Cytokines play a very important role in our immune system by acting as mediators to put up a coordinated defense against foreign elements in our body. Elevated levels of cytokines in the body can signal to an ongoing response of the immune system to some abnormality. Thus, the quantification of a panel of cytokines can provide valuable information regarding the diagnosis of specific diseases and state of overall health of an individual. Conventional Enzyme Linked Immunosorbent Assay (ELISA) is the gold-standard for quantification of cytokines, however the need for trained personnel and expensive equipment limits its application to centralized laboratories only. In this context, there is a lack of simple, low-cost and portable devices which can allow for quantification of panels of cytokines at point-of-care and/or resource limited settings.

Here, we report the development of a versatile, low-cost and portable bead-based centrifugal microfluidic platform allowing for multiplexed detection of cytokines with minimal hands-on time and an integrated colorimetric signal readout without the need for any external equipment. As a model, multiplexed colorimetric quantification of three target cytokines i.e., Tumor necrosis factor alpha (TNF-α), Interferon gamma (IFN-γ) and Interleukin-2 (IL-2) was achieved in less than 30 min with limits of detection in ng/mL range. The developed platform was further evaluated using spiked-in plasma samples to test for matrix interference. The ease of use, low-cost and portability of the developed platform highlight its potential to serve as a sample-to-answer solution for detection of cytokine panels in resource limited settings.

Microfluidics has emerged rapidly over the past 20 years and has been investigated for a variety of applications from life sciences to environmental monitoring. Although continuous-flow microfluidics is ubiquitous, segmented-flow or droplet microfluidics offers several attractive features. Droplets can be independently manipulated and analyzed with very high throughput. Typically, microfluidics is carried out within planar networks of microchannels, namely, microfluidic chips. We propose that fibers offer an interesting alternative format with key advantages for enhanced optical coupling. Herein, we demonstrate the generation of monodisperse droplets within a uniaxial optofluidic Lab-in-a-Fiber scheme. We combine droplet microfluidics with laser-induced fluorescence (LIF) detection achieved through the development of an optical side-coupling fiber, which we term a periscope fiber. This arrangement provides stable and compact alignment. Laser-induced fluorescence offers high sensitivity and low detection limits with a rapid response time making it an attractive detection method for in situ real-time measurements. We use the well-established fluorophore, fluorescein, to characterize the Lab-in-a-Fiber device and determine the generation of similar to 0.9 nL droplets. We present characterization data of a range of fluorescein concentrations, establishing a limit of detection (LOD) of 10 nM fluorescein. Finally, we show that the device operates within a realistic and relevant fluorescence regime by detecting reverse-transcription loop-mediated isothermal amplification (RT-LAMP) products in the context of COVID-19 diagnostics. The device represents a step towards the development of a point- of-care droplet digital RT-LAMP platform.

This systematic review aims to present an overview of the current aerosol sampling methods (and equipment) being used to investigate the presence of SARS-CoV-2 in the air, along with the main parameters reported in the studies that are essential to analyze the advantages and disadvantages of each method and perspectives for future research regarding this mode of transmission. A systematic literature review was performed on PubMed/MEDLINE, Web of Science, and Scopus to assess the current air sampling methodologies being applied to SARS-CoV-2. Most of the studies took place in indoor environments and healthcare settings and included air and environmental sampling. The collection mechanisms used were impinger, cyclone, impactor, filters, water-based condensation, and passive sampling. Most of the reviewed studies used RT-PCR to test the presence of SARS-CoV-2 RNA in the collected samples. SARS-CoV-2 RNA was detected with all collection mechanisms. From the studies detecting the presence of SARS-CoV-2 RNA, fourteen assessed infectivity. Five studies detected viable viruses using impactor, water-based condensation, and cyclone collection mechanisms. There is a need for a standardized protocol for sampling SARS-CoV-2 in air, which should also account for other influencing parameters, including air exchange ratio in the room sampled, relative humidity, temperature, and lighting conditions.

Background: Although an increasing body of data reports the detection of SARS-CoV-2 RNA in air, this does not correlate to the presence of infectious viruses, thus not evaluating the risk for airborne COVID-19. Hence there is a marked knowledge gap that requires urgent attention. Therefore, in this systematic review, viability/stability of airborne SARS-CoV-2, SARS-CoV and MERS-CoV viruses is discussed. Methods: A systematic literature review was performed on PubMed/MEDLINE, Web of Science and Scopus to assess the stability and viability of SARS-CoV, MERS-CoV and SARS-CoV-2 on air samples. Results and discussion: The initial search identified 27 articles. Following screening of titles and abstracts and removing duplicates, 11 articles were considered relevant. Temperatures ranging from 20 °C to 25 °C and relative humidity ranging from 40% to 50% were reported to have a protective effect on viral viability for airborne SARS-CoV and MERS-CoV. As no data is yet available on the conditions influencing viability for airborne SARS-CoV-2, and given the genetic similarity to SARS-CoV and MERS-CoV, one could extrapolate that the same conditions would apply. Nonetheless, the effect of these conditions seems to be residual considering the increasing number of cases in the south of USA, Brazil and India, where high temperatures and humidities have been observed. Conclusion: Higher temperatures and high relative humidity can have a modest effect on SARS-CoV-2 viability in the environment, as reported in previous studies to this date. However, these studies are experimental, and do not support the fact that the virus has efficiently spread in the tropical regions of the globe, with other transmission routes such as the contact and droplet ones probably being responsible for the majority of cases reported in these regions, along with other factors such as human mobility patterns and contact rates. Further studies are needed to investigate the extent of aerosol transmission of SARS-CoV-2 as this would have important implications for public health and infection-control policies.

The up- and down- regulation of inflammatory biomarkers such as cytokines can be indicative of several diseases such as primary cancers and/or metastatic tumors, as well as less serious conditions. For point-of-care clinical applications, the detection of these biomarkers requires a combination of a sensitive assay and multiplexing capabilities, together with fit-for-purpose signal transduction strategies. Here, we report the development of a versatile and cost-effective integrated centrifugal microfluidic platform compatible with resource-limited settings using nanoporous microbeads for immunoaffinity-based profiling of cytokines. With an automated colorimetric readout at the end, the platform allows for profiling of cytokines in < 30 mins.

Sensitive viral diagnostic methods are increasingly in demand to tackle emerging epidemics. The Zika virus (ZIKV) is particularly relevant in tropical resource limited settings (RLS) and is associated with intermittent epidemics such as the recent 2016 ZIKV outbreak in South America, wherein Zika fever was classified by WHO as a public health emergency of international concern. Thus, there is an urgent need for widespread Zika fever diagnostics and efficient drug therapies. ZIKV diagnostics are typically performed using RT-qPCR in centralized laboratories. While extremely sensitive, RT-qPCR requires rapid heating-cooling cycles, combined with continuous fluorescence measurements to allow quantification, implying high costs and limiting availability of molecular diagnostics in RLS. Here, we report isothermal amplification of ZIKV cDNA using padlock probes followed by two rounds of Rolling Circle Amplification (RCA), termed as circle-to-circle amplification (C2CA), combined with a microfluidic affinity chromatography enrichment (mu ACE) platform. This platform allowed the detection of <17 vRNA copies per reaction mixture, equivalent to similar to 3 aM, showed a positive correlation with RT-qPCR in both average (r = 0.80) and discrete (r = 0.95) signal modes, and was validated for drug efficiency tests using in vitro infected peripheral blood mononuclear cells from 3 healthy donors. This performance shows significant promise towards highly sensitive, albeit simple and cost-effective point-of-care viral diagnostics.

Silica fibers and capillaries offer opportunities for compact integration of optics with microfluidics while adding advantages such as; flexibility within a high aspect ratio format, uniaxial arrangements, and measurement-at-a-distance. Here, we describe droplet microfluidics-based nucleic acid detection of SARS-CoV-2 in a lab-in-a-fiber platform. The fiber component integrates three modules with key functions: droplet generation, incubation, and fluorescence detection. Within the scope of this work, we developed the component specifically to target the quantification of SARS-CoV-2 viral RNA through reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The all-fiber component could successfully generate uniform droplets and differentiate pre-amplified positive LAMP reaction from negative sample.

Since the COVID-19 outbreak was declared a pandemic by the World Health Organization (WHO) in March 2020, ongoing efforts have been made to develop sensitive diagnostic platforms. Detection of viral RNA provides the highest sensitivity and specificity for detection of early and asymptomatic infections. Thus, this work aimed at developing a label-free genosensor composed of graphene as a working electrode that could be embedded into a flex printed circuit board (FPCB) for the rapid, sensitive, amplification-free and label-free detection of SARS-CoV-2. To facilitate liquid handling and ease of use, the developed biosensor was embedded with a user-friendly reservoir chamber. As a proof-of-concept, detection of a synthetic DNA strand matching the sequence of ORF1ab was performed as a two-step strategy involving the immobilization of a biotinylated complementary sequence on a streptavidin-modified surface, followed by hybridization with the target sequence recorded by the differential pulse voltammetric (DPV) technique in the presence of a ferro/ferricyanide redox couple. The effective design of the sensing platform improved its selectivity and sensitivity and allowed DNA quantification ranging from 100 fg/mL to 1 mu g/mL. Combining the electrochemical technique with FPCB enabled rapid detection of the target sequence using a small volume of the sample (5-20 mu L). We achieved a limit-of-detection of 100 fg/mL, whereas the predicted value was similar to 33 fg/mL, equivalent to approximately 5 x 10(5) copies/mL and comparable to sensitivities provided by isothermal nucleic acid amplification tests. We believe that the developed approach proves the ability of an FPCB-implemented DNA sensor to act as a potentially simpler and more affordable diagnostic assay for viral infections in Point-Of-Care (POC) applications.