High-efficiency monitoring of respiratory viruses in a single exhaled breath

High-efficiency monitoring of respiratory viruses in a single exhaled breath | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article High-efficiency monitoring of respiratory viruses in a single exhaled breath Quanjun Liu, Weihao Li, Han Gao, Mengqing Cheng, Chaoyi Yin, Manman Lv, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5708899/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Mar, 2026 Read the published version in Analytical Chemistry → Version 1 posted You are reading this latest preprint version Abstract Exhaled breath (EB), as a non-invasive biological sample, has garnered attention for diagnosis and monitoring respiratory diseases. However, efficiently collecting EB viruses for downstream detection remains a key challenge. This study introduced a novel Phase-change Drywall Cyclone Sampler (PDC-sampler), which was designed and optimized through airflow control to integrate condensation with drywall cyclone gas-liquid separation. The PDC-sampler efficiently collects viral particles from EB (8-10 μL per single EB (SEB)) without requiring a sampling liquid solution. When combined with the gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR), it enabled routine positive detection in infected individuals (≥ 10 3 copies/SEB). By coupling with the low-cost microfluidic electrophoretic virus enrichment chip or the high-cost and highly sensitive droplet digital PCR (ddPCR), the detection limit of EB viruses was reduced to 5-9 copies/SEB, which is an order of magnitude lower than the minimum viral concentration required for infection (~ 10 2 copies/SEB). Notably, tests on coronavirus disease 2019 (COVID-19) and influenza-infected volunteers demonstrated performance comparable to or better than throat swabs, validating the PDC-sampler’s effectiveness in real-world applications. This study provides an efficient, automated, and non-invasive solution for the early diagnosis, transmission mechanism study, and large-scale screening and monitoring of respiratory diseases. Biological sciences/Microbiology/Infectious-disease diagnostics Health sciences/Diseases/Respiratory tract diseases/Respiratory distress syndrome Health sciences/Medical research/Epidemiology Health sciences/Diseases/Infectious diseases/Influenza virus exhaled breath respiratory viruses condensing drywall cyclone sampler microfluidic electrophoretic virus enrichment chip droplet digital PCR Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Exhaled breath (EB), as a non-invasive and contact-free biological sample, had attracted significant attention in recent years for the diagnosis and monitoring of respiratory diseases 1 , 2 . Compared to invasive sampling methods such as throat swabs 2 , 3 , nasal swabs 4 , or blood collection 5 , 6 , EB sampling offered a simpler and safer process that could reflect diverse physiological and pathological conditions 7 , 8 . However, the tidal volume of EB in a normal adult is approximately 10 L/min with 20 breaths 9 , and studies had shown that the concentration of viruses in the EB of infected individuals was generally low 10 , ranging from approximately 10 3 to 10 7 copies/SEB 2,11,12 . The concentration required to cause infection was estimated at approximately 10 2 copies/SEB 13 . Efficient collection of viral particles from EB has thus become a critical challenge for respiratory virus detection and monitoring. Respiratory viruses, including influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were highly infectious and toxic, posing serious threats to human health 14 – 16 . According to the World Health Organization (WHO), the coronavirus disease 2019 (COVID-19) pandemic infected over 770 million individuals and caused approximately 7 million deaths globally 17 . These viruses were transmitted through droplets or aerosols expelled by infected individuals during breathing, speaking, coughing, or sneezing 18 – 21 . Smaller aerosol particles (diameter < 5 µm) carried higher viral loads and served as critical transmission vectors 16 , 22 – 24 . Thus, the development of devices capable of efficiently and rapidly capturing viruses from EB, providing high-concentration liquid samples for downstream detection, became essential to overcoming the challenges of rapid and highly sensitive detection respiratory virus. Such a solution was critical for the rapid diagnosis of infected individuals, elucidating viral transmission mechanisms, and providing real-time evaluations of prevention strategies. Despite various efforts, the collection and analysis of EB virus samples faced significant limitations. First, conventional devices that relied on low-temperature condensation for liquefying EB showed inadequate efficiency in capturing small aerosol virus particles, as well as limited automation and applicability, which restricted the sensitivity of downstream detection and applicability across multiple scenarios 25 , 26 . Second, biological aerosol wet-wall cyclone samplers, which were more efficient in capturing viral particles, required the pre-addition and continuous supply of milliliter-scale sampling liquid solution 27 . They also involved large sample volumes and prolonged collection times, leading to insufficient viral concentration, which prevented direct application for human EB sampling and combination with the gold-standard RT-qPCR detection method 28 , 29 . Lastly, mask-based virus capture and detection methods, which emerged in response to the COVID-19 pandemic, demonstrated inadequate detection stability, leading to elevated false-positive rates and limiting their rapid implementation in real-world applications 24 , 30 – 32 . In this study, a novel EB virus sampler, named the Phase-change Drywall Cyclone Sampler (PDC-sampler), was innovatively designed and optimized to address these challenges. This device employed airflow control technology to innovatively integrate and optimize the condensation with a drywall cyclone gas-liquid separation structure. Unlike existing methods, the PDC-sampler operated without requiring additional collection liquids 33 , 34 . The PDC-sampler efficiently collected and enriched small aerosol viral particles from EB, providing 8–10 µL of liquid sample per single EB (SEB) with high viral concentration for downstream detection. When combined with RT-qPCR, the PDC-sampler achieved positive detection in infected individuals with viral loads (≥ 10 3 copies/SEB) 12,35 . Furthermore, When combined with the low-cost microfluidic electrophoretic virus enrichment chip or the high-cost ddPCR, the detection limit was further reduced to 5–9 copies/SEB, which is an order of magnitude lower than the minimum viral concentration required for infection (~ 10 2 copies/SEB) 13,36 . The device was tested and validated in multiple experimental scenarios, including EB sampling from healthy volunteers and virus-infected individuals, effectively demonstrating its practicality for respiratory virus detection. This study highlights the potential of EB sampling in clinical diagnostics and public health surveillance, advancing the application of non-invasive diagnostic platforms for respiratory disease. 2. Materials and methods 2.1 The PDC-sampler and enrichment detection system: design and working principle The PDC-sampler collection and enrichment system consist of a condensation module, a drywall cyclone gas-liquid separator, and a microfluidic electrophoretic virus enrichment device (Fig. 1 a). The drywall cyclone gas-liquid separator (Fig. S1 ) is connected at the inlet to the condensation module (Fig. S2), with the top gas outlet linked to an air pump. The bottom sample collection port is connected via a peristaltic pump (Danhao, OL280A, China) to a microfluidic virus enrichment chip (Fig. S3). Prior to sampling, the condensation module was pre-cooled for 2–3 min, reducing the condensation zone temperature to -15 ± 1°C. The air pump was then activated, and EB was collected at a flow rate of 26 ± 1 L/min. The negative pressure facilitated rapid intake of EB into the system, causing viral particles quickly collide and fuse with the liquefied tiny droplets within the condensation zone, gradually forming larger mixed droplets and completing the transfer of viral particles from the gas phase to the liquid phase. These droplets then entered the drywall cyclone gas-liquid separator, where they were tangentially impacted with the inner wall, and under the influence of gravity, surface tension, and stable cyclonic forces, rapidly fused and spiraled downward. The collected viral particles were transferred into the liquid medium, yielding 8–10 µL/SEB of liquid sample. Uncaptured viral particles were expelled from the top outlet and captured by a HEPA filter. Figure 1 b showed the 3D structural layout of the PDC-sampler. Subsequently, the liquid sample was transferred to the microfluidic electrophoretic virus enrichment chip using a peristaltic pump for further enrichment, providing higher-concentration samples for downstream detection. The microfluidic electrophoretic virus enrichment chip consisted of a sample channel, filter membrane, enrichment channel, and electrodes (Fig. S3). Following this, virus liquid samples were enriched by setting the voltage to 30 V, the sample flow rate to 200 µL/h, and the enrichment liquid flow rate to 10 µL/h. Under the influence of electrophoretic force, negatively charged viral particles shifted from the sample channel to the enrichment channel and were concentrated. To enhance the chip's stability and durability, glass was used as the base material, and Polydimethylsiloxane (PDMS) materials were added to connect the inlet and outlet. 2.2 Optimization of structural design and condition selection To achieve optimal structural design and condition control, computational fluid dynamics (CFD) simulations were conducted. Theoretical evaluations were conducted on the airflow control in the drywall cyclone gas-liquid separator of the PDC-sampler and the enrichment conditions of the microfluidic electrophoretic virus enrichment chip. Gradient simulations were conducted on the airflow velocity, collection inlet angle, inlet/outlet diameter ratio, outlet position, and sidewall tilt angle of the drywall cyclone gas-liquid separator (Supplementary Method 1), leading to the development of an optimal and controllable airflow structure design. To theoretically assess the enrichment efficiency of negatively charged viral particles in the microfluidic electrophoretic virus enrichment chip, simulations were conducted at voltages of 1 V, 3 V, 5 V, 10 V, 20 V, and 30 V, with sample flow rates of 200 µL/h, 400 µL/h, 600 µL/h, 800 µL/h, 1000 µL/h, and 1500 µL/h. A control group with 0 V was included for comparison (Supplementary Method 2). 2.3 Preparation of virus samples In the experimental tests, inactivated SARS-CoV-2 and H1N1 were selected as representative viruses. Their size and characteristics met the requirements for studying bioaerosols, and they posed no risk to laboratory personnel during testing. The inactivated SARS-CoV-2 virus samples were commercially purchased (Fubaiao Biomedical Technology Co Ltd., Jiangsu, China), with an initial liquid concentration of 4.32 × 10 7 copies/µL. The simulated EB SARS-CoV-2 virus concentrations were categorized into high concentration (3.60 × 10 7 copies/SEB and 3.60 × 10 6 copies/SEB), medium concentration (3.60 × 10 5 copies/SEB and 3.60 × 10 4 copies/SEB), and low concentration (3.60 × 10 3 copies/SEB and 3.60 × 10 2 copies/SEB). InfA/H1N1 (PR8) was provided by the Key Laboratory of Jiangsu Preventive Veterinary Medicine (Yangzhou, China). The H1N1 virus was propagated following standard methods in accordance the World Health Organization guidelines for culturing in chicken embryo cells 37 . The liquid samples were centrifuged at 8000 revolutions per minute (RPM) for 2 min. The viral titer was quantified using a hemagglutination assay, yielding hemagglutination units (HAU), with an initial liquid concentration of 3.72 × 10 7 copies/µL. The simulated EB H1N1 virus concentrations were categorized as high concentration (3.10 × 10 7 copies/SEB and 3.10 × 10 6 copies/SEB), medium concentration (3.10 × 10 5 copies/SEB and 3.10 × 10 4 copies/SEB), and low concentration (3.10 × 10 3 copies/SEB and 3.10 × 10 2 copies/SEB). For all liquid sample preparations, 10-fold serial dilutions were performed using 1 × PBS. 1 × PBS was used as a negative control. 2.4 Characterization of simulated virus-containing EB EB was simulated using a bioaerosol generator, with 50 mL centrifuge tubes replacing sample vials. A fixed sample volume of 15 mL was used for each experiment, and the airflow rate was set to 10 L/min. To ensure that the particle size distribution and quantity of the simulated EB closely resembled those of adult EB, an Aerodynamic Particle Sizer Spectrometer (APS, TSI model 3321) was used for characterization. The results indicated that the droplets in the simulated EB were predominantly small particles, with more than 50% of the particles having a diameter of D p < 1.50 µm and more than 80% having a diameter of D p < 3.00 µm (Supplementary methods 3), consistent with the study by Pan et al. 38 , 39 . 2.5 Collection and enrichment of EB virus First, tests were conducted in the laboratory on the PDC-sampler and the microfluidic electrophoretic virus enrichment chip. A bioaerosol generator was used to simulate the EB of individuals infected with influenza and COVID-19. The PDC-sampler and a commercial bioaerosol sampler (ASE-200p, Shenzhen, China) were employed to collect EB viruses, and the collection efficiency was compared. The microfluidic electrophoretic virus enrichment chip was used to enrich the collected liquid virus samples, increasing the liquid sample concentration and enhancing the sensitivity of virus detection. All experiments were repeated three times. Between samplings, a nebulizer was used to aerosolize hypochlorous acid cleaning solution, ensuring that the cleaning solution was applied to the entire channel in the same manner as the EB collection. Detailed collection procedures are provided in Supplementary Method 4. Subsequently, the performance of the PDC-sampler was tested in real-world applications. The device was used to collect EB from volunteers, including healthy individuals, COVID-19 patients, and H1N1 patients. The collection flow rate was adjusted to 26 ± 1 L/min, and the condensation zone temperature was lowered to -15 ± 1°C. To meet the requirements for downstream RT-qPCR and ddPCR detection, the collecting time was set to 1 min per session, yielding approximately 170 µL of liquid sample. 2.6 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) The liquid samples were quantitatively analyzed by RT-qPCR. RNA was extracted from 50 µL of viral liquid samples using the AxyPrep Small Volume Kit for Body Fluid Virus DNA/RNA (AxyPrep), eluted with 30 µL of Buffer TE, and 1 µL of RNA was prepared with TaqMan® Fast Virus 1-Step Master Mix (ThermoFisher) assay reagent in a total amount of 20 µL of the reaction system, and the reaction was thermally cycled in a real-time fluorescence quantitative PCR instrument (QuantStudio 3, ThermoFisher). The primers and probes used for detection were designed using Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ), and synthesized by Shanghai Bioengineering Corporation (Shanghai China), and the validity of the primers was confirmed by qPCR analysis of the solvent curves. The comparative cycle threshold (Ct) value method was used to calculate mRNA levels to determine the amount of virus collected and enriched. For more detailed relevant details, see Supplementary Methods 5. 2.7 Droplet digital PCR (ddPCR) The low viral concentration EB collection liquid samples were subjected to ddPCR assay at DynaTech Biotech (Wuhan, China), using the same primers and probes as those employed in the RT-qPCR assay. See Supplementary Methods 6 for specific detailed assay details. The results of the assay were meticulously analyzed with SightPro software, which set thresholds between positive and negative droplets, and confirmed by manual inspection. 2.8 Statistical analysis All data were expressed as mean ± standard deviation (SD, n ≥ 3) and plotted using Origin 9 (Origin 9 Software, USA). Statistical analysis was conducted using the One-Way Analysis of Variance in SPSS software, considering p-values less than 0.05 as significant. Asterisks in the figures indicate statistical differences: “*” p < 0.05, “**” p < 0.01, “***” p < 0.001, “****” p 0.05 indicating no statistical significance (N.S.). 3. Results 3.1 Determination of EB virus collection and enrichment conditions To achieve optimal EB collection and enrichment efficiency, the best conditions for the PDC-sampler were tested and analyzed. Figure 2 a showed that the EB-to-liquid conversion efficiency was approximately 60% when the collection flow rate was 24 L/min, 26 L/min, or 28 L/min, and the collection distance was 1 mm (close but not touching). However, a significant decrease in conversion efficiency was observed when the collection flow rate was reduced to 22 L/min or increased to 30 L/min, as well as when the collection distance was increased. Detailed procedures are provided in Supplementary Methods 7. The actual collection efficiency of the PDC-sampler was tested using standardized PSL microspheres. Figure 2 b showed that the collection efficiency for 0.5 µm aerosol particles exceeded 60%, while it approached 100% for aerosol particles with a diameter of 2 µm. Detailed procedures are provided in Supplementary Methods 8. Next, the enrichment conditions for the microfluidic electrophoretic virus enrichment chip were tested using H1N1 virus with a concentration of 3.72 × 10 3 copies/µL. Figure 2 c showed that at a sample flow rate of 200 µL/h, the enrichment groups showed significantly higher values than the unenriched groups at gradient voltages ranging from 1 to 30 V. The enrichment efficiency increased with voltage, reaching the optimal level at 30 V. No signal was detected in the 0 V enrichment group or the negative control group (1 × PBS). Figure 2 d showed that at 30 V, with sample flow rates ranging from 200 µL/h to 1000 µL/h, the enrichment groups remained significantly higher than the unenriched groups, although enrichment efficiency decreased as sample flow rate increased. No signal was detected in the negative control group (1 × PBS). Detailed concentration processes are provided in Supplementary Methods 9. 3.2 Performance of the PDC-sampler for EB virus collection To evaluate the performance of the PDC-sampler for collecting EB viruses, a bioaerosol generator was used in the laboratory to simulate EB containing varying concentrations of viruses. EB virus was collected using the PDC-sampler and a bioaerosol sampler, respectively. As shown in Fig. 3 a, the collected liquid samples were analyzed using RT-qPCR. The results showed that when using the PDC-sampler to collect EB containing SARS-CoV-2 or H1N1 viruses, the mean Ct values from the high-concentration to the low-concentration groups were: 23.93, 26.77, 29.14, 34.02, 36.24 for SARS-CoV-2, and 25.62, 27.02, 30.98, 35.25, 36.58 for H1N1, respectively. In contrast, other liquid samples had viral concentrations below the detection limit. When the bioaerosol sampler was used to collect EB containing SARS-CoV-2 or H1N1 viruses, the mean Ct values from the high-concentration to the low-concentration groups were 29.79, 33.16, 35.05 for SARS-CoV-2, and 33.98 for H1N1. Other liquid samples had viral concentrations below the detection limit. Statistical analysis revealed that the virus concentrations in liquid samples collected using the PDC-sampler were significantly higher than those collected using the bioaerosol liquid sampler. However, in the low-concentration groups for SARS-CoV-2 and H1N1 viruses, the virus concentrations in the collected liquid samples were below the RT-qPCR detection limit or showed weak positive results. 3.3 Virus concentration enhanced by microfluidic electrophoretic virus enrichment chip To evaluate the effectiveness of the PDC-sampler in collecting and enriching low-concentration EB viruses, the microfluidic electrophoretic virus enrichment chip was combined with the PDC-sampler. As shown in Fig. 3 b, the chip was used to enrich liquid samples containing H1N1 virus from EB, further increasing the liquid sample concentration for detection using the gold-standard RT-qPCR. The results indicated that in the high-virus-concentration group, the Ct values showed no significant enrichment effect compared to the unenriched group. However, as the concentration decreased, the enrichment effect became increasingly pronounced. In the medium-concentration group, statistical analysis demonstrated that the virus concentrations in the enriched group were significantly higher than those in the unenriched group. Notably, as the dilution factor increased further, and the virus concentration decreased to the low-concentration group, the unenriched group were below the RT-qPCR detection limit. In contrast, after enrichment using the chip, liquid sample concentrations were raised above the detection limit, enabling effective detection. No signal was detected in the negative control group (1 × PBS). These results demonstrated that the combination of the PDC-sampler and the microfluidic electrophoretic virus enrichment chip reduced the detectable concentration of EB viruses to approximately 31 copies/SEB. 3.4 Detection limit of PDC-sampler reduced by ddPCR To enhance the sensitivity and accuracy of detecting EB with low virus concentrations, the advanced and highly sensitive ddPCR technology was employed to analyze both the liquid virus samples before EB virus generation and the low-concentration liquid samples collected by the PDC-sampler. As shown in Fig. 3 c and 3 d, positive droplets were detected in both the liquid virus samples before EB virus generation and the liquid virus samples collected by the PDC-sampler for low-concentration groups containing SARS-CoV-2 (5 × 10 0 − 8.20 × 10 3 copies/SEB) or H1N1 virus (8.65 × 10 0 − 2.19 × 10 2 copies/SEB). As the dilution factor increased, liquid samples with concentrations below the ddPCR detection limit showed negative droplets. Negative droplets were detected in the negative control group (1 × PBS). These results demonstrated that combining the PDC-sampler with ddPCR technology reduced the EB virus detection limit to approximately 5–9 copies/SEB. Notably, low-concentration EB viruses collection samples that were negative by RT-qPCR were detected as positive using ddPCR, confirming that the PDC-sampler effectively collected low-concentration EB viruses. 3.5 Real-world testing of PDC-sampler To validate the real-world application of the PDC-sampler, respiratory infection patients (with symptoms such as fever, and muscle aches) were recruited as volunteers. Throat swab and EB virus liquid samples were collected and analyzed using RT-qPCR, with healthy volunteers serving as negative controls. As shown in Fig. 4 a, SARS-CoV-2 was detected in both the throat swab and EB liquid samples collected from Case 1 on the first day, with mean Ct values of 32.45 and 32.22, respectively, showing no statistical difference. On the second and third days, the viral concentrations in the liquid samples were below the detection limit. Throat swab and EB samples collected from this volunteer after recovery were used as negative controls. Fig. S9 showed the results of COVID-19 antigen tested using throat swabs from Case 1, which were consistent with the RT-qPCR results. As shown in Fig. 4 b, H1N1 virus was detected in both Case 2 and Case 3, with mean Ct values of 28.96 and 27.94 for throat swab samples, and 27.98 and 28.49 for EB liquid samples, respectively. The EB liquid sample concentration from Case 2 was significantly higher than that of the throat swab, while no statistical difference was observed for Case 3. In Cases 4 and 5, the throat swab samples tested negative, while EB liquid samples were weakly positive. Healthy volunteers served as negative controls. These findings indicated that the PDC-sampler's performance in collecting and detecting EB viruses was comparable to or better than throat swab sampling. 3.6 PDC-sampler: toward real-world deployment This study demonstrated an automated and rapid PDC-sampler designed to collect viruses from human EB, efficiently transferring them into a liquid medium to provide higher-concentration liquid samples for downstream detection. To evaluate the performance of the PDC-sampler in real-world applications, it was deployed in a hospital fever clinic with a collection time set to 1 min (~ 20 SEB). Liquid EB samples collected from five volunteers infected with either SARS-CoV-2 or H1N1 showed that the viral concentrations in liquid samples collected using the PDC-sampler were comparable to or higher than those in throat swab samples from the same individuals. However, when the PDC-sampler was combined with the microfluidic electrophoretic virus enrichment chip or ddPCR, the detection limit for EB viruses was further reduced to approximately 5–9 copies/SEB. Based on these findings, the real-world deployment of the PDC-sampler is envisioned as follows: First, during large-scale outbreaks, such as COVID-19, the PDC-sampler could rapidly and automatically collect liquid samples of EB viruses from infected individuals. This would reduce direct contact between healthcare workers and patients, minimizing unnecessary infection. Second, in hospital settings, such as respiratory outpatient clinics, pediatric emergency rooms, and ICUs, the PDC-sampler could automate the collection of respiratory viruses from patients with diseases like pneumonia or from difficult-to-sample populations such as infants. This would provide a non-contact, automated, and safe method for diagnosing respiratory infectious diseases. Third, in the diagnosis of specific respiratory infectious diseases such as COVID-19 or pneumonia, deploying the PDC-sampler in combination with the microfluidic electrophoretic virus enrichment chip or ddPCR could advance the diagnosis timeline to before EB virus concentrations reach levels capable of transmitting to others, enabling management personnel to implement timely isolation and other preventive measures to effectively halt the rapid spread of infectious diseases. 4. Discussion This study demonstrated a PDC-sampler specifically designed to collect viruses from human EB and transfer them into a liquid medium, providing liquid samples for downstream detection. To enhance the accuracy and sensitivity of detecting low-concentration EB viruses, the combination of the PDC-sampler with either the low-cost microfluidic electrophoretic virus enrichment chip or the high-cost ddPCR technology was proposed. The results revealed that the PDC-sampler possesses key advantages, including rapidity (SEB), non-invasiveness, high sensitivity, automation, and convenience, offering an innovative solution for the early diagnosis and monitoring of respiratory infectious diseases. Firstly, the structural design of the PDC-sampler demonstrated significant advantages in collecting small aerosol particles. Small aerosol particles are considered the primary transmission medium for high-concentration viruses 16 , 24 , yet traditional samplers, such as wet-wall cyclones, have limited efficiency in capturing these small particles 27 , 34 . This study showed that the PDC-sampler effectively captured and enriched small aerosol particles by rapidly combining them with microdroplets formed through condensation, followed by separation using a drywall cyclone gas-liquid separation structure. This design avoided the substantial dilution caused by the use of sampling liquid solution in traditional methods 28 , 29 and minimized the contamination from excessive saliva often collected by direct condensation-based samplers 12 , 40 , 41 . Furthermore, SEB yielded 8–10 µL of liquid sample with a high viral concentration. The collection efficiency for 0.5 µm aerosol particles exceeded 60%, while it approached 100% for aerosol particles with a diameter of 2 µm. These advantages make the PDC-sampler particularly suitable for studying and monitoring high-risk respiratory viruses, such as SARS-CoV-2 and H1N1, which are primarily transmitted through aerosols. The enhanced ability to collect small particles not only improved collection sensitivity but also provided opportunities to better understand virus transmission mechanisms. Secondly, the PDC-sampler demonstrated high efficiency of EB virus collection in experimental tests. When combined with the gold-standard RT-qPCR detection, the PDC-sampler successfully detected the minimum EB virus concentrations reported during the confirmed infectious period of SARS-CoV-2-infected individuals (~ 10 3 copies/SEB) 11,12 . This indicated that the PDC-sampler could provide effective liquid samples for detection during the critical phase of confirmed infections. Furthermore, when combined with microfluidic electrophoretic virus enrichment chip or ddPCR detection, the sensitivity for detecting EB viruses was significantly improved (5–9 copies/SEB), enabling detection of the minimum infectious concentration of viruses such as SARS-CoV-2 (10 2 copies/SEB) 13 . This demonstrated that the PDC-sampler performed well in collecting low-concentration EB viruses and, when combined with advanced detection technologies, could advance the diagnosis timeline to before the virus concentration reaches transmissible levels. This capability provided critical time for public health decisions, facilitating timely isolation and contact tracing measures to interrupt transmission chains effectively. Finally, this study evaluated the potential value of the PDC-sampler in real-world applications. In real-world scenarios, the collection of EB viruses from SARS-CoV-2 or H1N1 infected individuals showed performance comparable to or better than throat swab samples. Its non-contact, automated collection approach not only reduced direct contact between healthcare workers and infected individuals, lowering infection risks, but also enabled deployment in scenarios such as large-scale pandemic outbreaks, hospital respiratory wards, pediatric units, and ICUs. The automation of the PDC-sampler significantly improved collection efficiency and safety, especially in high-risk environments or for challenging groups like infants. Despite the notable advantages of the PDC-sampler demonstrated in this study, some limitations remain. Firstly, the actual collection efficiency might have been influenced by environmental factors such as humidity and temperature, necessitating more clinical samples to test its stability under varying conditions. Secondly, the compatibility between the microfluidic electrophoretic virus enrichment chip and the PDC-sampler requires further improvement, as the enrichment speed currently cannot fully match the collection rate while maintaining high enrichment efficiency. This limited the realization of a combined collection-enrichment system. Future research will focus on optimizing the device, validating its performance in real-world applications, and expanding its capability for detecting multiple viruses. The widespread application of the PDC-sampler is expected to drive innovation in respiratory infectious disease diagnostics and support the development of global public health systems. 5. Conclusion A novel PDC-sampler was designed and optimized in this study for efficient collection of EB viruses. The integration of condensation and drywall cyclone gas-liquid separation structure was optimized using airflow control technology, enabling automation SEB virus collection (8-10 μL) without the need for a sampling liquid solution. Combined with RT-qPCR, the PDC-sampler achieved routine positive detection in infected individuals. When combined with the low-cost microfluidic electrophoretic virus enrichment chip or the high-cost ddPCR, the detection limit was reduced to 5-9 copies/SEB, significantly lower than the minimum EB virus concentration reported in confirmed patients (10 3 copies/SEB). Validation with SARS-CoV-2 and H1N1 infected volunteers demonstrated that the PDC-sampler's performance matched or exceeded throat swabs, confirming its effectiveness in real-world applications. The non-invasive, automated design and high sensitivity of the PDC-sampler highlight its potential for deployment in hospitals, public health monitoring, and pandemic responses. This study provides a rapid and efficient solution for the diagnosis and monitoring of respiratory viruses, opening new possibilities for infectious disease control. Declarations Ethics declaration The experiments involving the collection and testing of throat swabs and EB liquid samples from volunteers were conducted in real-world settings with approval from the Ethics Committee of Clinical Research at Southeast University Zhongda Hospital (Project Number: 2024ZDSYLL210-P01). With assistance from hospital staff, throat swab samples were collected using commercial COVID-19 antigen test kits, and EB liquid samples were collected using the PDC-sampler in the respiratory outpatient clinic. Antigen tests were performed on-site, and liquid samples were inactivated before being transported to the laboratory for RT-qPCR analysis. Notably, all volunteers who provided samples signed informed consent forms, agreeing to the collection of throat swabs and EB samples. Data availability The source data are provided as a “Source Data” file. Source data are provided with this paper. Credit author statement Weihao Li designed the study, conducted the experiments and assay and wrote the manuscript. Han Gao assisted with some of the fluid dynamics simulations. Mengqing Cheng and Chaoyi Yin assisted in designing part of the experiments and revising the article. Manman Lv , Yemin Han , Haotian Yu , and Weiming Lin assisted with some of the experiments. Yan Huang , Qiang Zhang , Dianhuai Meng , Tian Weng , Zuhong Lu conducted writing-review and editing. Quanjun Liu guide the project design and supervised its progress. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 31872726, 61827814), the Shenzhen Fundamental Research Program (award number: JCYJ20230807114612024 and JCYJ20220530160416036), the Key Research and Development Project of Jiangsu Province (BE2022804 and BE2021012-4). References Hu Li et al. 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Guo Hao Zhang, Qiu Hong Zhu, Lei Zhang, Fang Yong & Guo Hong Tao. High-performance particulate matter including nanoscale particle removal by a self-powered air filter. Nat. Commun. 11 , 1653 (2020). Yuchen Yang et al. Ultrathin, ultralight dual-scale fibrous networks with high-infrared transmittance for high-performance, comfortable and sustainable PM0.3 filter. Nat. Commun. 15 , 1586 (2024). Bingfang Wang et al. Wearable bioelectronic masks for wireless detection of respiratory infectious diseases by gaseous media. Matter 5 , 4347-4362 (2022). YuSung Cho et al. Continuous Surveillance of Bioaerosols On-site by an Automated Bioaerosol Monitoring System. ACS Sen. 5 , 395-403 (2020). Hui Chena et al. Novel aerosol detection platform for SARS–CoV–2: Based on specific magnetic nanoparticles adsorption sampling and digital droplet PCR detection. Chinese Chem. Lett. 34 , 107701 (2022). Kristen K. Coleman et al. Viral Load of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Respiratory Aerosols Emitted by Patients With Coronavirus Disease 2019 (COVID-19) While Breathing, Talking, and Singing. Clin. Infect. Dis. 74 , 1722-1728 (2022). Xiaoxiang Zhou, Zhanping Li, Zhen Zhang, Libo Zhu & Liu, Q. A rapid and label-free platform for virus enrichment based on electrostatic microfluidics. Talanta 242 , 122989 (2022). Stuart-Harris, C. Epidemiology of influenza in man. Brit. Med. Bull. 35 , 3-8 (1979). Wenzhao Chen, Nan Zhang, Jianjian Wei, Hui Ling Yen & Li, Y. Short-range airborne route dominates exposure of respiratory infection during close contact. Build. Environ. 176 , 1-16 (2020). Shihai Pan, Chunwen Xu, Chuck Wah Francis Yu & Liu, L. Characterization and size distribution of initial droplet concentration discharged from human breathing and speaking. Indoor Built Environ. 32 , 2020-2033 (2023). Ying Huang et al. Environmental virus detection associated with asymptomatic SARS-CoV-2-infected individuals with positive anal swabs. Sci. Total Environ. 753 , 142289 (2020). Lian Zhou et al. Breath-, air- and surface-borne SARS-CoV-2 in hospitals. J. Aerosol Sci. 152 , 105693 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files VideoSEB.mp4 Video 1 Video1min.mp4 Video 2 supplementaryinformation.docx supplementary information GraphicalAbstract.png Cite Share Download PDF Status: Published Journal Publication published 02 Mar, 2026 Read the published version in Analytical Chemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5708899","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":395900706,"identity":"ba9ae694-eed6-4127-bd69-bdbe93c2f5cc","order_by":0,"name":"Quanjun Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqUlEQVRIiWNgGAWjYBACPgaGxAcQZgKRWtgYGJINSNbCJkGiFvaEZ5U/Kg4z8LPnGDD83EGMFp4Habd5zhxmkOx5Y8DYe4YYLRIJabcZ2w4zGNzIMWBmbCNSS+HPf4cZ7EnSwsDbALRFgmgtPA+SpXmOpfNInHlWcLCXGC3AgEr8+KPGWo6/PXnjg5/EaGFgyEkAkTwg4gBRGhgY0olVOApGwSgYBSMWAAAVXzDJPqTe+wAAAABJRU5ErkJggg==","orcid":"","institution":"southeast university","correspondingAuthor":true,"prefix":"","firstName":"Quanjun","middleName":"","lastName":"Liu","suffix":""},{"id":395900707,"identity":"c0120edc-20d3-4925-9f29-d83766e0c9ee","order_by":1,"name":"Weihao Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Weihao","middleName":"","lastName":"Li","suffix":""},{"id":395900708,"identity":"8e91aa05-732a-4be1-b901-41f3ba69c643","order_by":2,"name":"Han Gao","email":"","orcid":"","institution":"southeast university","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Gao","suffix":""},{"id":395900709,"identity":"1dc3f77a-cb6d-4942-94b4-6954c0ee63f0","order_by":3,"name":"Mengqing Cheng","email":"","orcid":"","institution":"southeast university","correspondingAuthor":false,"prefix":"","firstName":"Mengqing","middleName":"","lastName":"Cheng","suffix":""},{"id":395900710,"identity":"45bcd083-6026-4cdd-bd44-8c599d7428a9","order_by":4,"name":"Chaoyi Yin","email":"","orcid":"","institution":"southeast university","correspondingAuthor":false,"prefix":"","firstName":"Chaoyi","middleName":"","lastName":"Yin","suffix":""},{"id":395900711,"identity":"893c0135-bac0-4646-bf18-d8978fae0494","order_by":5,"name":"Manman Lv","email":"","orcid":"","institution":"southeast 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university","correspondingAuthor":false,"prefix":"","firstName":"Weiming","middleName":"","lastName":"Lin","suffix":""},{"id":395900715,"identity":"43c9d375-e062-4d17-a7bf-ffe4422f2b81","order_by":9,"name":"Yan Huang","email":"","orcid":"","institution":"State Key Laboratory of Digital Medical Engineering, School of Biological Science \u0026 Medical Engineering, Southeast University, Nanjing 210096, China.","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Huang","suffix":""},{"id":395900716,"identity":"6d2a95f1-3148-4e39-8883-7de68aeb24ba","order_by":10,"name":"Qaing Zhang","email":"","orcid":"","institution":"Southeast University Zhongda Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qaing","middleName":"","lastName":"Zhang","suffix":""},{"id":395900717,"identity":"e2146b59-76d8-487a-8ad6-c7559596b7a6","order_by":11,"name":"Dianhuai Meng","email":"","orcid":"","institution":"The First Affiliated Hospital with Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dianhuai","middleName":"","lastName":"Meng","suffix":""},{"id":395900718,"identity":"ebb2287c-e898-4ae2-9a5a-5690601dd29a","order_by":12,"name":"Tian Weng","email":"","orcid":"","institution":"Jiangsu Provincial Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Weng","suffix":""},{"id":395900719,"identity":"221be651-27e5-4ac6-8e37-599dbd9252c0","order_by":13,"name":"Zuhong Lu","email":"","orcid":"https://orcid.org/0000-0003-3332-2615","institution":"State Key Laboratory of Bioelectronics, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Zuhong","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2024-12-25 03:45:50","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5708899/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5708899/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1021/acs.analchem.5c06572","type":"published","date":"2026-03-03T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72671094,"identity":"260375c0-af33-4fb0-a570-b13ff148de93","added_by":"auto","created_at":"2024-12-31 04:54:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284832,"visible":true,"origin":"","legend":"\u003cp\u003eLayout of the PDC-sampler collection and enrichment system.\u003cstrong\u003e a\u003c/strong\u003eSchematic diagram of the PDC-sampler collection and enrichment system, which utilizes low-temperature condensation to liquefy and enrich viral particles in EB, forming microdroplets. The droplets are collected in the drywall cyclone gas-liquid separator, followed by further enrichment in the microfluidic electrophoretic virus enrichment chip. \u003cstrong\u003eb\u003c/strong\u003e 3D layout of the internal structure of the PDC-sampler and enrichment system, all dimensions are in millimeters (mm).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/891bc1c990b2420cb12fb45c.png"},{"id":72671095,"identity":"d8be6d74-bd5a-46a2-b7c7-0a2839bbd298","added_by":"auto","created_at":"2024-12-31 04:54:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129529,"visible":true,"origin":"","legend":"\u003cp\u003eTesting conditions of PDC-sampler and enrichment system. \u003cstrong\u003ea\u003c/strong\u003e The EB-to-liquid conversion efficiency of the PDC-sampler at 22-30 L/min collection volume and 1-50 mm collection position. \u003cstrong\u003eb\u003c/strong\u003e The collection volume of the PDC-sampler is 26 ± 1 L/min and the collection position is 1 mm, and the collection efficiency of PSL microspheres with different particle sizes is obtained. \u003cstrong\u003ec\u003c/strong\u003e Enrichment performance of the microfluidic electrophoretic virus enrichment chip for H1N1 virus at a flow rate of 200 μL/h under different voltages (30 v, 20 v, 10 v, 5 v, 1 v and 0 v). \u003cstrong\u003ed\u003c/strong\u003e Enrichment performance of the chip for H1N1 virus at a voltage of 30 V under different sample flow rates (200 μL/h, 400 μL/h, 600 μL/h, 800 μL/h and 1000 μL/h,). Each experiment was repeated at least three times (n ≥ 3), and data are presented as mean ± SD. 1 × PBS was used as the negative control. In the figure, \"P.C.\" represents the positive control, \"N.C.\" represents the negative control, and statistical significance levels are indicated as “*” p \u0026lt; 0.05, “**” p \u0026lt; 0.01, “***” p \u0026lt; 0.001, and “****” p \u0026lt; 0.0001, and p \u0026gt; 0.05 indicating no statistical significance (N.S.).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/5f09c367702c5e72b5493536.png"},{"id":72672254,"identity":"ea24aa62-73e0-4640-93d8-46aa6dea6478","added_by":"auto","created_at":"2024-12-31 05:10:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":525821,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance evaluation of the PDC-sampler and enrichment platform in the laboratory. \u003cstrong\u003ea\u003c/strong\u003e RT-qPCR analysis of EB virus liquid samples collected by the PDC-sampler compared to the bioaerosol sampler. EB contained SARS-CoV-2 or H1N1 viruses at gradient concentrations (high, medium, and low concentration groups). The left Y-axis indicated the SEB virus concentration, while the right Y-axis showed the RT-qPCR Ct values. \u003cstrong\u003eb\u003c/strong\u003e RT-qPCR results of H1N1 virus liquid samples enriched using the microfluidic electrophoretic virus enrichment chip. EB containing H1N1 viruses at gradient concentrations (high, medium, and low concentration groups) was tested for enrichment. \u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e ddPCR analysis showed statistical results and images of EB virus liquid samples collected by the PDC-sampler. EB contained SARS-CoV-2 or H1N1 viruses at gradient concentrations (low-concentration groups). Each experiment was repeated at least three times (n ≥ 3), and data are presented as mean ± SD. 1 × PBS was used as the negative control. In the figure, \"N.C.\" represents the negative control, \"N.S.\" indicates no statistical difference, and statistical significance levels are indicated as “*” p \u0026lt; 0.05, “**” p \u0026lt; 0.01, “***” p \u0026lt; 0.001, and “****” p \u0026lt; 0.0001, and p \u0026gt; 0.05 indicating no statistical significance (N.S.).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/0a253c29c253d7c29f489c5f.png"},{"id":72671100,"identity":"177de63d-c400-47ce-8ae1-d363cddbe25f","added_by":"auto","created_at":"2024-12-31 04:54:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":86538,"visible":true,"origin":"","legend":"\u003cp\u003eReal-world Testing of the PDC-sampler. \u003cstrong\u003ea\u003c/strong\u003eRT-qPCR results of EB liquid samples collected by the PDC-sampler over three consecutive days from a COVID-19 patient, compared with throat swab sampling results. Samples collected after the patient fully recovered served as a negative control. \u003cstrong\u003eb\u003c/strong\u003e RT-qPCR results of EB liquid samples collected by the PDC-sampler from four influenza patients, compared with throat swab sampling results. Healthy volunteers were used as a negative control. Each experiment was repeated at least three times (n ≥ 3), and data are presented as mean ± SD. In the figure, \"N.S.\" indicates no significant difference, and statistical significance levels are indicated as “*” p \u0026lt; 0.05, “**” p \u0026lt; 0.01, “***” p \u0026lt; 0.001, and “****” p \u0026lt; 0.0001, and p \u0026gt; 0.05 indicating no statistical significance (N.S.).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/ab77a28c92da987ed5f380b1.png"},{"id":104260316,"identity":"f38306e3-787c-4622-b775-0a1e3ee2b3ca","added_by":"auto","created_at":"2026-03-09 18:10:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1784134,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/9e5b7605-37ff-47ca-8b6e-a9c1e7656236.pdf"},{"id":72671096,"identity":"e6d9a24c-5a1b-4c4f-a6ba-e948f1c7b124","added_by":"auto","created_at":"2024-12-31 04:54:37","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4530002,"visible":true,"origin":"","legend":"Video 1","description":"","filename":"VideoSEB.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/b0898e64705e0625b9ee0c84.mp4"},{"id":72671102,"identity":"cb996e4f-9cbc-4fad-b312-71420d1201fe","added_by":"auto","created_at":"2024-12-31 04:54:37","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12829706,"visible":true,"origin":"","legend":"Video 2","description":"","filename":"Video1min.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/77bdd9e8b5c80f5af5d1feb5.mp4"},{"id":72671104,"identity":"8767bec8-2dc4-492f-b8c6-81c38c064480","added_by":"auto","created_at":"2024-12-31 04:54:37","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3369760,"visible":true,"origin":"","legend":"supplementary information","description":"","filename":"supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/274742a6f88aa68c17d355bb.docx"},{"id":72671297,"identity":"94853b98-a749-4f3f-b05d-3fab39c9a652","added_by":"auto","created_at":"2024-12-31 05:02:37","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":173893,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-5708899/v1/1ba177b1ca086bd073bc3dab.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"High-efficiency monitoring of respiratory viruses in a single exhaled breath","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eExhaled breath (EB), as a non-invasive and contact-free biological sample, had attracted significant attention in recent years for the diagnosis and monitoring of respiratory diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Compared to invasive sampling methods such as throat swabs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, nasal swabs\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, or blood collection\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, EB sampling offered a simpler and safer process that could reflect diverse physiological and pathological conditions\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, the tidal volume of EB in a normal adult is approximately 10 L/min with 20 breaths\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and studies had shown that the concentration of viruses in the EB of infected individuals was generally low\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, ranging from approximately 10\u003csup\u003e3\u003c/sup\u003e to 10\u003csup\u003e7\u003c/sup\u003e copies/SEB\u003csup\u003e2,11,12\u003c/sup\u003e. The concentration required to cause infection was estimated at approximately 10\u003csup\u003e2\u003c/sup\u003e copies/SEB\u003csup\u003e13\u003c/sup\u003e. Efficient collection of viral particles from EB has thus become a critical challenge for respiratory virus detection and monitoring.\u003c/p\u003e \u003cp\u003eRespiratory viruses, including influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were highly infectious and toxic, posing serious threats to human health\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. According to the World Health Organization (WHO), the coronavirus disease 2019 (COVID-19) pandemic infected over 770\u0026nbsp;million individuals and caused approximately 7\u0026nbsp;million deaths globally\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These viruses were transmitted through droplets or aerosols expelled by infected individuals during breathing, speaking, coughing, or sneezing\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Smaller aerosol particles (diameter\u0026thinsp;\u0026lt;\u0026thinsp;5 \u0026micro;m) carried higher viral loads and served as critical transmission vectors\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Thus, the development of devices capable of efficiently and rapidly capturing viruses from EB, providing high-concentration liquid samples for downstream detection, became essential to overcoming the challenges of rapid and highly sensitive detection respiratory virus. Such a solution was critical for the rapid diagnosis of infected individuals, elucidating viral transmission mechanisms, and providing real-time evaluations of prevention strategies.\u003c/p\u003e \u003cp\u003eDespite various efforts, the collection and analysis of EB virus samples faced significant limitations. First, conventional devices that relied on low-temperature condensation for liquefying EB showed inadequate efficiency in capturing small aerosol virus particles, as well as limited automation and applicability, which restricted the sensitivity of downstream detection and applicability across multiple scenarios\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Second, biological aerosol wet-wall cyclone samplers, which were more efficient in capturing viral particles, required the pre-addition and continuous supply of milliliter-scale sampling liquid solution\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. They also involved large sample volumes and prolonged collection times, leading to insufficient viral concentration, which prevented direct application for human EB sampling and combination with the gold-standard RT-qPCR detection method\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Lastly, mask-based virus capture and detection methods, which emerged in response to the COVID-19 pandemic, demonstrated inadequate detection stability, leading to elevated false-positive rates and limiting their rapid implementation in real-world applications\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, a novel EB virus sampler, named the Phase-change Drywall Cyclone Sampler (PDC-sampler), was innovatively designed and optimized to address these challenges. This device employed airflow control technology to innovatively integrate and optimize the condensation with a drywall cyclone gas-liquid separation structure. Unlike existing methods, the PDC-sampler operated without requiring additional collection liquids\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The PDC-sampler efficiently collected and enriched small aerosol viral particles from EB, providing 8\u0026ndash;10 \u0026micro;L of liquid sample per single EB (SEB) with high viral concentration for downstream detection. When combined with RT-qPCR, the PDC-sampler achieved positive detection in infected individuals with viral loads (\u0026ge;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e copies/SEB)\u003csup\u003e12,35\u003c/sup\u003e. Furthermore, When combined with the low-cost microfluidic electrophoretic virus enrichment chip or the high-cost ddPCR, the detection limit was further reduced to 5\u0026ndash;9 copies/SEB, which is an order of magnitude lower than the minimum viral concentration required for infection (~\u0026thinsp;10\u003csup\u003e2\u003c/sup\u003e copies/SEB)\u003csup\u003e13,36\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe device was tested and validated in multiple experimental scenarios, including EB sampling from healthy volunteers and virus-infected individuals, effectively demonstrating its practicality for respiratory virus detection. This study highlights the potential of EB sampling in clinical diagnostics and public health surveillance, advancing the application of non-invasive diagnostic platforms for respiratory disease.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 The PDC-sampler and enrichment detection system: design and working principle\u003c/h2\u003e \u003cp\u003eThe PDC-sampler collection and enrichment system consist of a condensation module, a drywall cyclone gas-liquid separator, and a microfluidic electrophoretic virus enrichment device (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The drywall cyclone gas-liquid separator (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) is connected at the inlet to the condensation module (Fig. S2), with the top gas outlet linked to an air pump. The bottom sample collection port is connected via a peristaltic pump (Danhao, OL280A, China) to a microfluidic virus enrichment chip (Fig. S3). Prior to sampling, the condensation module was pre-cooled for 2\u0026ndash;3 min, reducing the condensation zone temperature to -15\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The air pump was then activated, and EB was collected at a flow rate of 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1 L/min. The negative pressure facilitated rapid intake of EB into the system, causing viral particles quickly collide and fuse with the liquefied tiny droplets within the condensation zone, gradually forming larger mixed droplets and completing the transfer of viral particles from the gas phase to the liquid phase. These droplets then entered the drywall cyclone gas-liquid separator, where they were tangentially impacted with the inner wall, and under the influence of gravity, surface tension, and stable cyclonic forces, rapidly fused and spiraled downward. The collected viral particles were transferred into the liquid medium, yielding 8\u0026ndash;10 \u0026micro;L/SEB of liquid sample. Uncaptured viral particles were expelled from the top outlet and captured by a HEPA filter. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb showed the 3D structural layout of the PDC-sampler.\u003c/p\u003e \u003cp\u003eSubsequently, the liquid sample was transferred to the microfluidic electrophoretic virus enrichment chip using a peristaltic pump for further enrichment, providing higher-concentration samples for downstream detection. The microfluidic electrophoretic virus enrichment chip consisted of a sample channel, filter membrane, enrichment channel, and electrodes (Fig. S3). Following this, virus liquid samples were enriched by setting the voltage to 30 V, the sample flow rate to 200 \u0026micro;L/h, and the enrichment liquid flow rate to 10 \u0026micro;L/h. Under the influence of electrophoretic force, negatively charged viral particles shifted from the sample channel to the enrichment channel and were concentrated. To enhance the chip's stability and durability, glass was used as the base material, and Polydimethylsiloxane (PDMS) materials were added to connect the inlet and outlet.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Optimization of structural design and condition selection\u003c/h2\u003e \u003cp\u003eTo achieve optimal structural design and condition control, computational fluid dynamics (CFD) simulations were conducted. Theoretical evaluations were conducted on the airflow control in the drywall cyclone gas-liquid separator of the PDC-sampler and the enrichment conditions of the microfluidic electrophoretic virus enrichment chip. Gradient simulations were conducted on the airflow velocity, collection inlet angle, inlet/outlet diameter ratio, outlet position, and sidewall tilt angle of the drywall cyclone gas-liquid separator (Supplementary Method 1), leading to the development of an optimal and controllable airflow structure design. To theoretically assess the enrichment efficiency of negatively charged viral particles in the microfluidic electrophoretic virus enrichment chip, simulations were conducted at voltages of 1 V, 3 V, 5 V, 10 V, 20 V, and 30 V, with sample flow rates of 200 \u0026micro;L/h, 400 \u0026micro;L/h, 600 \u0026micro;L/h, 800 \u0026micro;L/h, 1000 \u0026micro;L/h, and 1500 \u0026micro;L/h. A control group with 0 V was included for comparison (Supplementary Method 2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of virus samples\u003c/h2\u003e \u003cp\u003eIn the experimental tests, inactivated SARS-CoV-2 and H1N1 were selected as representative viruses. Their size and characteristics met the requirements for studying bioaerosols, and they posed no risk to laboratory personnel during testing. The inactivated SARS-CoV-2 virus samples were commercially purchased (Fubaiao Biomedical Technology Co Ltd., Jiangsu, China), with an initial liquid concentration of 4.32 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e copies/\u0026micro;L. The simulated EB SARS-CoV-2 virus concentrations were categorized into high concentration (3.60 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e copies/SEB and 3.60 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e copies/SEB), medium concentration (3.60 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e copies/SEB and 3.60 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e copies/SEB), and low concentration (3.60 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e copies/SEB and 3.60 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e copies/SEB).\u003c/p\u003e \u003cp\u003e InfA/H1N1 (PR8) was provided by the Key Laboratory of Jiangsu Preventive Veterinary Medicine (Yangzhou, China). The H1N1 virus was propagated following standard methods in accordance the World Health Organization guidelines for culturing in chicken embryo cells\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The liquid samples were centrifuged at 8000 revolutions per minute (RPM) for 2 min. The viral titer was quantified using a hemagglutination assay, yielding hemagglutination units (HAU), with an initial liquid concentration of 3.72 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e copies/\u0026micro;L. The simulated EB H1N1 virus concentrations were categorized as high concentration (3.10 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e copies/SEB and 3.10 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e copies/SEB), medium concentration (3.10 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e copies/SEB and 3.10 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e copies/SEB), and low concentration (3.10 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e copies/SEB and 3.10 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e copies/SEB). For all liquid sample preparations, 10-fold serial dilutions were performed using 1 \u0026times; PBS. 1 \u0026times; PBS was used as a negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization of simulated virus-containing EB\u003c/h2\u003e \u003cp\u003eEB was simulated using a bioaerosol generator, with 50 mL centrifuge tubes replacing sample vials. A fixed sample volume of 15 mL was used for each experiment, and the airflow rate was set to 10 L/min. To ensure that the particle size distribution and quantity of the simulated EB closely resembled those of adult EB, an Aerodynamic Particle Sizer Spectrometer (APS, TSI model 3321) was used for characterization. The results indicated that the droplets in the simulated EB were predominantly small particles, with more than 50% of the particles having a diameter of \u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e \u0026lt; 1.50 \u0026micro;m and more than 80% having a diameter of \u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e \u0026lt; 3.00 \u0026micro;m (Supplementary methods 3), consistent with the study by Pan et al.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Collection and enrichment of EB virus\u003c/h2\u003e \u003cp\u003eFirst, tests were conducted in the laboratory on the PDC-sampler and the microfluidic electrophoretic virus enrichment chip. A bioaerosol generator was used to simulate the EB of individuals infected with influenza and COVID-19. The PDC-sampler and a commercial bioaerosol sampler (ASE-200p, Shenzhen, China) were employed to collect EB viruses, and the collection efficiency was compared. The microfluidic electrophoretic virus enrichment chip was used to enrich the collected liquid virus samples, increasing the liquid sample concentration and enhancing the sensitivity of virus detection. All experiments were repeated three times. Between samplings, a nebulizer was used to aerosolize hypochlorous acid cleaning solution, ensuring that the cleaning solution was applied to the entire channel in the same manner as the EB collection. Detailed collection procedures are provided in Supplementary Method 4.\u003c/p\u003e \u003cp\u003eSubsequently, the performance of the PDC-sampler was tested in real-world applications. The device was used to collect EB from volunteers, including healthy individuals, COVID-19 patients, and H1N1 patients. The collection flow rate was adjusted to 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1 L/min, and the condensation zone temperature was lowered to -15\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. To meet the requirements for downstream RT-qPCR and ddPCR detection, the collecting time was set to 1 min per session, yielding approximately 170 \u0026micro;L of liquid sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eThe liquid samples were quantitatively analyzed by RT-qPCR. RNA was extracted from 50 \u0026micro;L of viral liquid samples using the AxyPrep Small Volume Kit for Body Fluid Virus DNA/RNA (AxyPrep), eluted with 30 \u0026micro;L of Buffer TE, and 1 \u0026micro;L of RNA was prepared with TaqMan\u0026reg; Fast Virus 1-Step Master Mix (ThermoFisher) assay reagent in a total amount of 20 \u0026micro;L of the reaction system, and the reaction was thermally cycled in a real-time fluorescence quantitative PCR instrument (QuantStudio 3, ThermoFisher). The primers and probes used for detection were designed using Primer-BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and synthesized by Shanghai Bioengineering Corporation (Shanghai China), and the validity of the primers was confirmed by qPCR analysis of the solvent curves. The comparative cycle threshold (Ct) value method was used to calculate mRNA levels to determine the amount of virus collected and enriched. For more detailed relevant details, see Supplementary Methods 5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Droplet digital PCR (ddPCR)\u003c/h2\u003e \u003cp\u003eThe low viral concentration EB collection liquid samples were subjected to ddPCR assay at DynaTech Biotech (Wuhan, China), using the same primers and probes as those employed in the RT-qPCR assay. See Supplementary Methods 6 for specific detailed assay details. The results of the assay were meticulously analyzed with SightPro software, which set thresholds between positive and negative droplets, and confirmed by manual inspection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD, n\u0026thinsp;\u0026ge;\u0026thinsp;3) and plotted using Origin 9 (Origin 9 Software, USA). Statistical analysis was conducted using the One-Way Analysis of Variance in SPSS software, considering p-values less than 0.05 as significant. Asterisks in the figures indicate statistical differences: \u0026ldquo;*\u0026rdquo; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026ldquo;**\u0026rdquo; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u0026ldquo;***\u0026rdquo; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u0026ldquo;****\u0026rdquo; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, and p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 indicating no statistical significance (N.S.).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Determination of EB virus collection and enrichment conditions\u003c/h2\u003e \u003cp\u003eTo achieve optimal EB collection and enrichment efficiency, the best conditions for the PDC-sampler were tested and analyzed. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea showed that the EB-to-liquid conversion efficiency was approximately 60% when the collection flow rate was 24 L/min, 26 L/min, or 28 L/min, and the collection distance was 1 mm (close but not touching). However, a significant decrease in conversion efficiency was observed when the collection flow rate was reduced to 22 L/min or increased to 30 L/min, as well as when the collection distance was increased. Detailed procedures are provided in Supplementary Methods 7. The actual collection efficiency of the PDC-sampler was tested using standardized PSL microspheres. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb showed that the collection efficiency for 0.5 \u0026micro;m aerosol particles exceeded 60%, while it approached 100% for aerosol particles with a diameter of 2 \u0026micro;m. Detailed procedures are provided in Supplementary Methods 8.\u003c/p\u003e \u003cp\u003eNext, the enrichment conditions for the microfluidic electrophoretic virus enrichment chip were tested using H1N1 virus with a concentration of 3.72 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e copies/\u0026micro;L. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec showed that at a sample flow rate of 200 \u0026micro;L/h, the enrichment groups showed significantly higher values than the unenriched groups at gradient voltages ranging from 1 to 30 V. The enrichment efficiency increased with voltage, reaching the optimal level at 30 V. No signal was detected in the 0 V enrichment group or the negative control group (1 \u0026times; PBS). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed showed that at 30 V, with sample flow rates ranging from 200 \u0026micro;L/h to 1000 \u0026micro;L/h, the enrichment groups remained significantly higher than the unenriched groups, although enrichment efficiency decreased as sample flow rate increased. No signal was detected in the negative control group (1 \u0026times; PBS). Detailed concentration processes are provided in Supplementary Methods 9.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Performance of the PDC-sampler for EB virus collection\u003c/h2\u003e \u003cp\u003eTo evaluate the performance of the PDC-sampler for collecting EB viruses, a bioaerosol generator was used in the laboratory to simulate EB containing varying concentrations of viruses. EB virus was collected using the PDC-sampler and a bioaerosol sampler, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the collected liquid samples were analyzed using RT-qPCR. The results showed that when using the PDC-sampler to collect EB containing SARS-CoV-2 or H1N1 viruses, the mean Ct values from the high-concentration to the low-concentration groups were: 23.93, 26.77, 29.14, 34.02, 36.24 for SARS-CoV-2, and 25.62, 27.02, 30.98, 35.25, 36.58 for H1N1, respectively. In contrast, other liquid samples had viral concentrations below the detection limit. When the bioaerosol sampler was used to collect EB containing SARS-CoV-2 or H1N1 viruses, the mean Ct values from the high-concentration to the low-concentration groups were 29.79, 33.16, 35.05 for SARS-CoV-2, and 33.98 for H1N1. Other liquid samples had viral concentrations below the detection limit. Statistical analysis revealed that the virus concentrations in liquid samples collected using the PDC-sampler were significantly higher than those collected using the bioaerosol liquid sampler. However, in the low-concentration groups for SARS-CoV-2 and H1N1 viruses, the virus concentrations in the collected liquid samples were below the RT-qPCR detection limit or showed weak positive results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Virus concentration enhanced by microfluidic electrophoretic virus enrichment chip\u003c/h2\u003e \u003cp\u003eTo evaluate the effectiveness of the PDC-sampler in collecting and enriching low-concentration EB viruses, the microfluidic electrophoretic virus enrichment chip was combined with the PDC-sampler. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, the chip was used to enrich liquid samples containing H1N1 virus from EB, further increasing the liquid sample concentration for detection using the gold-standard RT-qPCR. The results indicated that in the high-virus-concentration group, the Ct values showed no significant enrichment effect compared to the unenriched group. However, as the concentration decreased, the enrichment effect became increasingly pronounced. In the medium-concentration group, statistical analysis demonstrated that the virus concentrations in the enriched group were significantly higher than those in the unenriched group. Notably, as the dilution factor increased further, and the virus concentration decreased to the low-concentration group, the unenriched group were below the RT-qPCR detection limit. In contrast, after enrichment using the chip, liquid sample concentrations were raised above the detection limit, enabling effective detection. No signal was detected in the negative control group (1 \u0026times; PBS). These results demonstrated that the combination of the PDC-sampler and the microfluidic electrophoretic virus enrichment chip reduced the detectable concentration of EB viruses to approximately 31 copies/SEB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Detection limit of PDC-sampler reduced by ddPCR\u003c/h2\u003e \u003cp\u003eTo enhance the sensitivity and accuracy of detecting EB with low virus concentrations, the advanced and highly sensitive ddPCR technology was employed to analyze both the liquid virus samples before EB virus generation and the low-concentration liquid samples collected by the PDC-sampler. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, positive droplets were detected in both the liquid virus samples before EB virus generation and the liquid virus samples collected by the PDC-sampler for low-concentration groups containing SARS-CoV-2 (5 \u0026times; 10\u003csup\u003e0\u003c/sup\u003e \u0026minus;\u0026thinsp;8.20 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e copies/SEB) or H1N1 virus (8.65 \u0026times; 10\u003csup\u003e0\u003c/sup\u003e \u0026minus;\u0026thinsp;2.19 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e copies/SEB). As the dilution factor increased, liquid samples with concentrations below the ddPCR detection limit showed negative droplets. Negative droplets were detected in the negative control group (1 \u0026times; PBS). These results demonstrated that combining the PDC-sampler with ddPCR technology reduced the EB virus detection limit to approximately 5\u0026ndash;9 copies/SEB. Notably, low-concentration EB viruses collection samples that were negative by RT-qPCR were detected as positive using ddPCR, confirming that the PDC-sampler effectively collected low-concentration EB viruses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Real-world testing of PDC-sampler\u003c/h2\u003e \u003cp\u003eTo validate the real-world application of the PDC-sampler, respiratory infection patients (with symptoms such as fever, and muscle aches) were recruited as volunteers. Throat swab and EB virus liquid samples were collected and analyzed using RT-qPCR, with healthy volunteers serving as negative controls. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, SARS-CoV-2 was detected in both the throat swab and EB liquid samples collected from Case 1 on the first day, with mean Ct values of 32.45 and 32.22, respectively, showing no statistical difference. On the second and third days, the viral concentrations in the liquid samples were below the detection limit. Throat swab and EB samples collected from this volunteer after recovery were used as negative controls. Fig. S9 showed the results of COVID-19 antigen tested using throat swabs from Case 1, which were consistent with the RT-qPCR results. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, H1N1 virus was detected in both Case 2 and Case 3, with mean Ct values of 28.96 and 27.94 for throat swab samples, and 27.98 and 28.49 for EB liquid samples, respectively. The EB liquid sample concentration from Case 2 was significantly higher than that of the throat swab, while no statistical difference was observed for Case 3. In Cases 4 and 5, the throat swab samples tested negative, while EB liquid samples were weakly positive. Healthy volunteers served as negative controls. These findings indicated that the PDC-sampler's performance in collecting and detecting EB viruses was comparable to or better than throat swab sampling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 PDC-sampler: toward real-world deployment\u003c/h2\u003e \u003cp\u003eThis study demonstrated an automated and rapid PDC-sampler designed to collect viruses from human EB, efficiently transferring them into a liquid medium to provide higher-concentration liquid samples for downstream detection. To evaluate the performance of the PDC-sampler in real-world applications, it was deployed in a hospital fever clinic with a collection time set to 1 min (~\u0026thinsp;20 SEB). Liquid EB samples collected from five volunteers infected with either SARS-CoV-2 or H1N1 showed that the viral concentrations in liquid samples collected using the PDC-sampler were comparable to or higher than those in throat swab samples from the same individuals. However, when the PDC-sampler was combined with the microfluidic electrophoretic virus enrichment chip or ddPCR, the detection limit for EB viruses was further reduced to approximately 5\u0026ndash;9 copies/SEB.\u003c/p\u003e \u003cp\u003eBased on these findings, the real-world deployment of the PDC-sampler is envisioned as follows: First, during large-scale outbreaks, such as COVID-19, the PDC-sampler could rapidly and automatically collect liquid samples of EB viruses from infected individuals. This would reduce direct contact between healthcare workers and patients, minimizing unnecessary infection. Second, in hospital settings, such as respiratory outpatient clinics, pediatric emergency rooms, and ICUs, the PDC-sampler could automate the collection of respiratory viruses from patients with diseases like pneumonia or from difficult-to-sample populations such as infants. This would provide a non-contact, automated, and safe method for diagnosing respiratory infectious diseases. Third, in the diagnosis of specific respiratory infectious diseases such as COVID-19 or pneumonia, deploying the PDC-sampler in combination with the microfluidic electrophoretic virus enrichment chip or ddPCR could advance the diagnosis timeline to before EB virus concentrations reach levels capable of transmitting to others, enabling management personnel to implement timely isolation and other preventive measures to effectively halt the rapid spread of infectious diseases.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study demonstrated a PDC-sampler specifically designed to collect viruses from human EB and transfer them into a liquid medium, providing liquid samples for downstream detection. To enhance the accuracy and sensitivity of detecting low-concentration EB viruses, the combination of the PDC-sampler with either the low-cost microfluidic electrophoretic virus enrichment chip or the high-cost ddPCR technology was proposed. The results revealed that the PDC-sampler possesses key advantages, including rapidity (SEB), non-invasiveness, high sensitivity, automation, and convenience, offering an innovative solution for the early diagnosis and monitoring of respiratory infectious diseases.\u003c/p\u003e \u003cp\u003eFirstly, the structural design of the PDC-sampler demonstrated significant advantages in collecting small aerosol particles. Small aerosol particles are considered the primary transmission medium for high-concentration viruses\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, yet traditional samplers, such as wet-wall cyclones, have limited efficiency in capturing these small particles\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This study showed that the PDC-sampler effectively captured and enriched small aerosol particles by rapidly combining them with microdroplets formed through condensation, followed by separation using a drywall cyclone gas-liquid separation structure. This design avoided the substantial dilution caused by the use of sampling liquid solution in traditional methods\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and minimized the contamination from excessive saliva often collected by direct condensation-based samplers\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Furthermore, SEB yielded 8\u0026ndash;10 \u0026micro;L of liquid sample with a high viral concentration. The collection efficiency for 0.5 \u0026micro;m aerosol particles exceeded 60%, while it approached 100% for aerosol particles with a diameter of 2 \u0026micro;m. These advantages make the PDC-sampler particularly suitable for studying and monitoring high-risk respiratory viruses, such as SARS-CoV-2 and H1N1, which are primarily transmitted through aerosols. The enhanced ability to collect small particles not only improved collection sensitivity but also provided opportunities to better understand virus transmission mechanisms.\u003c/p\u003e \u003cp\u003eSecondly, the PDC-sampler demonstrated high efficiency of EB virus collection in experimental tests. When combined with the gold-standard RT-qPCR detection, the PDC-sampler successfully detected the minimum EB virus concentrations reported during the confirmed infectious period of SARS-CoV-2-infected individuals (~\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e copies/SEB)\u003csup\u003e11,12\u003c/sup\u003e. This indicated that the PDC-sampler could provide effective liquid samples for detection during the critical phase of confirmed infections. Furthermore, when combined with microfluidic electrophoretic virus enrichment chip or ddPCR detection, the sensitivity for detecting EB viruses was significantly improved (5\u0026ndash;9 copies/SEB), enabling detection of the minimum infectious concentration of viruses such as SARS-CoV-2 (10\u003csup\u003e2\u003c/sup\u003e copies/SEB)\u003csup\u003e13\u003c/sup\u003e. This demonstrated that the PDC-sampler performed well in collecting low-concentration EB viruses and, when combined with advanced detection technologies, could advance the diagnosis timeline to before the virus concentration reaches transmissible levels. This capability provided critical time for public health decisions, facilitating timely isolation and contact tracing measures to interrupt transmission chains effectively.\u003c/p\u003e \u003cp\u003eFinally, this study evaluated the potential value of the PDC-sampler in real-world applications. In real-world scenarios, the collection of EB viruses from SARS-CoV-2 or H1N1 infected individuals showed performance comparable to or better than throat swab samples. Its non-contact, automated collection approach not only reduced direct contact between healthcare workers and infected individuals, lowering infection risks, but also enabled deployment in scenarios such as large-scale pandemic outbreaks, hospital respiratory wards, pediatric units, and ICUs. The automation of the PDC-sampler significantly improved collection efficiency and safety, especially in high-risk environments or for challenging groups like infants.\u003c/p\u003e \u003cp\u003eDespite the notable advantages of the PDC-sampler demonstrated in this study, some limitations remain. Firstly, the actual collection efficiency might have been influenced by environmental factors such as humidity and temperature, necessitating more clinical samples to test its stability under varying conditions. Secondly, the compatibility between the microfluidic electrophoretic virus enrichment chip and the PDC-sampler requires further improvement, as the enrichment speed currently cannot fully match the collection rate while maintaining high enrichment efficiency. This limited the realization of a combined collection-enrichment system. Future research will focus on optimizing the device, validating its performance in real-world applications, and expanding its capability for detecting multiple viruses. The widespread application of the PDC-sampler is expected to drive innovation in respiratory infectious disease diagnostics and support the development of global public health systems.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eA novel PDC-sampler was designed and optimized in this study for efficient collection of EB viruses. The integration of condensation and drywall cyclone gas-liquid separation structure was optimized using airflow control technology, enabling automation SEB virus collection (8-10 μL) without the need for a sampling liquid solution. Combined with RT-qPCR, the PDC-sampler achieved routine positive detection in infected individuals. When combined with the low-cost microfluidic electrophoretic virus enrichment chip or the high-cost ddPCR, the detection limit was reduced to 5-9 copies/SEB, significantly lower than the minimum EB virus concentration reported in confirmed patients (10\u003csup\u003e3\u003c/sup\u003e copies/SEB). Validation with SARS-CoV-2 and H1N1 infected volunteers demonstrated that the PDC-sampler's performance matched or exceeded throat swabs, confirming its effectiveness in real-world applications. The non-invasive, automated design and high sensitivity of the PDC-sampler highlight its potential for deployment in hospitals, public health monitoring, and pandemic responses. This study provides a rapid and efficient solution for the diagnosis and monitoring of respiratory viruses, opening new possibilities for infectious disease control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics declaration\u003c/p\u003e\n\u003cp\u003eThe experiments involving the collection and testing of throat swabs and EB liquid samples from volunteers were conducted in real-world settings with approval from the Ethics Committee of Clinical Research at Southeast University Zhongda Hospital (Project Number: 2024ZDSYLL210-P01). With assistance from hospital staff, throat swab samples were collected using commercial COVID-19 antigen test kits, and EB liquid samples were collected using the PDC-sampler in the respiratory outpatient clinic. Antigen tests were performed on-site, and liquid samples were inactivated before being transported to the laboratory for RT-qPCR analysis. Notably, all volunteers who provided samples signed informed consent forms, agreeing to the collection of throat swabs and EB samples.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe source data are provided as a \u0026ldquo;Source Data\u0026rdquo; file. Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003eCredit author statement\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWeihao Li\u003c/strong\u003e designed the study, conducted the experiments and assay and wrote the manuscript. \u003cstrong\u003eHan Gao\u003c/strong\u003e assisted with some of the fluid dynamics simulations.\u0026nbsp;\u003cstrong\u003eMengqing Cheng\u003c/strong\u003e and \u003cstrong\u003eChaoyi Yin\u003c/strong\u003e assisted in designing part of the experiments and revising the article. \u003cstrong\u003eManman Lv\u003c/strong\u003e, \u003cstrong\u003eYemin Han\u003c/strong\u003e, \u003cstrong\u003eHaotian Yu\u003c/strong\u003e, and \u003cstrong\u003eWeiming Lin\u003c/strong\u003e assisted with some of the experiments. \u003cstrong\u003eYan Huang\u003c/strong\u003e, \u003cstrong\u003eQiang Zhang\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Dianhuai Meng\u003c/strong\u003e, \u003cstrong\u003eTian Weng\u003c/strong\u003e, \u003cstrong\u003eZuhong Lu\u003c/strong\u003e conducted writing-review and editing. \u003cstrong\u003eQuanjun Liu\u003c/strong\u003e guide the project design and supervised its progress.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 31872726, 61827814), the Shenzhen Fundamental Research Program (award number: JCYJ20230807114612024 and JCYJ20220530160416036), the Key Research and Development Project of Jiangsu Province (BE2022804 and BE2021012-4).\u003cbr\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHu Li\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Wireless, battery-free, multifunctional integrated bioelectronics for respiratory pathogens monitoring and severity evaluation. \u003cem\u003eNat. 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Aerosol Sci.\u003c/em\u003e \u003cstrong\u003e152\u003c/strong\u003e, 105693 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"exhaled breath, respiratory viruses, condensing drywall cyclone sampler, microfluidic electrophoretic virus enrichment chip, droplet digital PCR","lastPublishedDoi":"10.21203/rs.3.rs-5708899/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5708899/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExhaled breath (EB), as a non-invasive biological sample, has garnered attention for diagnosis and monitoring respiratory diseases. However, efficiently collecting EB viruses for downstream detection remains a key challenge. This study introduced a novel Phase-change Drywall Cyclone Sampler (PDC-sampler), which was designed and optimized through airflow control to integrate condensation with drywall cyclone gas-liquid separation. The PDC-sampler efficiently collects viral particles from EB (8-10 μL per single EB (SEB)) without requiring a sampling liquid solution. When combined with the gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR), it enabled routine positive detection in infected individuals (≥ 10\u003csup\u003e3\u003c/sup\u003e copies/SEB). By coupling with the low-cost microfluidic electrophoretic virus enrichment chip or the high-cost and highly sensitive droplet digital PCR (ddPCR), the detection limit of EB viruses was reduced to 5-9 copies/SEB, which is an order of magnitude lower than the minimum viral concentration required for infection (~ 10\u003csup\u003e2\u003c/sup\u003e copies/SEB). Notably, tests on coronavirus disease 2019 (COVID-19) and influenza-infected volunteers demonstrated performance comparable to or better than throat swabs, validating the PDC-sampler’s effectiveness in real-world applications. This study provides an efficient, automated, and non-invasive solution for the early diagnosis, transmission mechanism study, and large-scale screening and monitoring of respiratory diseases.\u003c/p\u003e","manuscriptTitle":"High-efficiency monitoring of respiratory viruses in a single exhaled breath","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-31 04:54:32","doi":"10.21203/rs.3.rs-5708899/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd55c94f-a143-45c5-98bd-e3149edeb01e","owner":[],"postedDate":"December 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42174644,"name":"Biological sciences/Microbiology/Infectious-disease diagnostics"},{"id":42174645,"name":"Health sciences/Diseases/Respiratory tract diseases/Respiratory distress syndrome"},{"id":42174646,"name":"Health sciences/Medical research/Epidemiology"},{"id":42174647,"name":"Health sciences/Diseases/Infectious diseases/Influenza virus"}],"tags":[],"updatedAt":"2026-03-09T18:09:16+00:00","versionOfRecord":{"articleIdentity":"rs-5708899","link":"https://doi.org/10.1021/acs.analchem.5c06572","journal":{"identity":"analytical-chemistry","isVorOnly":true,"title":"Analytical Chemistry"},"publishedOn":"2026-03-03 00:00:00","publishedOnDateReadable":"March 3rd, 2026"},"versionCreatedAt":"2024-12-31 04:54:32","video":"","vorDoi":"10.1021/acs.analchem.5c06572","vorDoiUrl":"https://doi.org/10.1021/acs.analchem.5c06572","workflowStages":[]},"version":"v1","identity":"rs-5708899","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5708899","identity":"rs-5708899","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}