key: cord-0772901-ieczs1a1 authors: Soto, Fernando; Ozen, Mehmet Ozgun; Guimarães, Carlos F.; Wang, Jie; Hokanson, Kallai; Ahmed, Rajib; Reis, Rui L.; Paulmurugan, Ramasamy; Demirci, Utkan title: Wearable Collector for Noninvasive Sampling of SARS-CoV-2 from Exhaled Breath for Rapid Detection date: 2021-08-24 journal: ACS Appl Mater Interfaces DOI: 10.1021/acsami.1c09309 sha: 3cca7d437e433b34677d65a19ec7f86af4814866 doc_id: 772901 cord_uid: ieczs1a1 [Image: see text] Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics. Diagnosis of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that is responsible for the coronavirus pandemic (COVID-19) remains time-consuming and a challenge for clinicians and researchers. 1−4 Most of the patients are asymptomatic until advanced stages when individuals experience severe symptoms. 5−7 However, in the early stages, when the infected patients are asymptomatic, the virus spreads through breathing without awareness. Without the capacity to accurately and rapidly quantify and identify infections, governments have established large-scale quarantine measures with catastrophic effects on the global economy. 8, 9 Continuous screening in the general population and healthcare professionals would potentially enable early detection of asymptomatic patients. The current gold standard method to detect SARS-CoV-2 infection relies on amplifying the virus' genetic material using quantitative reverse transcription polymerase chain reaction (RT-PCR). 10−12 Other approaches include the use of lateral flow immunoassays based on the detection of anticoronavirus antibodies (IgG and/or IgM) 13−15 or CRISPR technology 16, 17 to detect SARS-CoV-2. Nevertheless, the antibody-based methods cannot detect the status of infection, while CRISPR-based technology is not implemented for routine diagnosis. Recent studies have reported the development of portable sensors for saliva or nasopharyngeal swab-based detection. 18−22 One of the main bottlenecks when screening patients is the type and method of sampling. Current sampling methodologies include collecting nasopharyngeal swabs, blood or saliva, in sterile containers. 23, 24 These sampling methods can cause patient discomfort (nasal swabs and needles) and, in some cases, require extra processing steps for isolating the few viruses present in the saliva. On the other hand, aerosol or filter samplers rely on long-term enrichment for sample collection, leading to time-dependent virus collection and enrichment. 25, 26 Such sampling devices have been crucial to understanding virus generation due to their high sensitivity and isolation capabilities. However, most of these collectors are bulky, have long sampling time requirements, and require trained professionals to perform sampling. 26 Here, we report the development of a point-of-care sampling platform that can potentially be used for the collection of SARS-CoV-2 from exhaled breath. We engineered an affordable, flexible, and detachable virus sample collector based on medical tape and a porous polycarbonate membrane for sample enrichment (Figure 1a ). This wearable breath-based sampler allows collecting large quantities of virus over prolonged periods of time such as during breathing through a regular mask or on a surface. Although mask samplers have been proposed, based on charged fibers or hydrogel collectors, 27, 28 they are designed to work only with compatible and rigid mask designs that would limit their rapid adoption and hinder their scalability. On the other hand, our flexible device is compatible with commonly used masks or protective equipment (e.g., N95, KN95, surgical, and textile masks) and can sustain constant mechanical deformation and be made in large quantities with batch fabrication. Apart from SARS-CoV-2, we evaluated particles exposed to a mask surface via spraying to mimic droplet-based exhalation in breathing. We assessed and modeled the wearable collection capabilities for viruses. After virus collection, the sample was eluted to isolate and recover the trapped viruses, followed by analytical sensing, where we tested the collector performance for COVID-19 detection using RT-PCR, loop-mediated isothermal amplifica-tion (LAMP), and dot blot assays. The use of protective masks is likely to become ubiquitous as a preventive tool; thus, their integration with user-friendly virus collectors can potentially increase sampling, enabling increased testing for new airborne viruses. A virus-concentrator-based wearable collector has been designed to combine the resilience and ease of use of a disposable test. The prototype integrates a detachable medical adhesive and a porous polycarbonate membrane (200 nm) for target enrichment in the sample. This wearable breath-based sampler is attached to the inner surface of the face covering. A porous polycarbonate membrane was chosen as the collection surface due to its large surface area and ease of integration with commonly used fluidic systems such as reusable syringe filters. 29 The porous material permits the airflow to pass, avoiding any blocking of the air of passage. Figure 1b qualitatively illustrates the scanning electron micrography (SEM) of 120 nm polymeric particles that are within the size range of SARS-CoV-2, that is, 60−120 nm in diameter, 30 collected on the exterior surface and interior pores of the collector membrane. These particles showed a brighter signal than the background due to surface charging of uncoated polymeric materials during SEM imaging. To provide an affordable platform, we used a large-scale compatible fabrication protocol ( Figure S1a ). Rolls of flexible medical adhesive ARcare@90445 were die-cut into single adhesive units to form the virus collector base. The adhesive plastic consisted of a 25 μm transparent polyester base layer coated on both sides with a 28 μm medical grade adhesive. The doublefaced adhesive thin plastic was protected by a 50 μm polyester release liner. This fabrication method could enable highthroughput fabrication due to the ease of use and the low Research Article expense of roll die cutting approach. The materials for the mask collector include the polycarbonate filter membrane and a plastic adhesive; hence, such a device can be made inexpensively, and the cost does not increase significantly compared to that of a protective mask. The wearable collector was built by integrating the flexible plastic adhesive film and a porous polycarbonate collector ( Figure S1b ). The purpose of using two different adhesive sections is to facilitate the recovery of the collector from the mask, reducing potential contamination. Thus, two adhesive patterns were designed using computer-assisted design: (1) a ring and L-shape design containing a 20 mm diameter hollow circle to hold and provide mechanical stability to the polycarbonate porous membrane and (2) a T-and ringshape design to interface the porous membrane. This Trectangle shape at the top of the ring enables the attachment of the wearable collector to the mask and remains unbound to the bottom side, which confers free mobility of the flexible membrane and allows adherence to irregular shapes. The final assembly is completed by removing the external protective liner and integrating the two adhesives with the (3) polycarbonate membrane. Both the adhesive and the polycarbonate membrane are flexible; thus, they can adapt, enabling close conformation to the surfaces of diverse types of commonly used mask protectors and face coverings. Figure 1c shows the adhesion of the collector on N-95, KN-95, surgical, and textile-based masks. The collector is located near the mouth with a distance lower than 5 mm to maximize the number of collected viruses while enhancing further downstream analysis. Although the primary purpose of this work is to create a wearable adhesive collector for masks, the sampling can be expanded by placing the collector on other regions of potential interest such as outside the mask, face cover, or computer monitor toward creating an external environmental sampler for sample collection ( Figure S2 ). The mask collector's ability to maintain close attachment to a mask was tested by subjecting it to continuous physical and mechanical stress. The collector resisted continuous mechanical deformation steps including twisting, bending, and indentation in a sequential order ( Figure 1d ) as seen in photographs taken from Video S1. Moreover, the collector remained attached after 200 continuous bending deformations (n = 3), illustrating the ability of the collector to endure continuous deformation, although we expect that the mask collector would only be bent during application and removal. The wearable collector could capture a larger number of viruses and other biologics from the exhaled breath depending on the amount of the released viral particles. The exhalation of viruses varies greatly based on different parameters such as age, disease stage, and other physiological parameters, 31 making it hard to establish a homogenous prediction of how much time is needed to collect enough virus for further downstream analysis. Therefore, the potential number of exhaled viruses was estimated using an established SARS-CoV-2 model reported from infected patients (ranging from 200 to 1400 viruses exhaled per minute). 32, 33 Using this model, we calculated the range of airborne viruses potentially exhaled after different time periods (Figure 2a ). This prediction indicates that a short sampling time might limit the quantity of collected virus, thus increasing the possibility of obtaining false negatives. On the contrary, prolonged sampling times enable the collection of increased virus quantities. Therefore, after 4 h, between 48,000 and 336,000 viruses are estimated to be exhaled. Further, to evaluate the ability to capture the threshold number of viral copies to perform downstream analytical analysis, a numerical model 34, 35 was created to understand the virus capture ability of the wearable virus collector (Figure 2b ). Although these models have not been fully clinically validated for SARS-CoV-2, they are based on previous models with other viruses which share similarities in terms of size and transport by droplets in breathing. Thus, we expect that the result of our model is representative for SARS-CoV-2 viral load that the mask would capture. This model estimates the amount of captured virus on the surface of the collector over 1 h time increments based on low and high airborne virus generated per hour. The details of the model are provided and explained in the Supporting Information (Section 2: Modeled collection of viruses). Airborne viruses exhaled from the mouth can land on the mask interior as well as the collector. When depositing on the collector, the particles land on the surface or within the pores. The calculations were simplified by reducing the face and mask to flat surfaces and assuming gravity effects to be negligible and viruses only to present within respiratory droplets. From this model, it was estimated that approximately 13% of exhaled viruses land on the surface of the collector, resulting in a potential collection between 6,240 and 43,680 viruses in 4 h. To evaluate the collector's capability to capture sprayed targets, we generated an aerosol of droplets with a spray atomizer. Although air-based methods are commonly used to study and evaluate a protective mask's ability to retain small particulates, 36 the liquid atomizer was used to mimic the liquid droplets that carry along with the exhaled breath and would later land on the collector. We loaded the atomizer with a liquid fluorescent dye and evaluated the mist dispersion and droplet size distribution. Spraying at different distances resulted in different droplet spreads collected at a surface. When the atomizer was placed more than 15 cm far from the target wall, the resulting droplet size on the surface was smaller than millimeter in size (0.2 ± 0.1 mm), while the closer proximity (<10 cm) between the dispenser and the collector resulted in the formation of larger droplets (1.9 ± 0.6 mm) as shown in Figure S3 and Video S2. The use of a porous membrane enabled collecting a larger volume of liquid when compared to a nonporous surface due to the higher surface area of the porous membrane ( Figure S4 ). SARS-CoV-2 is spread through airborne transmission of virus carrying droplets of saliva in the micrometer range. Thus, we sprayed 2 μm fluorescent tracer particles (255 ± 38 particles/μL) onto a collector to simulate the distribution of the viruses on the mask collector surface. This visual model enabled the rapid identification and location of the microparticles after spraying them onto the mask collector. The collection capability was analyzed by measuring the number of particles in the 200 × 200 μm 2 area located at the center of the collector under different conditions, including (i) before spraying (0), (ii) after spraying (61 ± 11), (iii) after washing (17 ± 6), and (iv) measuring the resuspended isolated particles (27 ± 9 recovered particles concentrated into 50 μL) as illustrated in the overlapped bright-field and fluorescent micrograph in Figure 2c . The measurement of particles in each condition indicates the ability to capture and recover the sprayed microbeads (Figure 2d ). The wearable collector enables to preconcentrate the isolated microparticles in small volumes using a reusable syringe filter ( Figure S5) . To evaluate the compatibility of the collector with the gold standard SARS-CoV-2 sensing platforms, we evaluated the ability of our wearable collector to capture, isolate, and transfer the captured virus (Figure 3a) . 15,000 heat-inactivated SARS-CoV-2 were sprayed onto the mask collector. After spraying, the detached membrane was eluted using a commercially available extraction kit, and the viral RNA was extracted and preconcentrated in 30 μL. Finally, a qualitative colorimetric RT-LAMP and quantitative real time RT-PCR were also evaluated to measure the recovered viral load (Figure 3b,c) . A commercial RT-LAMP test (RayBiotech) was used to detect SARS-CoV-2. RT-LAMP relies on the amplification of specific sequences of RNA at constant temperature (60°C). 37 A reverse transcriptase, a DNA polymerase, and four specific primers (F3, B3, FIB, and BIP) were included in the mix. In the presence of a few RNA template molecules, a chain reaction amplifies the fragments and produces changes in magnesium levels. These changes were detected by a colorimetric readout using hydroxynaphthol blue, a metal-ion indicator, which allows the formation of the Mg 2 P 2 O 7 complexes to change color from purple to blue when the reaction is positive. We incubated the extracted RNA from our sprayed collector and incubated it with the correspondent primers and master mix (see Methods section). Negative (purple) and positive (blue) controls were performed in parallel along with three samples recovered from the collector (Figure 3b) . A positive response (blue) was detected for the three recovered samples indicating the presence of SARS-CoV-2 viral RNA. A control experiment was also performed using a virus T4 bacteriophage. In all cases, the recovered samples from the collectors containing T4-bacteriophage showed negative results in the colorimetric LAMP tests where the color change to purple indicates that the performed assay is specific to SARS-CoV-2 (positive for blue color). This method illustrates a simple and rapid qualitative tool that provides a visual method of detection. RT-PCR quantitative analysis was also performed due to the higher sensitivity when compared to the RT-LAMP technique. PCR has high sensitivity being able to detect very few viral copies per milliliter. In this direction, our collector aims to reach the limit of detection of PCR samples, thus minimizing false negative test results. RT-PCR relies on the amplification of a specific sequence and monitoring of this amplification via fluorescent readout. Figure 3c illustrates the curves for the analysis of negative (only buffer), stock (solution with a known viral concentration), and recovered (virus collected from the mask collector). As an extra control and to prove the selectivity, we added an influenza (H3N2) virus (10:1; influenza/SARS-CoV-2). The cycle of threshold (Ct) values taken from the RT-PCR applied for stock (26.8 ± 0.1), recovered (29.1 ± 0.2), negative (34.8 ± 1.4), and influenza H3N2 (33.9 ± 1.3) are shown in Figure 3d . This Ct value is translated as a recovery of ∼585 viruses from the original stock solution of 15,000 viruses. Moreover, the commercially available test distinguished against the influenza virus, thus illustrating the potential of this sampling methodology to be integrated into the common testing workflow. Finally, to demonstrate the versatility of the virus collector, we developed an alternative collector design using a nitrocellulose membrane toward performing a dot blot-based assay on colorimetric detection of SARS-CoV-2, which detects surface proteins rather than extracted viral RNA. The dot blot assay is a simplified version of western blotting, where individual protein solutions are deposited on a surface such as a nitrocellulose membrane into individual dots (circular regions). The exposure of these dots to antibodies can be used to detect and semiquantitatively assess the presence of proteins of interest by color changes inside the dot regions. 38 In this direction, an adhesive tape layer containing six circular holes was adhered to a nitrocellulose membrane to fabricate a wearable collector which allowed to detect colorimetric changes following dot plot protocols (Figure 4a ). Analyzing the protein content directly over the collector enhanced the number of viruses trapped in the nitrocellulose surface as no viruses are potentially lost during isolation and recovery steps. The nitrocellulose membrane is not as flexible as the polycarbonate, but the plastic improved mechanical stability, enabling the attachment to a surgical mask (Figure 4b) . Similarly, the utility of having six collection points is illustrated in the fluorescent photograph showing the collector with individual collection point stained with fluorescent dye sprayed microdroplets. We note that the sprayed droplets cover the whole collector surface, but the protective layer with opening in the collection sites was removed, showing only the collection points (Figure 4c ). The membranes exposed to different conditions were processed in a typical dot blot workflow, blocked against nonspecific binding, and incubated with a first anti-SARS-CoV-2 spike antibody solution overnight. Following a secondary incubation with an alkaline-phosphatase (AP)-conjugated secondary antibody, the colorimetric readout was obtained by providing membranes with the AP substrate and observing the color change produced by the 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium reaction with the AP antibody. Figure 4d shows the actual images of the collectors under multiple control parameters, including blank control, a negative control consisting of spraying 100,000 T4 bacteriophage, and a positive control consisting of spraying 100,000 heat-inactivated SARS-CoV-2. We plotted the intensity (gray value) of the multiple dots using image analysis tools, obtaining a semiquantitative comparison as illustrated in the bar graph in Figure 4e . The images illustrate a color signal only on the positive sample, providing a qualitative indication of the presence of SARS-CoV-2. We have demonstrated a proof-of-concept disposable and inexpensive airborne biological collector. The wearable collectors can be fabricated, adaptable to mass fabrication methods, and incorporate biocompatible materials commonly used in medical devices. The collectors can be applied to a wide range of mask protectors and face coverings, potentially enabling rapid translation. The collector was able to withstand mechanical deformation and remained attached for prolonged periods, representing its potential to be used for a whole working day. This device does not potentially present an increased health risk to the wearer. Any reaerosolization of viral particles collected due to breathing or contact is similar to those by the use of a mask protector by itself. We used models and performed experiments to understand both the virus collection and isolation, supporting the prolonged use for a working day. Moreover, we showed the compatibility of our collector with the state-of-the-art existing analytical methods. We demonstrated the collection of airborne RNA quantification by RT-LAMP via a color change in solution and real-time quantification via RT-PCR. We also demonstrate the ability to measure the SARS-CoV-2 spike proteins using a dot blot assay. The use of colorimetric assays (RT-LAMP and dot blot assay) can expand testing in the field or remote locations where centralized testing facilities and PCR equipment are not available. There are additional challenges in incorporating this method into the clinic and validating its daily use. Clinical models have reported that infected patients exhale viruses ranging from 200 to 1400 copies per minute, 32, 33 indicating that even if a small percentage of those viruses are captured, the number of viruses collected on our device is within the limit of detection of PCR analysis. In the next phase, we plan to test and validate the collector with participants to evaluate its effectiveness to detect airborne diseases. Although we focused on SARS-CoV-2, we expect our collection platform to be broadly applicable to other diseases and airborne infectious agents, such as influenza. The sensitivity, selectivity, and reproducibility of the detection will rely on the existing diagnostic test used for analyzing the samples isolated from the collector. Increased period between collector removal from the mask and the analytical technique to quantify the collected virus is another critical parameter that could change the sample composition, reducing the test's efficacy. We expect that the mask collector would follow similar handling and disposal protocols as those for protective masks as the collector would contain similar viral quantities as those present in a common face covering after use. The use of wearable virus collectors will provide a new approach to complement sampling methodologies, such as nasopharyngeal swabs and saliva, and monitor patients for airborne diseases adequately without invasive or costly methodologies. We expect that the presented approach could translate to a universal and ubiquitous sampling platform that could scale up COVID-19 testing capabilities. The collector can be easily integrated with a sensor system on the mask itself, which marks the future development potential for our work. 39 We envision that the work proposed here will provide the foundation for a rapidly adaptable platform capable of sampling and quick identification of novel airborne viruses once they emerge in future pandemics and serve as a widely deployable, costeffective, early alert, and viral surveillance tool. The wearable collector was built by integrating a commercially available flexible plastic adhesive film ARcare@90445 (Adhesives Research) and a 200 nm porous polycarbonate collector (Whatman, 25 mm diameter, 10 μm thickness). A die cutting Cricut Maker (Cricut) machine was used to fabricate the adhesive components of the virus collector. Sterilization of the whole device was performed under UV for 5 min prior to use. In the case of the dot blot experiments, a nitrocellulose membrane was cut into 25 mm diameter circles and used instead of the polycarbonate membrane. After collection, the collector was placed in a reusable syringe filter (Cole Palmer, 06623-62), and an approximate volume of 10 mL of phosphate buffer solution was passed through to recover the samples from the collector. A 2 mL sprayer atomizer was used to generate the droplets. 120 nm PS particles were used to mimic the virus sizes. The morphology of the mask with beads was characterized by filedemission scanning electron microscopy (JSM-7500F, JEOL). UV green dye (Bitspower) dye was used for fluorescence experiments. We purchased heat-inactivated (Catalog # NR-52286, Isolate USA-WA1/2020) SARS-CoV-2 from BEI resources (order number: IC2020-1784). The SARS-CoV-2 isolate was isolated from an oropharyngeal swab from a patient with a respiratory illness. The complete genome of SARS-CoV-2, USA-WA1/2020 has been sequenced (the isolateGenBank: MN985325 and GISAID: EPI_ISL_404895 and after one passage in Vero cellsGenBank: MT020880) after four passages in Vero cells in collaboration with Database for Reference Grade Microbial Sequences (FDA-ARGOS; GenBank: MT24667). 15,000 viruses/μL heat-inactivated SARS-coV-2 were sprayed onto the mask collector (50 μL) and let dry in a fume hood. Next, the membrane was placed in a reusable syringe filter adapter. 10 mL of deionized water was filtered through the system to recover the virus collected at the porous membrane and stored in a centrifuge tube. Next, a commercially available RNA extraction kit (miRNeasy Kit, Qiagen) was used to extract and preconcentrate the RNA viral processed content into 30 μL of DNAse free solution. After the RNA extraction protocol, we performed RT-LAMP assay using a SARS-CoV-2 RNA isothermal amplification colorimetric assay kit (RayBiotech) and a RT-PCR test kit PCR-COV-HTOS, COVID-19 1-step high throughput PCR kit (RayBiotech) to verify the ability to detect SARS-CoV-2 content. The test followed manufacturer's protocols. Control experiments were compared to Escherichia coli bacteriophage T4 (ATCC 11303-B4, 1.2 × 10 9 PFU/mL) and influenza H3N2 virus (Bioworld, Recombinant 5.3 × 10 7 PFU/mL) to evaluate the specificity. For RT-PCR analysis, we used the RT-PCR assay kit from RayBiotec using the CFX96 BioRad PCR system. In order to evaluate the affinity of the anti-SARS-spike antibody to bind to and detect the presence of SARS-CoV-2 viruses in the nitrocellulose membrane, a standard dot blot approach was followed. First, different dilutions of the virus solution were blotted in a nitrocellulose membrane (BioRad, 1620113) by pipetting 2 μL drops of each condition in individual locations (∼4 mm diameter membranes). After drying, the membranes were blocked by ACS Applied Materials & Interfaces www.acsami.org Research Article incubation with 5% bovine serum albumin (Sigma-Aldrich) in TBS (Tris-buffered saline)-T0.05% Tween20 in TBS(Sigma-Aldrich) for 30 min at room temperature. After blocking, the membranes were incubated with the primary antibody (mouse anti-spike, 1:500 dilution) overnight at 4°C. Afterward, the membranes were washed three times with TBS-T and were then incubated with the secondary antibody (goat anti-mouse, AP-conjugate, Invitrogen, 1:1000 dilution) for 1 h at room temperature. Membranes were then washed three times with TBS-T and posteriorly revealed with the APconjugate kit (BioRad, 1706432). The reaction was stopped once the dots were visible, and the membranes were then converted to eight-bit digital images. Using ImageJ blot tools, semiquantitative analysis was performed to compare the different dots' intensity values between dilutions and sample types. The same protocol was applied for the membranes exposed to virus spraying within the mask collector. ■ ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.1c09309. Additional figures and expanded speed modeling of virus collection (PDF) Resiliency to external mechanical strain of a wearable collector attached to a surgical mask (AVI) Fluorescent aerosolized droplet generation using a spray atomizer directed at the collector attached to a surgical mask (AVI) focusing on developing microfluidic sorters using magnetic levitation, and (iv) Hillel Inc., a company bringing microfluidic cell phone tools to home settings. UD's interests were viewed and managed in accordance with the conflict-of-interest policies. Prof. Utkan Demirci (U.D.) is a founder of and has an equity interest in (i) DxNow Inc., a company that is developing microfluidic IVF tools and imaging technologies, (ii) Koek Biotech, a company that is developing microfluidic technologies for clinical solutions, (iii) Levitas Inc., a company focusing on developing microfluidic sorters using magnetic levitation, and (iv) Hillel Inc., a company bringing microfluidic cell phone tools to home settings. The interests of U.D. were viewed and managed in accordance with the conflict-of-interest policies. 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