Mayaro virus detection by integrating sample preparation with isothermal amplification in portable devices.

Mayaro virus (MAYV) is an emerging mosquito-borne alphavirus that causes clinical symptoms similar to those caused by Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV). To differentiate MAYV from these viruses diagnostically, we have developed a portable device that integrates sample preparation with real-time, reverse-transcription, loop-mediated isothermal amplification (rRT-LAMP). First, we designed a rRT-LAMP assay targeting MAYV's non-structural protein (NS1) gene and determined the limit of detection of at least 10 viral genome equivalents per reaction. The assay was specific for MAYV, without cross-reactions with CHIKV, DENV, or ZIKV. The rRT-LAMP assay was integrated with a sample preparation device (SPD) wherein virus lysis and RNA enrichment/purification were carried out on the spot, without requiring pipetting, while subsequent real-time amplification device (RAD) enables virus detection at the point of care (POC). The functions of our platform were demonstrated using purified MAYV RNA or blood samples containing viable viruses. We have used the devices for detection of MAYV in as short as 13 min, with limit of detection to as low as 10 GEs/reaction.

Viable SARS-CoV-2 Delta variant detected in aerosols in a residential setting with a self-isolating college student with COVID-19.

The B.1.617.2 (Delta) variant of SARS-CoV-2 emerged in India in October of 2020 and spread widely to over 145 countries, comprising over 99% of genome sequence-confirmed virus in COVID-19 cases of the United States (US) by September 2021. The rise in COVID-19 cases due to the Delta variant coincided with a return to in-person school attendance, straining COVID-19 mitigation plans implemented by educational institutions. Some plans required sick students to self-isolate off-campus, resulting in an unintended consequence: exposure of co-inhabitants of dwellings used by the sick person during isolation. We assessed air and surface samples collected from the bedroom of a self-isolating university student with mild COVID-19 for the presence of SARS-CoV-2. That virus' RNA was detected by real-time reverse-transcription quantitative polymerase chain reaction (rRT-qPCR) in air samples from both an isolation bedroom and a distal, non-isolation room of the same dwelling. SARS-CoV-2 was detected and viable virus was isolated in cell cultures from aerosol samples as well as from the surface of a mobile phone. Genomic sequencing revealed that the virus was a Delta variant SARS-CoV-2 strain. Taken together, the results of this work confirm the presence of viable SARS-CoV-2 within a residential living space of a person with COVID-19 and show potential for transportation of virus-laden aerosols beyond a designated isolation suite to other areas of a single-family home.

SARS-CoV-2 in residential rooms of two self-isolating persons with COVID-19.

Individuals with COVID-19 are advised to self-isolate at their residences unless they require hospitalization. Persons sharing a dwelling with someone who has COVID-19 have a substantial risk of being exposed to the virus. However, environmental monitoring for the detection of virus in such settings is limited. We present a pilot study on environmental sampling for SARS-CoV-2 virions in the residential rooms of two volunteers with COVID-19 who self-quarantined. Apart from standard surface swab sampling, based on availability, four air samplers positioned 0.3-2.2 m from the volunteers were used: a VIable Virus Aerosol Sampler (VIVAS), an inline air sampler that traps particles on polytetrafluoroethylene (PTFE) filters, a NIOSH 2-stage cyclone sampler (BC-251), and a Sioutas personal cascade impactor sampler (PCIS). The latter two selectively collect particles of specific size ranges. SARS-CoV-2 RNA was detected by real-time Reverse-Transcription quantitative Polymerase Chain Reaction (rRT-qPCR) analyses of particles in one air sample from the room of volunteer A and in various air and surface samples from that of volunteer B. The one positive sample collected by the NIOSH sampler from volunteer A's room had a quantitation cycle (Cq) of 38.21 for the N-gene, indicating a low amount of airborne virus [5.69E-02 SARS-CoV-2 genome equivalents (GE)/cm3 of air]. In contrast, air samples and surface samples collected off the mobile phone in volunteer B's room yielded Cq values ranging from 14.58 to 24.73 and 21.01 to 24.74, respectively, on the first day of sampling, indicating that this volunteer was actively shedding relatively high amounts of SARS-CoV-2 at that time. The SARS-CoV-2 GE/cm3 of air for the air samples collected by the PCIS was in the range 6.84E+04 to 3.04E+05 using the LED-N primer system, the highest being from the stage 4 filter, and similarly, ranged from 2.54E+03 to 1.68E+05 GE/cm3 in air collected by the NIOSH sampler. Attempts to isolate the virus in cell culture from the samples from volunteer B's room with the aforementioned Cq values were unsuccessful due to out-competition by a co-infecting Human adenovirus B3 (HAdVB3) that killed the Vero E6 cell cultures within 4 days of their inoculation, although Cq values of 34.56-37.32 were measured upon rRT-qPCR analyses of vRNA purified from the cell culture medium. The size distribution of SARS-CoV-2-laden aerosol particles collected from the air of volunteer B's room was >0.25 µm and >0.1 µm as recorded by the PCIS and the NIOSH sampler, respectively, suggesting a risk of aerosol transmission since these particles can remain suspended in air for an extended time and travel over long distances. The detection of virus in surface samples also underscores the potential for fomite transmission of SARS-CoV-2 in indoor settings.

Phase II Study of 5-Fluorouracil, Oxaliplatin plus Dasatinib (FOLFOX-D) in First-Line Metastatic Pancreatic Adenocarcinoma.

LESSONS LEARNED: Preclinical studies have demonstrated that Src inhibition through dasatinib synergistically enhances the antitumor effects of oxaliplatin. In this phase II, single-arm study, FOLFOX with dasatinib in previously untreated patients with mPC only showed only modest clinical activity, with a progressive-free survival of 4 months and overall survival of 10.6 months. Continued investigation is ongoing to better understand the role of Src inhibition with concurrent 5-fluorouracil and oxaliplatin in a subset of exceptional responders. BACKGROUND: Src tyrosine kinase activity is overexpressed in many human cancers, including metastatic pancreatic cancer (mPC). Dasatinib is a potent inhibitor of Src family of tyrosine kinases. This study was designed to investigate whether dasatinib can synergistically enhance antitumor effects of FOLFOX regimen (FOLFOX-D). METHODS: In this single-arm, phase II study, previously untreated patients received dasatinib 150 mg oral daily on days 1-14, oxaliplatin 85 mg/m2 intravenous (IV) on day 1 every 14 days, leucovorin (LV) 400 mg/m2 IV on day 1 every 14 days, 5-fluorouracil (5-FU) bolus 400 mg/m2 on day 1 every 14 days, and 5-FU continuous infusion 2,400 mg/m2 on day 1 every 14 days. Primary endpoint was progression-free survival (PFS) with preplanned comparison to historical controls. RESULTS: Forty-four patients enrolled with an estimated median PFS of 4.0 (95% confidence interval [CI], 2.3-8.5) months and overall survival (OS) of 10.6 (95% CI, 6.9-12.7) months. Overall response rate (ORR) was 22.7% (n = 10): one patient (2.3%) with complete response (CR) and nine patients (20.5%) with partial response (PR). Fifteen patients (34.1%) had stable disease (SD). Nausea was the most common adverse event (AE) seen in 35 patients (79.5%). CONCLUSION: The addition of dasatinib did not appear to add incremental clinical benefit to FOLFOX in untreated patients with mPC.

Microfluidics-based device for the measurement of blood viscosity and its modeling based on shear rate, temperature, and heparin concentration.

Blood viscosity measurements are crucial for the diagnosis and understanding of a range of hematological and cardiovascular diseases. Such measurements are heavily used in monitoring patients during and after surgeries, which necessitates the development of a highly accurate viscometer that uses a minimal amount of blood. In this work, we have designed and implemented a microfluidic device that was used to measure fluid viscosity with a high accuracy using less than 10 µl of blood. The device was further used to construct a blood viscosity model based on temperature, shear rate, and anti-coagulant concentration. The model has an R-squared value of 0.950. Finally, blood protein content was changed to simulate diseased conditions and blood viscosity was measured using the device and estimated using the model constructed in this work. Simulated diseased conditions were clearly detected when comparing estimated viscosity values using the model and the measured values using the device, proving the applicability of the setup in the detection of rheological anomalies and in disease diagnosis.

Integration of Lateral Filter Arrays with Immunoaffinity for Circulating-Tumor-Cell Isolation.

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size-based separation with immunoaffinity-based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two-dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.

An efficient virus aerosol sampler enabled by adiabatic expansion.

Protection of public health against pathogenic viruses transmitted through the airborne route requires effective sampling of airborne viruses for determination of their concentration and distribution. However, sampling viable airborne viruses is challenging as conventional bioaerosol sampling devices operate on inertia-based mechanisms that inherently have low sampling efficiency for virus aerosols in the ultrafine size range (< 100 nm). Herein, a Batch Adiabatic-expansion for Size Intensification by Condensation (BASIC) approach was developed for efficient sampling of virus aerosols. The BASIC utilizes adiabatic expansion in a supersaturated container to activate condensation of water vapor onto virus aerosol particles, thus amplifying the size of the particles by orders of magnitude. Using aerosolized MS2 bacteriophage, the BASIC's performance was evaluated and optimized both from the perspectives of physical size amplification as well as preservation of the viability of the MS2 bacteriophage. Experimental results show that one compression/expansion (C/E) cycle under a compression pressure of 103.5 kPa and water temperature of 25 °C was sufficient to increase the particle diameter from < 100 nm to > 1 µm; further increases in the number of C/E cycles neither increased particle number concentration nor diameter. An increase in compression pressure was associated with physical size amplification and a higher concentration of collected viable MS2. Water temperature of 40 °C was found to be the optimal for size amplification as well as viability preservation. No significant effect on particle size enlargement was observed by changing the dwell time after expansion. The results illustrate the BASIC's capability as a simple, quick and inexpensive tool for rapid sampling of viable airborne viruses.

Valve-Enabled Sample Preparation and RNA Amplification in a Coffee Mug for Zika Virus Detection.

The recent outbreaks of Zika virus (ZIKV) infection represent a public health challenge. Rapid, cost-effective, and reliable diagnostic tools for ZIKV detection at the point of care (POC) are highly desirable, especially for resource-limited nations. To address the need, we have developed an integrated device to achieve sample-to-answer ZIKV detection. The device features innovative ball-based valves enabling the storage and sequential delivery of reagents for virus lysis and a paper-based unit for RNA enrichment and purification. The paper unit is placed in a commercially available coffee mug that provides a constant temperature for reverse transcription loop-mediated isothermal amplification (RT-LAMP), followed by colorimetric detection by naked eye or a cellphone camera. Using the device, we demonstrated the reproducible detection of ZIKV in human urine and saliva samples.

Evaporation-Driven Bioassays in Suspended Droplets.

The microtiter plate has been an essential tool for diagnostics, high-throughput screening, and biological assays. We present an alternative platform to perform bioassays in a microplate format that exploits evaporation to drive assay reactions. Our method consists of droplets suspended on plastic pillars; reactions occur in these droplets instead of the wells. The pillars are fabricated by milling, and the rough surface created by this fabrication method pins the droplet to a constant contact line during the assay and also acts as a hydrophobic surface. Upon evaporation, natural convection arising from Marangoni currents mixes solutions in the droplet, which speeds up assay reactions, decreases assay times, and increases limits of detection. As a proof of concept we implemented two colorimetric assays to detect glucose and proteins in only 1.5 µL, without any external devices for mixing and with a digital microscope as a readout mechanism. Our platform is an ideal alternative to the microtiter plate, works with different volumes, is compatible with commercially available reagent dispensers and plate-readers, and could have broad applications in diagnostics and high-throughput screening.

Use of RNA amplification and electrophoresis for studying virus aerosol collection efficiency and their comparison with plaque assays.

The spread of virus-induced infectious diseases through airborne routes of transmission is a global concern for economic and medical reasons. To study virus transmission, it is essential to have an effective aerosol collector such as the growth tube collector (GTC) system that utilizes water-based condensation for collecting virus-containing aerosols. In this work, we characterized the GTC system using bacteriophage MS2 as a surrogate for a small RNA virus. We investigated using RNA extraction and reverse transcription- polymerase chain reaction (RT-PCR) to study the total virus collection efficiency of the GTC system. Plaque assays were also used to enumerate viable viruses collected by the GTC system compared to that by a commercially available apparatus, the SKC® Biosampler. The plaque assay counts were used to enumerate viable viruses whereas RT-PCR provides a total virus count, including those viruses inactivated during collection. The effects of relative humidity (RH) and other conditions on collection efficiency were also investigated. Our results suggest that the GTC has a collection efficiency for viable viruses between 0.24 and 1.8% and a total virus collection efficiency between 18.3 and 79.0%, which is 1-2 orders of magnitude higher than that of the SKC® Biosampler. Moreover, higher RH significantly increases both the viable and total collection efficiency of the GTC, while its effect on the collection efficiency of the SKC® Biosampler is not significant.

A universal tumor cell isolation method enabled by fibrin-coated microchannels.

We report a simple but effective strategy to capture tumor cells using fibrin-immobilized microchannels. It is a universal method since it shows an ability to capture both epithelial and mesenchymal tumor cells. The cell capture efficiency is up to 90%.

Optimization of a miniaturized fluid array device for cell-free protein synthesis.

Cell-free protein synthesis (CFPS), which entails synthesizing proteins outside of intact cells, is conducted in several formats with the continuous-exchange cell-free (CECF) format generally having the greatest protein expression yields. With this format, continuous chemical exchange occurs through a dialysis membrane separating a reaction solution from a feeding solution containing supplemental nutrient/energy molecules. Here, we describe the optimization of the miniaturized fluid array device (µFAD) by studying the effects of structural and experimental parameters responsible for the heightened chemical exchange across the dialysis membranes and enhanced protein expression capabilities of the high-throughput device. The interface area and number between the reaction and feeding solutions have a direct impact on protein expression, with a 1.6% enhancement in protein expression yield with each square millimeter increase in area and a 20% decrease with each additional interface. For nutrient/energy availability, an increasing solution volume ratio and height difference increase protein expression yield until the expression yield plateaus at a volume ratio of 20 to 1 (feeding to reaction solution) and a solution height difference of 2 mm. This yield can be further increased by 7% every 30 min with feeding solution replacement. Of the studied experimental factors (feeding solution stirring, device shaking, and temperature increase), feeding solution stirring has a significant effect on protein expression in this device. In the optimized system, green fluorescent protein (GFP), ß-glucuronidase (GUS), ß-galactosidase (LacZ), luciferase, and tissue plasminogen activator (tPA) expression increased 77.8-, 212-, 3.66-, 463-, and 5.43-fold, respectively, compared to the conventional batch format in a standard microplate. These results highlight the significance of structural/experimental conditions on the productive expression of proteins in the CECF format.

Mixing in microfluidic devices and enhancement methods.

Mixing in microfluidic devices presents a challenge due to laminar flows in microchannels, which result from low Reynolds numbers determined by the channel's hydraulic diameter, flow velocity, and solution's kinetic viscosity. To address this challenge, novel methods of mixing enhancement within microfluidic devices have been explored for a variety of applications. Passive mixing methods have been created, including those using ridges or slanted wells within the microchannels, as well as their variations with improved performance by varying geometry and patterns, by changing the properties of channel surfaces, and by optimization via simulations. In addition, active mixing methods including microstirrers, acoustic mixers, and flow pulsation have been investigated and integrated into microfluidic devices to enhance mixing in a more controllable manner. In general, passive mixers are easy to integrate, but difficult to control externally by users after fabrication. Active mixers usually take efforts to integrate within a device and they require external components (e.g. power sources) to operate. However, they can be controlled by users to a certain degree for tuned mixing. In this article, we provide a general overview of a number of passive and active mixers, discuss their advantages and disadvantages, and make suggestions on choosing a mixing method for a specific need as well as advocate possible integration of key elements of passive and active mixers to harness the advantages of both types.

The use of polyurethane as an elastomer in thermoplastic microfluidic devices and the study of its creep properties.

We report using polyurethane (PU) as an elastomer in microvalves integrated with thermoplastic microfluidic devices. Elastomer-based microvalves have been used in a number of applications and the elastomer often used is PDMS. Although it is a convenient material for prototyping, PDMS has been recognized to possess shortcomings such as solvent incompatibility and unfavorable manufacturability. We investigated the use of PU as an elastomer to address the challenges. A reliable method was developed to bond hybrid materials such as PU and cyclic olefin copolymer. The film thickness from 3.5 to 24.5 µm was studied to identify an appropriate thickness of PU films for desirable elasticity in microvalves. We integrated PU with thermally actuated, elastomer-based microvalves in thermoplastic devices. Valve actuations were demonstrated, and the relationship between the valve actuation time and heater power was studied. We compared PU with PDMS in terms of their microvalve performance. Valves with PDMS failed to function after two weeks since the thermal-sensitive solution evaporated through porous PDMS membrane, whereas the same valve with PU functioned properly after eight months. In addition, we evaluated the creep and creep recovery of PU, which is a common phenomenon of viscoelastic materials and is related to the long-term elastic property of PU after prolonged use.

Thermal lens microscopy as a detector in microdevices.

The small length scales that make microfluidics attractive are also the source of some very stringent constraints, especially with respect to the detection approach used. The low concentrations often analyzed in microfluidic devices require highly sensitive detection methods that are effective even in vanishingly small sample volumes. Over the years, many detection approaches have been developed for microfluidics. The majority of these methods rely upon optical phenomena, with the most common being fluorescence detection. Fluorescence detection is well suited to microfluidics because it is both flexible and sensitive; however, it does have shortcomings. Weak fluorescence of targets, autofluorescence of materials, and photobleaching are a few of the issues that have to be dealt with when working with fluorescence detection. Another option that eliminates all of these problems is thermal lens microscopy (TLM), a photothermal spectroscopy technique. TLM is a flexible, sensitive detection approach for nonfluorescent molecules that is capable of carrying out single-molecule detection to label-free in vivo quantification. Despite the potential benefits of TLM, it is still an underutilized detection approach. We hope this review will help broaden the use of TLM for microchip-based CE, as well as a host of other microfluidic applications.

Sample preparation and detection methods in point-of-care devices towards future at-home testing.

Timely and accurate diagnosis is critical for effective healthcare, yet nearly half the global population lacks access to basic diagnostics. Point-of-care (POC) testing offers partial solutions by enabling low-cost, rapid diagnosis at the patient's location. At-home POC devices have the potential to advance preventive care and early disease detection. Nevertheless, effective sample preparation and detection methods are essential for accurate results. This review surveys recent advances in sample preparation and detection methods at POC. The goal is to provide an in-depth understanding of how these technologies can enhance at-home POC devices. Lateral flow assays, nucleic acid tests, and virus detection methods are at the forefront of POC diagnostic technology, offering rapid and sensitive tools for identifying and measuring pathogens, biomarkers, and viral infections. By illuminating cutting-edge research on assay development for POC diagnostics, this review aims to accelerate progress towards widely available, user-friendly, at-home health monitoring tools that empower individuals in personalized healthcare in the future.

A Capillary-Force-Driven, Single-Cell Transfer Method for Studying Rare Cells.

The characterization of individual cells within heterogeneous populations (e.g., rare tumor cells in healthy blood cells) has a great impact on biomedical research. To investigate the properties of these specific cells, such as genetic biomarkers and/or phenotypic characteristics, methods are often developed for isolating rare cells among a large number of background cells before studying their genetic makeup and others. Prior to using real-world samples, these methods are often evaluated and validated by spiking cells of interest (e.g., tumor cells) into a sample matrix (e.g., healthy blood) as model samples. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. People often circumvent the problem by diluting a solution of high-concentration cells, but the concentration becomes inaccurate after series dilution due to the fact that a cell suspension solution can be inhomogeneous, especially when the cell concentration is very low. We report on an alternative method for low-cost, accurate, and reproducible low-concentration cell spiking without the use of external pumping systems. By inducing a capillary force from sudden pressure drops, a small portion of the cellular membrane was aspirated into the reservoir tip, allowing for non-destructive single-cell transfer. We investigated the surface membrane tensions induced by cellular aspiration and studied a range of tip/tumor cell diameter combinations, ensuring that our method does not affect cell viability. In addition, we performed single-cell capture and transfer control experiments using human acute lymphoblastic leukemia cells (CCRF-CEM) to develop calibrated data for the general production of low-concentration samples. Finally, we performed affinity-based tumor cell isolation using this method to generate accurate concentrations ranging from 1 to 15 cells/mL.

Investigating surface proteins and antibody combinations for detecting circulating tumor cells of various sarcomas.

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.

Air Change Rate and SARS-CoV-2 Exposure in Hospitals and Residences: A Meta-Analysis.

As SARS-CoV-2 swept across the globe, increased ventilation and implementation of air cleaning were emphasized by the US CDC and WHO as important strategies to reduce the risk of inhalation exposure to the virus. To assess whether higher ventilation and air cleaning rates lead to lower exposure risk to SARS-CoV-2, 1274 manuscripts published between April 2020 and September 2022 were screened using key words "airborne SARS-CoV-2 or "SARS-CoV-2 aerosol". Ninety-three studies involved air sampling at locations with known sources (hospitals and residences) were selected and associated data were compiled. Two metrics were used to assess exposure risk: SARS-CoV-2 concentration and SARS-CoV-2 detection rate in air samples. Locations were categorized by type (hospital or residence) and proximity to the sampling location housing the isolated/quarantined patient (primary or secondary). The results showed that hospital wards had lower airborne virus concentrations than residential isolation rooms. A negative correlation was found between airborne virus concentrations in primary-occupancy areas and air changes per hour (ACH). In hospital settings, sample positivity rates were significantly reduced in secondary-occupancy areas compared to primary-occupancy areas, but they were similar across sampling locations in residential settings. ACH and sample positivity rates were negatively correlated, though the effect was diminished when ACH values exceeded 8. While limitations associated with diverse sampling protocols exist, data considered by this meta-analysis support the notion that higher ACH may reduce exposure risks to the virus in ambient air.