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Highly enriched, controllable, continuous aerosol sampling using inertial microfluidics and its application to real-time detection of airborne bacteria Jeongan Choi, Seung Chan Hong, Woojin Kim, and Jaehee Jung ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00753 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 25, 2017

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Highly enriched, controllable, continuous aerosol sampling using inertial microfluidics and its application to real-time detection of airborne bacteria Jeongan Choi†, 1,3Seung Chan Hong†, 4Woojin Kim*, and 1Jae Hee Jung**

1,2

1

Center for Environment, Health, and Welfare Research, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea 2

Department of Mechanical Science and Engineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States

3

Department of Mechanical and Aerospace Engineering, Seoul National University, Seoul 08826, Republic of Korea

4

Technology Convergence R&BD Group, Korea Institute of Industrial Technology, Daegu 42994, Republic of Korea

ABSTRACT: We report a novel microfluidic technique for sampling of aerosols into liquids. The two-phase fluid, sampling air and collecting liquid, forms a stratified flow in the curved microchannel. By passing fluids through the curved region, the particles are transferred from air into the liquid phase by the particle centrifugal and drag forces. This microfluidic-based aerosol-into-liquid sampling system, called the MicroSampler, is driven by particle inertial differences. To evaluate the physical particle collection efficiency of the MicroSampler, we used standard polystyrenelatex (PSL) particles ranging in size from 0.6 to 2.1 µm and measured particle concentrations upstream and downstream of the MicroSampler with an aerodynamic particle sizer. The cut-off diameter of particle collection was selected controlling the air flow velocity (microfluidic air flow of 0.6 L/min showed a particle collection efficiency of ~98% at a particle diameter of 1 µm), and continuous enriched particle sampling was possible for real-time post-processing application.

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With regard to biological collection efficiency, the MicroSampler showed superior microbial recovery (Staphylococcus epidermidis) compared to the conventional BioSampler technique. These results indicate that our MicroSampler can be used as a portable, cost-effective, simple and continuous airborne microorganism collector for applications in real-time bioaerosol detection. Keywords: Bioaerosol, inertial microfluidics, bacteria, continuous sampling, two-phase flow

The management of indoor air quality (IAQ) is a significant issue, and there is much research interest in the presence and detection of bioaerosols, which are airborne particulate matter of biological origin, in relation to their adverse health effects.1-2 In particular, bioaerosols, such as pathogenic viruses, bacteria, and fungal spores, have been a major focus of attention due to their hazardous effects on human health, including allergies, infectious diseases, asthma and pneumonia.3-5 There have been a number of recent outbreaks of Middle East respiratory syndrome coronavirus (MERS) and severe acute respiratory syndrome (SARS) in the Middle East and Asia, which are believed to be transmitted by aerosol.6-7 Not only epidemic viruses but toxic bacteria, such as Bacillus anthracis, can cause adverse health effects with high morbidity. The Centers for Disease Control (CDC) reported that the inhalation LD50 (median lethal dose) of B. anthracis is ~ 100 ng.8 High concentrations of toxic bioaerosols can have detrimental effects on human health, because microorganisms can travel freely through air and spread quickly over a wide area. Therefore, for active disease control and to minimize bioaerosol exposure risk, there is a requirement for effective bioaerosol monitoring systems, including continuous bioaerosol sampling and rapid analysis.9-11 Bioaerosol sampling is based on the same principles as those for non-biological aerosols (i.e., gravitational sedimentation, inertial impaction, centrifugation, filtering, and electrical or thermal precipitation).12 A fundamental requirement for microbial sampling is that the sampling technique should have high efficiency for both collection and microbial recovery.13 However, as available devices differ in a number of operating parameters and environments (i.e., temperature, humidity, sampling time, and flow rate), airborne microorganisms are exposed to various physical and metabolic stresses during the sampling process that can affect the viability and culturability of cells, as well as possibly affecting the integrity of their genomic material (e.g., DNA).14

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Two main types of bioaerosol sampling method have been studied: (i) bioaerosol collection onto a solid substrate or filter medium and (ii) direct sampling into liquid collection media. In the first types of bioaerosol sampling technique, the collected samples are extracted and resuspended using an appropriate solution (e.g., pure water or phosphate-buffered saline) and then analyzed. However, some of the bioaerosols may rebound from the collecting surface (in the case of inertial impactors). If the amount of sample overloads the collector, incomming aerosol particles may bounce off the collected particles. Desiccation of the collecting medium surface may occur during sampling, which would reduce the microbial recovery of microorganisms. In the second group with use of a liquid collection medium for bioaerosol sampling, the aerosol is collected directly into liquid medium for subsequent biochemical analysis. The wet-cyclone and BioSampler (or impinger) are well-known liquid-based bioaerosol samplers.14-16 This method is useful for sampling airborne viruses and bacteria which require gentle handling and for the recovery of soluble materials (e.g., mycotoxins, antigens, endotoxins). In particular, these methods can eliminate the additional sample extraction procedures required in the first group of bioaerosol sampling methods. However, current liquid-based bioaerosol sampling techniques14-17 are not well-suited for use with other cutting-edge techniques for real-time, continuous bioaerosol monitoring. It is therefore important to develop a bioaerosol sampling system that is capable of delivering collected airborne samples to a sensor device in real-time,18 and there is also increasing interest in the development of continuous and enriched aerosol-to-liquid sampling techniques. As an example, the microfluidic electrostatic sampler described by Ma et al.18 provides continuous sampling to deliver the collected sample to other sensing devices. However, these systems are still complex and require an additional particle charging system, such as a high-voltage source. As a simple detection method, Kang et al. and Liu et al. presented real-time airborne microorganism detection using microfluidic enrichment systems.19-21 However, it is still difficult to maintain cell viability and integrate with other detection systems, such as PCR and biochemical analyses. Hong et al. examined separation of airborne particles by their differences in inertia using a micro-size device, and reported efficient size separation of aerosols and bioaerosols in real-time. However, the separator did not perform aerosol collection and therefore could not be used as an aerosol sampler.6 Here, we present a novel bioaerosol sampling technique based on inertial microfluidics with two-phase continuous flow. This two-phase fluid, sampling air and collecting liquid, stably forms a stratified flow in the simple curved-microfluidic channel composed of one curve with an

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angle of 180°, two inlets, and two outlets in a single microchip. The collecting liquid covers the outer wall of the channel during bioaerosol sampling. For collection, the particles are transferred from air to the liquid phase by centrifugal and drag forces by passing fluids through the curved region. This microfluidic-based aerosol-into-liquid sampling system, called the MicroSampler, is driven by particle inertial differences. The operating performance of MicroSampler was evaluated using standard polystyrene-latex (PSL) particles and a real-time particle analyzer. For application to the bacterial bioaerosol sampling (Staphylococcus epidermidis), the various characteristics of the MicroSampler system with regard to physical collection capability and microbial recovery (i.e., culturability) were investigated in comparison with conventional bioaerosol samplers, such as gelatin filters and BioSampler.22 The MicroSampler makes it possible to perform size-selective particle sampling with high collection efficiency and rapid recognition with a low air sample volume. Our results indicate that the MicroSampler system represents significant progress in the development of a simple, inexpensive, portable and continuous sample collector for real-time bioaerosol detection.

THEORY AND EXPERIMENTAL A gas-liquid stratified flow was introduced into the curved microchannel for bioaerosol sampling. This two-fluid flow occurs mainly in hydrophobic microchannels via moderation of the gas and liquid flow rates.23 The working principle of the MicroSampler can be explained by simple aerodynamics. Figure 1(a) shows schematics of particle migration due to the inertial force in the curved channel. While traveling around the curved channel, particles are gradually pushed outward radially due to a centrifugal force with velocity, Vr, perpendicular to the tangential velocity, Ua (Fig. 1(b)). The particle’s radial travel distance from the original streamline, L, can be expressed as6, 24 =

( ),

(1)

where W, θ, and Stk indicate the channel width, channel curve angle, and Stokes number, respectively. The Stokes number, which indicates the inertial strength of airborne particles when encountering an obstacle, is defined as25 =

,

(2)

where ρp and dp indicate the density and diameter of the particle, and Ua and µa are the velocity and viscosity of air, respectively. Cc represents the Cunningham slip correction factor, defined as 4


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= 1 + 0.5 [2.34 + 1.05 exp'−0.195 *],

(3)

where Knp, defined as 2λ/dp, is the Knudsen number of the particle, and λ represents the mean free path of the working fluid.25 Because the particle’s radial travel distance is proportional to the Stokes number (Eq. 1), larger particles travel farther in the radial direction, and are finally collected by the liquid flow. Figure 1(c) shows a schematic of the Dean flow generated in a typical curved channel, as well as the resulting particle trajectory. The schematic is a numerical simulation of air flow in a curved channel using commercial computational fluid dynamics software (CFD-ACE; ESI US R&D Inc., Huntsville, AL, USA). The air flow velocity at the center of the curved channel is faster than that near the wall due to the no-slip condition of the fluid. This velocity difference results in a pressure gradient between the center and wall of the channel, generating the Dean flow.19, 26 Thus, the movement of particles radially outward increases toward the center of the channel, whereas the movement of particles at the top and bottom of the channel is disturbed because the particles move in the opposite direction of the Dean flow. Furthermore, if the intensities of the drag forces (FD) due to the Dean flow are stronger than the centrifugal force (FC), particles at the top and bottom of the channel also show inward radial movement. Therefore, excessively strong Dean flow prevents the efficient collection of airborne particles in a curved channel, and the intensity of the Dean flow should be adjusted to lower levels for more efficient particle collection.6 The strength of the Dean flow can be represented using a dimensionless Dean number, De, which is a function of the Reynolds number, Re, and the radius of curvature for a curved channel, R.27 0

,- = .-/ 1 , 2

.- =

01 ,

(4) (5)

Here, ρa is the density of air and Dh is the hydraulic diameter of the channel, defined as 2WH/(W+H). The strength of the Dean flow is also influenced by the channel aspect ratio, which is the ratio of the height and width of the channel (H/W).19 As the Reynolds number decreases, collection efficiency is reduced due to a decrease in the radial displacement of the particles. The collection efficiency is also affected by the Dean vortices.28 Weaker Dean vortices are preferable because strong Dean vortices cause particle deposition at the top and bottom walls of the channel, rather than on the liquid film.19 As the channel aspect ratio decreases, the intensity of the Dean flow also decreases. Therefore, particle movement due to Dean flow can be controlled by modifying the Dean number and channel aspect ratio. 5


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The MicroSampler system developed in this study is based on a simple curvedmicrofluidic platform fabricated from a single-layered polydimethylsiloxane (PDMS) channel using soft lithography process29 with two inlets (for aerosol gas and collecting liquid) and two outlets for separating the collecting liquid and gas flows after particle sampling. For weak Dean vortices, as mentioned above, the MicroSampler has a channel size of 1,000 µm (W) × 100 µm (H) (i.e., aspect ratio of 0.1) and the curved channel region has a radius angle of 180° and radius curvature of 5,300 µm (Fig. 1(d)). Figure 2 shows a schematic diagram of the experimental setup for performance evaluation of the MicroSampler in airborne particle sampling into liquid. To generate test aerosols (i.e., PSL, fluorescent PSL, or bacterial particles), filtered dehydrated air from a diffusion dryer and HEPA filter entered a one-jet Collison nebulizer at a rate of 1 L/min. The particles were passed through a diffusion dryer and

210

Po neutralizer, sequentially to remove

moisture and electrical charge from the aerosols before injection into the MicroSampler. The flow rate of sampling gas was set from 0.2 (Re = 381, De = 51.4) to 0.6 L/min (Re = 1,144, De = 154.2) with a mass flow controller (FC-280S; Mykrolis Corp., Billerica, MA, USA). In all experiments, the air flow rates of both inlet and outlet parts of the MicroSampler were checked using a mass flow meter (Mass flowmeter 4100; TSI Inc., Shoreview, MN, USA). Sterilized distilled water (SDW) was used as the collecting liquid for particle sampling. Using a syringe pump (KDS200; KD Scientific Inc., Holliston, MA, USA), the collecting liquid was injected into the inlet of the MicroSampler at a flow rate of 0.1 – 0.3 mL/min and formed a liquid film in the microchannel by centrifugal force covering the outer wall of the channel. To evaluate the physical collection efficiency, the particle number concentrations at the inlet and outlet were measured with an aerodynamic particle sizer (APS 3321; TSI Inc., Shoreview, MN, USA). The microscopy counting method was also used to enumerate sampled particles in the collecting liquid. As an application of the MicroSampler to airborne microorganism sampling, we evaluated the physical and biological collection efficiency of S. epidermidis. The morphology of collected bacteria was obtained by scanning electron microscopy (Nova Nano SEM 200; FEI Co., Hillsboro, OR, USA). For microbial recovery of sampled bacterial particles, we investigated the bacterial culturability as quantified by the colony counting method in comparison with conventional

bioaerosol

sampling

methods,

i.e.,

gelatin

filters

and

BioSampler

(http://www.skcinc.com). In the experiments, a gelatin filter and BioSampler were installed in parallel with the MicroSampler. The air samples from the Collison nebulizer were collected simultaneously by the three sampling systems.

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RESULTS AND DISCUSSION Two-phase flow in a curved microchannel Surface tension is a predominant force in microfluidics and significantly reduces slip velocity. It renders gas-liquid flow independent of gravity and achieves stratified flow, in contrast to the characteristics of large dimension channels.30 In this experiment, gas-liquid stratified flow in the microchannel was formed for sampling bioaerosols into the collecting liquid. To obtain stratified flow conditions, this two-phase flow was controlled precisely by adjusting the gas and liquid flow rates. Figure 3(a-1) shows the operating flow regime of the MicroSampler, distinguished as sprayed and stratified regions in terms of flow rates of air and water in the microchannel. In this study, we adjusted the air and water flow rates from 0.1 to 0.6 L/min and 0.1 to 0.5 mL/min, respectively; the gas to liquid ratio of flow rate is on the order of 103. For dimensionless analysis in stratified and sprayed regimes, the ratio of the velocities of air and water (Ua/Uw) and the modified Weber number (We) are introduced in Figure 3(a-2). The modified Weber number, We, is defined as We =

3 43 , 53

(6)

where 67 is the water density, 7 is the thickness of the water film, and 87 is the surface tension of the water. The two regimes, as represented by non-dimensional parameters, are shown in Figure 3(a-2). To visualize the behavior of two-phase flow in the microchannel, the dye-stained SDW was injected into the inlet and imaged under a microscope. In the stratified microfluidic region, the water flow formed a thin film along the channel wall, as shown in Figure 3(b-1). Airborne particles flowed along the air stream and were forced radially outward under the particle centrifugal and drag forces by passing fluids in the curved region, then finally collected to the water flow. Particle transfer from the air layer to the water layer is governed by the interplay of centrifugal force, drag force, and surface tension force at the air-water interface. The net resultant force from centrifugal and drag forces should overcome the surface tension force for particle migration across the interface.31 However, in the sprayed flow region (Fig. 3(b-2)), the water was separated from the surface and sprayed into the surrounding air stream. The water separated from the surface when the We exceeded some value. The surface tension of the liquid acts against the formation of small droplets generated by the interfacial pressure fluctuations of the air stream, and passing into the atomization regime.32-33 In this case, the

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volume of collecting liquid covering the outer wall can be reduced by spraying fluid. This sprayed flow condition results in unstable flow on the liquid surface, which interrupts particle capture. This phase-change is introduced in Movie S1 in the Supplemental Information. In this study, all experiments were performed in the stratified microfluidic region. This liquid flow can not only act as a continuously moving collection medium for airborne particles, but can also reduce the particle rebound effect. Thus, the MicroSampler system showed superior particle collection efficiency and greater microbial recovery of biological samples compared to conventional methods. Physical collection efficiency of the MicroSampler The collection efficiency (η) of standard-sized PSL particles is calculated using the following equation: η (%) = (1– Nout/Nin) × 100,

(7)

where Nin and Nout are the particle number concentrations at the inlet and outlet, respectively. The collection efficiency curves of PSL particles with various air flow rates (0.2 – 0.6 L/min) are shown in Figure 4(a). If this curve is extremely sharp, like a step-function curve, all particles above a certain cut-off diameter (d50) will be collected and all particles below this size will pass through. In practical application, the cut-off diameter is that which gives 50% collection efficiency of particles from the actual particle collection curve.25 In this study, the MicroSampler cut-off diameter was calculated as ~ 0.8 µm at an air flow rate of 0.2 L/min in Figure 4(a). The particle collection efficiency over the entire size range increases with air flow rate because the radial displacement (L) increases with centrifugal force (Eqs. 1 and 2). In particular, size-selective particle collection is possible according to the target particle size as the cut-off diameter can be shifted with air flow rate. For application of the MicroSampler system to bacterial bioaerosol sampling (i.e., S. epidermidis; single cell size of ~0.84 µm), we set the microfluidic operating conditions at an air flow rate of 0.6 L/min and liquid flow rate of 0.3 mL/min, which showed > 80% collection efficiency for particle diameters > 0.65 µm (Fig. 4(b)). Collection efficiency curves for general inertial particle samplers are often plotted as efficiency versus the Stk number, which is related to the particle size in Figure 4(a) (Eq. 2). As shown in Fig. S1 (Supplemental Information), the particle collection efficiency curves for three different Re numbers (381, 763, and 1144, related to air flow rates of 0.2, 0.4, and 0.6 L/min, respectively) were similar (Eq. 5). At an air flow rate of 0.6 L/min, the air flow velocity approached ~ 100 m/s in this system. In this case, the calculated Stk number and radial particle

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displacement (dp = 1 µm) are 0.632 and ~ 1,000 µm, respectively, when the air flow rate is 0.6 L/min in MicroSampler. Theoretically, therefore, the particle collection efficiency (dp = 1 µm) is ~100%. This theoretical calculation agrees well with the experimental results shown in Figures 4 (a) and (b). Microscopic counting of sample particles in liquid flow To verify the actual collected particles in the liquid flow, we used 1-µm fluorescent PSL (FPSL) particles and enumerated the total particle number by fluorescence microscopy and image processing. Two other conventional bioaerosol sampling methods (i.e., gelatin filters and BioSampler) were introduced for comparison with the particle collection characteristics of MicroSampler. According to the respective manufacturer’s protocol, these methods have collection efficiencies of ~100% and ~94.3%, respectively, for particle sizes of ~1 µm, as shown in Supplemental Information (Fig. S2). The details of the experimental conditions are listed in Table 1. Figure 4(c) shows the FPSL particle concentration, particle count (#) per unit volume (1 µL), of liquid samples after a sampling time of 10 minutes. The particle concentrations obtained with the gelatin filter, BioSampler, and MicroSampler were 9.83 ± 2.26, 8.66 ± 0.96, and 52 ± 9.85 #/µL, respectively. The results shown in Figure 4(c) indicate that the MicroSampler provides continuous and highly enriched particle collection performance from a high ratio of airto-liquid flow rate (~2 × 103) despite a short sampling time. In general, with conventional methods (i.e., gelatin filter and BioSampler) the enrichment of collected particles is adjusted by controlling sampling time. Therefore, for a long sampling period (> 1 hour under these experimental conditions), the particle concentrations obtained with the gelatin filter and BioSampler would be higher than that of MicroSampler (i.e., the sampling time of the MicroSampler does not affect the collected particle concentration in liquid flow) because the particle enrichment ratios of these methods are proportional to the sampling time, assuming a constant particle concentration in the air. In this study, the MicroSampler used the two-phase fluids, sampling air and collection liquid, to form a stable, stratified flow in the simple, curved microfluidic channel. In the particle sampling mechanism of the MicroSampler, airborne particles are collected directly from the air layer into the liquid medium layer by particle inertial force. This particle-containing liquid medium then exits the microchannel continuously for subsequent biochemical analysis. The liquid medium in MicroSampler thus plays a role not only as the particle collection medium, but also as the particle carrier medium. Due to this unique feature of

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the MicroSampler, the concentration of collected particles in the liquid medium does not change with sampling time, but rather with the ratio of air-to-liquid flow rate. As the MicroSampler requires only a very low sample volume, and can produce a highly enriched suspension within a short time compared with conventional methods (i.e., gelatin filters and BioSampler), it would be appropriate for use in rapid and continuous particle sampling and delivery, and would be advantageous for integration with real-time physico-biochemical analysis systems. To verify the particle loss in the above methods, we calculated the total particle number (#) (TPN) in collecting liquid with input of the same number of particles. To determine the relative particle collection efficiency (%), the TPN measured with each method was normalized relative to the TPN of gelatin filter because it has physical collection and extraction efficiency of 100% without any particle loss. Finally, the particle loss (%) can be calculated using the following equation: Particle loss (%) = 100 – relative particle collection efficiency. (8) As shown in Figure 4(d), BioSampler and MicroSampler have particle loss of 11.9% and 20.7%, respectively. Particle loss occurs in the sampling process when the particles adhere to the microchannel due to the gravitational force and diffusional deposition. The particle loss of the MicroSampler can be reduced by decreasing the particle residence time with faster air flow velocity (Ua) and smaller curve angle (θ), maintaining the particle radial displacement (L) (Eq. 1). Surface treatment of the inner channel can be useful for minimizing electrostatic adhesion in the microchannel. Application to detection of bacterial bioaerosol Using test bacterial bioaerosols of S. epidermidis, the microbial recovery of sampled bacterial particles on MicroSampler was evaluated in terms of colony culturability. The size distribution and cell morphology of airborne S. epidermidis are shown in Figure 5(a). S. epidermidis particles showed a unimodal curve with a specific geometric mean diameter (GMD), peak diameter, and geometric standard deviation (GSD) listed in Table 2. Scanning electron microscopy (SEM) showed that S. epidermidis cells were spherical and 0.6 – 1.0 µm in diameter. Figure 5(b) shows the collection efficiency curve of the airborne S. epidermidis in the MicroSampler. The maximum and minimum aerodynamic diameters of S. epidermidis were ~0.55 µm and ~1.3 µm, respectively. The collection efficiency curve of S. epidermidis was similar to that of standard-sized PSL particles (Fig. 4). In addition, the collection efficiency was >

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90% over the whole size range of S. epidermidis particles. To clarify the effects of bacterial shape, the size distribution and collection efficiency of rod-shaped bacteria (Escherichia coli and Bacillus subtilis) are shown in Figure S4. The collection efficiency of all test bacteria was constant in our MicroSampler and was only dependent on their aerodynamic particle diameter. After collecting S. epidermidis bioaerosols for a period of 10 minutes, 0.1 mL of the sample suspension was spread onto the agar substrate for colony counting assay. Figure 5(c) shows the colony concentration (colony forming units (CFU)/mL) of MicroSampler (918 ± 146.6 CFU/mL), which was higher than those of the gelatin filter (146 ± 39.3 CFU/mL) and the BioSampler (148 ± 44.9 CFU/mL). This bacterial colony counting result corresponds well with the FPSL particle counting result determined by microscopy, as shown in Figure 4(c). The bacterial colony culturability can be defined as the ratio of colony number concentration and the total number concentration of bacterial cells. The total counts of bacterial cells were measured by the staining of cells with the fluorescent dye SYTO82 and fluorescence microscopy. Finally, to determine the relative bacterial culturability (%) for comparison, the culturability with each method was normalized relative to the bacterial culturability with the MicroSampler system. Figure 5(d) shows that the relative culturability of samples from gelatin filter and BioSampler were ~84.81% and ~95.53%, respectively, compared with that of MicroSampler. The relative culturability of the gelatin filter was slightly lower than the other methods because the gelatin filter particle collection medium is different from the others, and is susceptible to harsh conditions in sampling, such as desiccation during sampling time and physical stress by agitation during the process of dissolution into phosphate-buffered saline (PBS) suspension. Therefore, the liquid-based bioaerosol sampling methods, such as MicroSampler and BioSampler, have advantages for microbial recovery of sampled cells. Our results showed that the MicroSampler system has greater cell enrichment performance over a short period and comparable microbial recovery to BioSampler. Furthermore, it is a real-time collection method with potential for use in continuous and online analysis tools. Potential for use in real environment Although the typical size of a single airborne bacterium is > 0.5 µm in size (Fig. S4), the airborne bacteria in the general atmosphere are combined with other particulate matter (e.g., dust) to form particles of about > 80%. The particle size of agglomerates has been reported to be > 2 µm.34 In this case, the particle collection efficiency of the MicroSampler will be much higher than that in the case of single bacterium collection because of the increase in particle size (dp).

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In this study, a bacteria concentration of ~ 4 × 105 CFU/m3 was used to clearly analyze the collection efficiency according to particle size in our MicroSampler. However, the system is designed to be fully usable in a real environment. Environmental institutes and governments around the world have established many maximum allowable limits to regulate bioaerosol. Suggested allowable limits by the World Health Organization (WHO) and other governmental institutes are < 500 – 1,000 CFU/m3.35-36 The MicroSampler produces a highly concentrated suspension within a short sampling time based on low flow rate of sampling liquid versus high air flow rate (gas to liquid concentration ratio of ~ 2 × 103). With this enrichment, it can collect 2 CFU per 1 mL of sampling liquid under atmosphere conditions of 1,000 CFU/m3. If the concentration of target bacteria is much lower than 1,000 CFU/m3, it can be measured by postenrichment processes, centrifugation or circulation of sampling liquid in the MicroSampler with a low flow rate pump. Currently, our research group is developing an integration system that can collect and detect airborne bacteria in real-time by connecting the MicroSampler to various in situ physicochemical analysis methods, such as continuous Raman analysis, single-cell flow cytometry, and bacterial ATP-luminescence detection microchip systems.37-38 When this advanced system is used for cell sensing applications based on microfluidics, it would be appropriate for rapid and continuous particle sampling and sensing in the real environment with real-time physico-biochemical analysis.

CONCLUSION We have developed a novel system for continuous and rapid sampling of airborne particles using inertial microfluidics, called MicroSampler. The performance of our device was investigated in terms of physical collection efficiency, cut-off particle diameter, particle collection enrichment, and microbial recovery for biological collection efficiency. MicroSampler makes it possible to perform size-selective particle sampling into collecting liquid in real-time. In addition, the MicroSampler showed superior microbial recovery (S. epidermidis) compared to conventional bioaerosol sampling techniques. This is first application of continuous bioaerosol sampling into liquid suspension using an inertial microfluidic platform. Our system has great potential for rapid detection because it requires only a very low sample volume and can produce a highly enriched suspension within a relatively short time compared with conventional methods (i.e., gelatin filter and BioSampler). More enriched particle 12


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collection is possible by integrating a virtual impactor system into the same microfluidic chip layer. In addition, the MicroSampler showed potential for use as a total airborne particle analyzer, as well as a portable sampler for continuous, real-time analysis of airborne particles and microorganisms. In future, we plan to integrate the MicroSampler with a real-time particle analysis system, such as a microfluidic flow cytometer, for quantitative and continuous particle characterization of airborne microorganisms.37 In this case, a new aerosol analysis system could be developed using various markers or dyes to target specific materials. For example, target-specific aptamers or antibodies can provide the physiochemical and biological properties of airborne particles.38

METHODS Test sample particles PSL particles (mono-sized standard spherical particle; 0.652, 0.8, 0.913, 1, 1.53, and 2.1 µm in diameter; refractive index 1.59; density 1.06 g/cm3; Duke Scientific Corporation, Palo Alto, CA, USA) and fluorescent PSL particles (Fluoro-Max™, 1.0 µm in diameter; Thermo Scientific, Waltham, MA, USA) were used to evaluate physical particle collection efficiency. The grampositive bacterium S. epidermidis (ATCC 12228) was used as a test airborne microorganism. The bacteria were cultivated at 37°C in nutrient broth (Becton Dickinson, Franklin Lakes, NJ, USA). The cultivated bacteria were collected using centrifugation (5,000 × g, 10 minutes). The PSL and bacteria suspensions were aerosolized using a one-jet Collison nebulizer (BGI Corp., Cambridge, MA, USA). Cell counting by fluorescence microscopy Aliquots (~ 10 µL) were injected into a disposable hemocytometer (DHC-N01; INCYTO, Cheonan, Korea). A fluorescence microscope (BX51; Olympus, Tokyo, Japan) with a U-MWG2 filter was used to detect fluorescence emission (> 590 nm) from bacteria excited by green light (510–550 nm). At least 16 microscopic fields were obtained by the CCD camera. Staining of bacterial samples SYTO82 dye (Molecular Probes Inc., Eugene, OR, USA) was used for nuclear staining of bacteria. It exhibits bright fluorescence upon binding to DNA or RNA,39-40 with excitation and emission wavelengths of 541 and 560 nm, respectively. 13


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Colony counting assay Collected bacterial suspensions were serially diluted. Aliquots of 100 µL were spread onto the surface of nutrient agar (Becton Dickinson) in Petri dishes. The resulting colonies were counted after incubation for 24 hours at 37°C.

Collection of airborne microorganisms using gelatin filter and BioSampler A gelatin filter was introduced for collecting test aerosol with a flow rate of 0.6 L/min for 10 minutes. After collection, the gelatin filter (SKC Inc., Eighty Four, PA, USA) was dissolved in 20 mL of PBS at pH 7.0. The BioSampler (SKC Inc.) collected bacterial bioaerosols into 20 mL of PBS at a nominal flow rate (sample flow and sheath flow) of 12.5 L/min for 10 minutes.

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available, free of charge, on the ACS Publications website at DOI: Collection efficiency in terms of Reynolds number, collection efficiency by gelatin filter and BioSampler, fluorescent PSL particle images, size distribution and shape of the test bacterial particles, and movie file description (PDF) Flow pattern of two-phase fluid in the microchannel (Movie S1, AVI)

AUTHOR INFORMATION Corresponding author * E-mail: [email protected]. Telephone: +82-54-336-9741. **E-mail: [email protected]. Telephone: +82-2-958-5718. Author Contributions †

J.C. and S.C.H. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was supported by the KIST Institutional Program and the Korea Institute of Industrial Technology (KITECH) through Research and Development programs. This research was also supported in part by the Ministry of Land, Infrastructure, and Transport (16RTRPB082501-03), the Ministry of Environment (2016000160008), and the Technology Support for Biomedical Industry in Gyeongbuk Province, Republic of Korea (IZ160025).

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Table 1. Experimental conditions for sampling with different sampling methods: gelatin filter, BioSampler, and MicroSampler. Experiment variables Sampling method Aerosol flow rate

Sampling time

Collection liquid

Gelatin filter

0.6 L/min

10, 60 min

20-mL PBSa

BioSampler

0.6 L/minb

10, 60 min

20-mL PBS

MicroSampler

0.6 L/min

N/Ac

0.3-mL/min PBSd

a

Phosphate-buffered saline (pH = 7.0)

b

with sheath air at a flow rate of 11.9 L/min

c

Sample enrichment is decided only by aerosol and collection liquid flow rate

d

Continuous collecting system

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Table 2. Size characteristics of test airborne bacterial particles. The geometric standard

deviation (GSD) is defined as exp 9/∑ ; 'ln>; − ln>? * /(A − 1)B, where dj is the diameter of an individual particle, nj is the number of particles in the jth group, N is the total number of particles, and lndg is the natural logarithm of the geometric mean diameter (GMD) of the particles, defined

as ∑ ; ln>; /A. Data are listed as the means of 10 repetitions ± standard deviation. Particle size distribution Bacterium

Peak diameter

GMD (µm) S. epidermidis

(µm)

0.79 ± 0.002

0.84 ± 0.026

20


GSD 1.18 ± 0.001

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Figure legends Fig. 1. (a) Schematic diagram of the principle of aerosol sampling in the curved microchannel. The gas-liquid fluid forms a stratified flow by covering the outer wall with liquid. Aerosols in the gas-flow move radially outward when passing through the MicroSampler due to centrifugal force. (b) Trace of the aerosol trajectory, indicating the direction of force applied to a single particle. (c) Schematic diagram of the Dean flow (secondary flow) in the curved microchannel. Dean flow is composed of two symmetric vortices at the top and bottom of the channel. The pressure gradient of the air flow between the center and wall of the channel leads to the Dean flow which influences the particle movement in the curved channel. (d) Photograph of the MicroSampler. Fig. 2. Schematic diagram of the experimental setup. Fig. 3. Two-phase flow in the MicroSampler, flow pattern transition in terms of air and water flow rate (a-1) and We and Ua/Uw (a-2). Flow pattern images in stratified region (b-1) and sprayed region (b-2). Error bars indicate standard deviations (n = 5). Fig. 4. Collection efficiency of polystyrene-latex (PSL) particles in terms of (a) different flow rates and (b) particle sizes. The air flow rates were 0.2, 0.4, and 0.6 L/min and the corresponding water flow rates were 0.1, 0.2, and 0.3 mL/min, respectively. The ratio of air to water flow rate was maintained at 2 × 103. (c) Collected particle concentration. Fluorescent PSL (FPSL) particles were collected using three types of sampler: gelatin filter, BioSampler, and MicroSampler. The sampling times for the gelatin filter and BioSampler were 10 and 60 minutes, respectively. (d) Relative collection efficiency. The collection efficiency was normalized relative to the count from the gelatin filter method. Error bars indicate standard deviations (n = 5). Fig. 5. (a) Size and morphology of S. epidermidis. The particle size distributions of S. epidermidis bioaerosols were determined. The number concentration of the particles in air was recorded for each aerodynamic particle sizer (APS) channel size, divided by the logarithmic interval of the corresponding particle size range and plotted as a function of the aerodynamic diameter. Scanning electron microscopy (SEM) images in the insets indicate that S. epidermidis cells were spherical. (b) Collection efficiency of S. 21


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epidermidis by the MicroSampler. (c) Colony concentration. Bioaerosols (S. epidermidis) were collected with three types of sampler: gelatin filter, BioSampler, and MicroSampler. The sampling time in gelatin filter and BioSampler was 10 minutes. (d) Relative culturability by gelatin filter, BioSampler, and MicroSampler. Culturability was normalized relative to that with the MicroSampler. Error bars indicate standard deviations (n = 5).

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Figure 1

Fig. 1. (a) Schematic diagram of the principle of aerosol sampling in the curved microchannel. The gas-liquid fluid forms a stratified flow by covering the outer wall with liquid. Aerosols in the gas-flow move radially outward when passing through the MicroSampler due to centrifugal force. (b) Trace of the aerosol trajectory, indicating the direction of force applied to a single particle. (c) Schematic diagram of the Dean flow (secondary flow) in the curved microchannel. Dean flow is composed of two symmetric vortices at the top and bottom of the channel. The pressure gradient of the air flow between the center and wall of the channel leads to the Dean flow which influences the particle movement in the curved channel. (d) Photograph of the MicroSampler.

23 ACS Paragon Plus Environment

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Figure 2

Fig. 2. Schematic diagram of the experimental setup.


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Figure 3

Fig. 3. Two-phase flow in the MicroSampler, flow pattern transition in terms of air and water flow rate (a-1) and We and Ua/Uw (a-2). Flow pattern images in stratified region (b-1) and sprayed region (b-2). Error bars indicate standard deviations (n = 5).


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Figure 4

Fig. 4. Collection efficiency of polystyrene-latex (PSL) particles in terms of (a) different flow rates and (b) particle sizes. The air flow rates were 0.2, 0.4, and 0.6 L/min and the corresponding water flow rates were 0.1, 0.2, and 0.3 mL/min, respectively. The ratio of air to water flow rate was maintained at 2 × 103. (c) Collected particle concentration. Fluorescent PSL (FPSL) particles were collected using three types of sampler: gelatin filter, BioSampler, and MicroSampler. The sampling times for the gelatin filter and BioSampler were 10 and 60 minutes, respectively. (d) Relative collection efficiency. The collection efficiency was normalized relative to the count from the gelatin filter method. Error bars indicate standard deviations (n = 5).


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Figure 5

Fig. 5. (a) Size and morphology of S. epidermidis. The particle size distributions of S. epidermidis bioaerosols were determined. The number concentration of the particles in air was recorded for each aerodynamic particle sizer (APS) channel size, divided by the logarithmic interval of the corresponding particle size range and plotted as a function of the aerodynamic diameter. Scanning electron microscopy (SEM) images in the insets indicate that S. epidermidis cells were spherical. (b) Collection efficiency of S. epidermidis by the MicroSampler. (c) Colony concentration. Bioaerosols (S. epidermidis) were collected with three types of sampler: gelatin filter, BioSampler, and MicroSampler. The sampling time in gelatin filter and BioSampler was 10 minutes. (d) Relative culturability by gelatin filter, BioSampler, and MicroSampler. Culturability was normalized relative to that with the MicroSampler. Error bars indicate standard deviations (n = 5). 27 ACS Paragon Plus Environment

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