtration efficiency for cell populations. The goal of this analysis is to use an unbiased approach to achieve dimensional reduction of deformability and other mechanical properties that affect cell capture. The PSC model provides a single calibrated principal size component that may be compared directly to device gap width, which is the critical dimension for cell filtration. The PSC model was evaluated experimentally using a tandem flexible micro spring array (tFMSA) device made of parylene filtration elements applied within micro-molded polydimethylsiloxane (PDMS) chambers. In the tFMSA device, a mixture of cells is sequentially passed through individual filters with decreasing gap widths to allow size-based selection. We applied this method to demonstrate viable separation of subgroups of cells with different mechanical properties from complex mixtures, including fractionation according to cancer cell type, cell cycle stage, cell viability status, and leukocyte nuclear phenotype. The PSC methodology and tFMSA device can advance a better understanding of complex factors affecting mechanical cell fractionation and provide a miniature platform for obtaining rationally designed cell fractions for biomedical applications.

Tunable and label-free virus enrichment for ultrasensitive virus detection using carbon nanotube arrays.

Viral infectious diseases can erupt unpredictably, spread rapidly, and ravage mass populations. Although established methods, such as polymerase chain reaction, virus isolation, and next-generation sequencing have been used to detect viruses, field samples with low virus count pose major challenges in virus surveillance and discovery. We report a unique carbon nanotube size-tunable enrichment microdevice (CNT-STEM) that efficiently enriches and concentrates viruses collected from field samples. The channel sidewall in the microdevice was made by growing arrays of vertically aligned nitrogen-doped multiwalled CNTs, where the intertubular distance between CNTs could be engineered in the range of 17 to 325 nm to accurately match the size of different viruses. The CNT-STEM significantly improves detection limits and virus isolation rates by at least 100 times. Using this device, we successfully identified an emerging avian influenza virus strain [A/duck/PA/02099/2012(H11N9)] and a novel virus strain (IBDV/turkey/PA/00924/14). Our unique method demonstrates the early detection of emerging viruses and the discovery of new viruses directly from field samples, thus creating a universal platform for effectively remediating viral infectious diseases.

Comparison of four molecular assays for the detection of Tembusu virus.

Tembusu virus (TMUV) belongs to the genus Flavivirus that may cause severe egg drop in ducks. In order to evaluate the most efficient TMUV detection method, the performances of a conventional RT-PCR (C-RT-PCR), a semi-nested PCR (SN-RT-PCR), a reverse-transcriptase real-time quantitative PCR (Q-RT-PCR), and a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) targeting the TMUV virus-specific NS5 gene were examined. In order to compare the sensitivity of these four techniques, two templates were used: (1) plasmid DNA that contained a partial region of the NS5 gene and (2) genomic RNA from TMUV-positive cell culture supernatants. The sensitivities using plasmid DNA detection by C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 2 × 10(4) copies/µL, 20 copies/µL, 2 copies/µL, and 20 copies/µL, respectively. The sensitivities using genomic RNA for the C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 100 pg/tube, 100, 10, and 100 fg/tube, respectively. All evaluated assays were specific for TMUV detection. The TMUV-specific RNA was detected in cloacal swabs from experimentally infected ducks using these four methods with different rates (52-92%), but not in the control (non-inoculated) samples. The sensitivities of RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs collected from suspected TMUV infected ducks within 2 weeks of severe egg-drop were 38/69 (55.1%), 52/69 (75.4%), 57/69 (82.6%), and 55/69 (79.7%), respectively. In conclusion, both RT-LAMP and Q-RT-PCR can provide a rapid diagnosis of TMUV infection, but RT-LAMP is more useful in TMUV field situations or poorly equipped laboratories.

Genomic characterization of a turkey reovirus field strain by Next-Generation Sequencing.

The genome of a turkey arthritis reovirus (TARV) field strain (Reo/PA/Turkey/22342/13), isolated from a turkey flock in Pennsylvania (PA) in 2013, has been sequenced using Next-Generation Sequencing (NGS) on the Illumina MiSeq platform. The genome of the PA TARV field strain was 23,496bp in length with 10 dsRNA segments encoding 12 viral proteins. The lengths of the genomic segments ranged from 1192bp (S4) to 3959bp (L1). The 5' and 3' conserved terminal sequences of the PA TARV field strain were similar to the two Minnesota (MN) TARVs (MN9 and MN10) published recently and avian orthoreovirus (ARV) reference strains. Phylogenetic analysis of the nucleotide sequences of all 10 genome segments revealed that there was a low to significant nucleotide sequence divergence between the PA TARV field strain and reference TARV and ARV strains. Analysis of the PA TARV sequence indicates that this PA TARV field strain is a unique strain and is different from the TARV MN9 or MN10 in M2 segment genes and ARV S1133 vaccine strain.

Point-of-care microdevices for blood plasma analysis in viral infectious diseases.

Each year, outbreaks of viral infections cause illness, disability, death, and economic loss. As learned from past incidents, the detrimental impact grows exponentially without effective quarantine. Therefore, rapid on-site detection and analysis are highly desired. In addition, for high-risk areas of viral contamination, close monitoring should be provided during the potential disease incubation period. As the epidemic progresses, a response protocol needs tobe rapidly implemented and the virus evolution fully tracked. For these scenarios, point-of-care microdevices can provide sensitive, accurate, rapid and low-cost analysis for a large population, especially in handling complex patient samples, such as blood, urine and saliva. Blood plasma can be considered as a mine of information containing sources and clues of biomarkers, including nucleic acids, immunoglobulin and other proteins, as well as pathogens for clinical diagnosis. However, blood plasma is also the most complicated body fluid. For targeted plasma biomarker detection or untargeted plasma biomarker discovery, the challenges can be as difficult as identifying a needle in a haystack. A useful platform must not only pursue single performance characteristics, but also excel at multiple performance parameters, such as speed, accuracy, sensitivity, selectivity, cost, portability, reliability, and user friendliness. Throughout the decades, tremendous progress has been made in point-of-care microdevices for viral infectious diseases. In this paper, we review fully integrated lab-on-chip systems for blood analysis of viral infectious disease.

Flexible micro spring array device for high-throughput enrichment of viable circulating tumor cells.

BACKGROUND: The dissemination of circulating tumor cells (CTCs) that cause metastases in distant organs accounts for the majority of cancer-related deaths. CTCs have been established as a cancer biomarker of known prognostic value. The enrichment of viable CTCs for ex vivo analysis could further improve cancer diagnosis and guide treatment selection. We designed a new flexible micro spring array (FMSA) device for the enrichment of viable CTCs independent of antigen expression. METHODS: Unlike previous microfiltration devices, flexible structures at the micro scale minimize cell damage to preserve viability, while maximizing throughput to allow rapid enrichment directly from whole blood with no need for sample preprocessing. Device performance with respect to capture efficiency, enrichment against leukocytes, viability, and proliferability was characterized. CTCs and CTC microclusters were enriched from clinical samples obtained from breast, lung, and colorectal cancer patients. RESULTS: The FMSA device enriched tumor cells with 90% capture efficiency, higher than 10(4) enrichment, and better than 80% viability from 7.5-mL whole blood samples in <10 min on a 0.5-cm(2) device. The FMSA detected at least 1 CTC in 16 out of 21 clinical samples (approximately 76%) compared to 4 out of 18 (approximately 22%) detected with the commercial CellSearch® system. There was no incidence of clogging in over 100 tested fresh whole blood samples. CONCLUSIONS: The FMSA device provides a versatile platform capable of viable enrichment and analysis of CTCs from clinically relevant volumes of whole blood.