A microfluidic system for rapid detection of airborne pathogenic fungal

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A microfluidic system for rapid detection of airborne pathogenic fungal spores Xiaoxu Li, Xinlian Zhang, Qi Liu, Wang Zhao, Sixiu liu, and Guodong Sui ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00615 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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A microfluidic system for rapid detection of airborne pathogenic

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fungal spores

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Xiaoxu Li, 1 Xinlian Zhang, 1 Qi Liu, 1 Wang Zhao,1 Sixiu Liu, 1 Guodong Sui, *, 1, 2, 3

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*To whom correspondence shall be addressed.

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of Environmental Science & Engineering, Fudan University, 220 Handan Road, Shanghai, 200433,

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P. R. China.

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Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department

Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, P. R. China

Institute of Biomedical Science, Fudan University, No. 138 Yixueyuan Road, Shanghai 200032, P.

R. China

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ACS Paragon Plus Environment

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ABSTRACT: Airborne fungi, including Aspergillus species, are the major causes of human

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asthma. Direct capture and analysis of pathogenic fungi in indoor air is important for disease

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prevention and control. In this paper, we demonstrated an integrated microfluidic system capable

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of enrichment and high-throughput detection for airborne fungal spores of Aspergillus niger, a

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well-known allergenic harmful species. The microfluidic system allowed semi-quantitative

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detection of Aspergillus niger spores based on immunofluorescence analysis. To assess its

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contaminated level, the whole analysis time could be completed in 2-3 hours including ~1h of

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enrichment and ~1h of target detection. The detection limit was ~20 spores, equivalent to ~300

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spores·m-3 of the concerned targets in air. In addition, the microfluidic system has integrated

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sampling and sample analysis to avoid additional sample concentration step, showing the potential

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for point-of-care detection for other pathogenic fungal spores.

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KEYWORDS: Microfluidic system, Pathogenic fungi, Aspergillus niger spores, Enrichment,

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Immunoassay analysis

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Fungi are commonly found indoors since they can colonize every inch of a room yet are scarcely

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associated with health effects in low concentration.1 In the presence of dampness, fungi are able to

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proliferate explosively to damage building materials,2-4 they can also invade and infect human

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body by means of releasing numerous spores as well as various harmful secondary metabolites

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such as carcinogenic mycotoxins into the air.1,2,5,6 Adverse effects of poor indoor air quality

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caused by fungal aerosol contamination on health have attracted much public attention, since

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people spend almost 90% of their time indoors.7-9 These undesirable bio-aerosols can lead to sick

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building syndrome (SBS) and building related illness (BRI),10 for instance, some sensory

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irritations of eyes and upper respiratory tract induced by volatile products of fungal

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metabolisms,11-13 and some respiratory infections and allergic reactions caused by airborne fungal

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spores13,14 including Penicillium, Aspergillus, Cladosporium, etc. Additionally, evidence showed

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that some pathogenic species can cause nosocomial infections which are always focal issues.15

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Especially for immunocompromised individuals such as transplant patients, most infections

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known as invasive fungal infections (IFIs) occur in their lungs because of inhalation of fungal

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spores among which Aspergillosis is the most common one.15-17 The IFIs can then spread to other

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systems of the body, and even lead to death.18,19 Among pathogenic Aspergillosis species,

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Aspergillus niger (A. niger) is one of the most common causes of IFIs. The previous studies have

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reported20 that the IFI caused by A. niger could infect a leukemic patient’s heart and develop into

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pericarditis and cardiac tamponade month after bone marrow transplantation. Moreover, the A.

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niger could also cause mycosis of ear and primary cutaneous aspergillosis and produce

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nephrotoxic and carcinogenic mycotoxin.21 However, evidence for the casual dose-response

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relationship between these molds and human health is still poorly documented.


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Due to high risks of fungal exposure, mold management has received extensive attentions,

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including regular inspection, elimination of moisture sources, removal of the viable mycelia from

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surfaces and filtration of airborne spores from indoor air to prevent indoor fungal growth.4,22

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Although eradication of fungal-polluted materials can stop the production of new spores and their

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dispersals, the residual fungal allergens settled in dust may cause episodic allergy symptom in

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sensitive individuals with a long duration.23 In addition, some fungi sources could hide in the

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building structure such as wall insulating layers to avoid elimination,24,25 furthermore, indoor

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microbiomes are usually a mixture of species resembling in outdoor environment. Overall, indoor

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microbiomes are usually changeable and complex mixture that are mainly influenced by dispersal

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from outdoor biomes.26 In densely populated buildings such as hospital, the microbial load,

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attributable to pathogenic problem species such as Penicillium or Aspergillus, is affected not only

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by nature factors, more often impacted by number of occupants, their activity, building ventilation,

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and so on.25,26 To completely avoid fungal exposure is not practical, instead, focusing on those

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problem species might be alternative to effectively reduce the risks of exposure.

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In order to effectively control pathogenic fungi exposure, airborne fungi assessment is considered

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as a reasonable means to directly evaluate the risks of fungal exposure and guide regular mold

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managements, in spite of the lack of information about dose-response relationship. Air samples are

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commonly used in various analytical assays to assess human exposure to fungi. Noticeably, air

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sample analyses are conducive to identify abnormal presence of fungi with hidden mold growth,

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to detect pathogenic spore releases from sources and to monitor the effects of mold managements.

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To our best knowledge, there are no standard sampling or analytical methods for airborne

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pathogenic fungi. Herein, we listed some typical techniques that have been used in studies for


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airborne fungi sampling (Table S1) and sample analysis (Table S2). Apart from the culture

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methods which are time-consuming and inadequate to cultivate all collected microorganisms,

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researchers have combined some commercial air samplers with advanced analytical techniques.

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For instance, the Coriolis µ air sampler combined with the quantitative polymerase chain reaction

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(qPCR) has been used for airborne fungi analysis. Luminex xMAP technique was also introduced

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in the high-throughput identification of fungi species in the air.27-29 In addition, several

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supersensitive sensor-based methods for pathogenic Aspergillus spore detection were reported,30,31

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but these methods could not directly analyze fungal spores from air.

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Herein, we presented a microfluidic system comprised of a sampling chip and an analysis chip

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capable of rapid detection of airborne pathogenic fungal spores of A. niger. The system showed

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high sensitivity and specificity towards specific fungal spores from indoor air. The staged

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herringbone mixer (SHM) structure was adopted in the sampling chip, which was successful used

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in the airborne bacteria collection.32-34 For the first time, SHM structure was utilized to capture

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airborne fungal spores (much larger than bacteria). By employing this technique, the volume of

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eluent from the sampling chip could be controlled in dozens of microliters, which makes it

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possible to directly introduce the sample into the downstream analysis chip without culturing or

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concentration steps. The analysis chip has a column structure, combining the nonspecific physical

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interpretation and specific immunocapture to enrich and identify the target spores. In order to

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assess the number of A. niger spores in air sample, the semi-quantitative standard curve of

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immunofluorescence intensity-number of spores were established. Furthermore, the sensitivity and

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specificity of the system was evaluated by mimic spore-aerosols. The system has potential to

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implement high-throughput detection of different specific fungal species by using corresponding


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immune labels.

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METHODS AND MATERIALS

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Materials and Reagents. Photoresists (SU-8 2005, SU-8 2025) were purchased from Microchem

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(USA). Photoresist (AZ-50XT) was purchased from AZ Electronic Materials USA Corp.

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Polydimethylsiloxane (PDMS, RTV-615-044) was purchased from Momentive Specialty

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Chemicals (USA). Carboxyl-modified microspheres (PS-COOH) were purchased from Bang’s

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Laboratory (USA). The fungal strain Aspergillus niger (A. niger, CGMCC No. 3.6469) and

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Aspergillus oryzae (A. oryzae, CGMCC No. 3.39544) were obtained from China General

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Microbiological Culture Collection Center (CGMCCC). Anti-Aspergillus niger polyclonal

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antibodies (IgG) were purchased from Virostat (Portland). Anti-Aspergillus niger monoclonal

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antibodies (MCA 2576) and fluorescein isothiocyanate-labeled (FITC) anti-Mouse secondary

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antibodies were purchased from Bio-Rad (USA). Sabouraud dextrose agar was purchased from

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Oxoid (UK). All other reagents were purchased from Sigma (USA).

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Microfluidic Chip Fabrication. The presented system is consisted of an enrichment chip for

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fungi spores collect from air and a detection chip for immunofluorescence analysis of collected

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pathogenic spores. Both chips were designed by AutoCAD 2014 software (Autodesk, USA) and

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fabricated following standard soft lithography. The silicon molds of the enrichment chip that

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patterned with fluidic channels and herringbone structures were made from SU-8 2025. For the

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detection chip fabrication, the silicon mold of the control layer was made from SU-8 2025 and the

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mold of the fluidic layer was made from SU-8 2005 and AZ-50XT. The PDMS polymer plates

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casted from the molds were assembled precisely to separately fabricate the enrichment chip and


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the detection chip. Inlet and outlet holes were punched for air or liquid flow. The channel surfaces

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of the enrichment chip were sequentially washed by HCl (1.0% v/v), H2O2 (1.0% v/v), and

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deionized water, then dried with clean nitrogen prior to the usage.

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Fungi Spore Culture and Collection. A. niger and A. oryzae were both grown at 37°C for 2-4

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days in Sabouraud dextrose agar. Fungal spores were harvested with sterile loops and washed

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twice with phosphate-buffered saline (PBS, pH 7.2). Harvested spores were suspended in 50 mM

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carbonate bicarbonate buffer (pH 9.6) to obtain the suspension of ~1.1 × 106 spores·mL-1 (~0.05

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mg·mL-1). The spore suspensions were stored at 4°C and used within 2 weeks.

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Spore-aerosol Generation and Spore Collect on the Enrichment Chip. To validate the

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performance of the enrichment chip, simulated spore-aerosols of A. niger was generated and

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monitored. The schematic diagram of the spore-aerosol generation system was shown in

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supplementary information Figure S1. A mini generator (DIONE N7, China) charged with 5 mL

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of prepared spore suspension (~1.1 × 106 spores·mL-1) was introduced to spray spore-aerosol

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droplets into a closed generation cube (750 mm × 450 mm × 450 mm) with an atomization rate of

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800 µL·min-1 (shown in Figure 3A). To rapidly distribute spore-aerosols, a turbulator was placed

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in the middle of the cube bottom. At 100 mm height above upper surface of the turbulator, a wind

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sensor (MIK-GWT 0-5 m·s-1, China) was set to measure its airflow speed. A particle matter

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monitor (PLANTOWER PMS5003T, China) was used to monitor particle concentration in

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real-time manner. For spore-aerosol generating, the mini generator which was loaded with 1.1 ×

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106 spores·mL-1 suspension executed the atomization for 1 min only at the beginning and then 10 s

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supplementary atomization every other 20 seconds. The generated aerosols were immediately

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dispersed by the 0.3 m·s-1 airflow blown from turbulator. The stability of generated aerosols was


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measured by APS (Aerodynamic Particle Sizer, TSI3321-APS, USA). Briefly, the sample mode of

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APS was a summed mode; sheath flowrate, 4.0 L·min-1; sample flown, 5.0 L·min-1; sample type,

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continuous; inlet pressure, 1000 Mbar; detection range, 3-20 µm (aerodynamic diameter);

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sampling time, 20 s. When collecting spores using the enrichment chip, two micro-vacuum air

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pumps (HARGRAVES, USA) assembled in parallel were connected to the airflow outlet of the

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enrichment chip and drew the aerosol into the enrichment chip.

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To evaluate the collection efficiency of the enrichment chip, the testing system (shown in Figure

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S2) was established to collect the uncollected spores in the filtered air. The traditional

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sedimentation method was used as control. Spores collecting by the enrichment and the traditional

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sedimentation method were both collected in PBS and diluted 100 times, respectively. Collected

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spores were then counted by the hemocytometer.

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To evaluate specificity of the detection chip for identifying A. niger spores, the A. niger

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suspension of 1.1 × 106 spores·mL-1 was mixed with A. oryzae suspension of 1.1 × 106

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spores·mL-1 in ratios of 3: 1 and 1: 3, respectively. Then the generator was charged with different

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suspensions containing 100%, 75%, and 25% A. niger spores, respectively. Later, different

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suspensions were atomized into the cube following the above generation steps. The sampling time

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of each spore-aerosol was 5 min (~500 spores could be collected). Spores collected by the

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enrichment chip were washed in 20-30 µL PBS for subsequent detections.

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Analysis on the Detection Chip. Microspheres of 20.38 µm diameter was adopted for both

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interception and immunocapture of specific spores. The interaction between PS-COOH and

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Rabbit polyclonal antibody is covalent. Figure 2A was illustrated the schema of immunoassay


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reactions for A. niger spores detection on microspheres. Following protocol from Bangs

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Laboratories, Inc., the carboxyl-modified microspheres were firstly immobilized with rabbit

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polyclonal antibodies (4-5 mg·ml-1) and the final microspheres with rabbit polyclonal antibodies

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were ~10 µg·µl-1. Then microspheres with rabbit polyclonal antibodies were loaded into reaction

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channels of the detection chip. For spore capture and analysis, 20 µL PBS-Tween-20 solution was

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used to rinse protective solution off before sample loading. The whole analysis procedure was

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schematically illustrated in Figure 2B. The spore analysis on the chip was conducted as following

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steps: (1) Sample loading. Washing buffer containing collected fungal spores was injected into the

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reaction channel via the inlet with the flowrate of 6 µL·min-1. (2) Blocking and specific marking.

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20 µL PBST solution containing 1% bull serum albumin (BSA) was used to wash and block the

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residual antibodies on the microspheres, and then 5 µL monoclonal antibody solution (1.0 mg·ml-1)

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was introduced into the reaction channel and the chip was incubated at 37°C for 15 min. (3)

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Washing and Blocking. Above washing and blocking step were repeated. (4) FITC labeling. 5 µL

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FITC labeled anti-mouse secondary antibody solution (2.0 mg·ml-1) was injected into the reaction

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channel and the chip was incubated in darkness at 37°C for 15 min. (5) Washing and analysis.

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PBST solution was used to continuously wash the microspheres for 5 min. Fluorescence images

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were obtained on the fluorescence microscope (Nikon, Eclipse Ti, Japan) and results were

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analyzed by the software MATLAB 2016 (MathWorks, USA).


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Figure 1. (A) Image of the microfluidic system for airborne spore-aerosol enrichment and

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detection: (a) airborne spore enrichment chip, (b) airborne spore detection chip. (B) Schematic

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illustration of the enrichment chip when sampling air. Air inlets and center outlet remained open.

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(C) Schematic illustration of the enrichment chip when eluting spores. The center outlet was

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closed. (D) Image of the detection chip and schematic illustration of the detection chip: (a) fluid

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layer, (b) control layer. (E) Image and illustration of the detection units on the chip.

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Figure 2. (A) Schematic illustration of immunoassay reactions for A. niger spore detection on


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microspheres. (B) Procedures of A. niger spore analysis on the detection chip.

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RESULTS AND DISCUSSION

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Design of spore-aerosol detection system. The system consists of an enrichment chip for

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airborne fungal spore sampling (Figure 1A-a) and a detection chip for analysis of specific

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pathogenic spore (Figure 1A-b).

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In the enrichment chip, sixteen identical channels (40 mm long × 0.6 mm wide × 0.04 mm high)

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with SHM structures were radially arranged from the center of the chip and shared the same center

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airflow outlet. Each channel had an inlet for air/liquid flow and a joint hole for liquid flow (shown

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as Figure 1B&C). When sampling air, 16 channels in the enrichment chip could work in parallel

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by drawing the air from the shared outlet (Figure 1B). The total flow rate of the enrichment chip

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was ~1.3-1.45 L·min-1 (80-90 ml·min-1 of each channel × 16 channels). The SHM structure was

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introduced for airborne spore enrichment, which has been demonstrated to efficiently capture

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airborne bacteria particles from air by adhering bacteria to surfaces of the channel.32,34 This

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structure could break the laminar airflow into chaotic flow when sampled air flow passing through

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the channel to improve the contact between fungal spores and inner surfaces of the channel.34,35

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When eluting spores, the center outlet was closed and 16 channels were connected in series, so

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that all the channels could be rinsed by injecting washing buffer for only one time (Figure 1C). By

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adopting this microfluidic chip, the volume of eluent consumed for sample collection could be

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minimized to 5-10 µl. With such a small volume, all fungal spores in the eluent could be directly

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analyzed in the downstream detection chip avoiding the traditional concentration operations such

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as centrifuging and culturing.36,37 In the presented system, the total volume of eluent was set to be


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20-30 µl due to the downstream detection chip could reconcentrate the target fungal spores to

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further increase the concentration of fungal spores in the eluent.

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Figure 3. (A). Spore-aerosol generation system. (B). Raw particle counts of A. niger

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spore-aerosols measured by APS (Generation from 1.1 × 106 cells·ml-1 of A. niger spore

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suspension). Values are the mean ± s.d. for n = 3.

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The detection chip (shown in Figure 1D-E) provided 10 identical units, which could analyze 10

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different samples separately by valve controls without cross contamination. The microfluidic

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immunoassay chip was 3×3 cm2 in size and composed of a fluidic layer (Figure 1D-a) and a

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control layer (Figure 1D-b). One outlet (black) was shared by ten channels (blue), while each

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channel has its own inlet (Figure 1D). The inlets and outlets of fluidic layer were 200µm width


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and the reaction column was 100µm width (Figure 1E). The inlet and reaction column of the

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fluidic layer were 30µm height and the outlet of the fluidic layer was 10µm height. The weir

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structure between inlet and reaction column can intercept 20.38µm-microsphere to stay in reaction

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column. Generally, the concentration of airborne spores in air can effectively reflect the fungal

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contamination. Analyses of nucleic acid are mostly used to identify microorganism species and

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estimate its quantities.38-40 However, different from bacterial cells, it is difficult to extract DNA

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from the fungal spores especially in a short time period because of their sturdy cell walls.41,42 Thus,

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for the rapid analysis of specific pathogenic spores, the detection chip adopted the

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immunofluorescence method to estimate the quantity of target spores instead of nucleic acid

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analysis. In the immunoassay process, the immunofluorescence assay efficiency could be directly

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influenced by the capture efficiency on the chip. Many studies demonstrated that it was an

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effective means to improve capture efficiency by increasing the contact between antigens and

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antibodies.43 For example, Wang et al.44 built hundreds of small columns in the microfluidic

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channels to increase antibody-modified area of inner channel surface and demonstrated the good

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performance of this structure for circular tumor cell capture. As reported by Wang and Han,45 the

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reaction kinetics of bead-based antibody-antigen binding can be enhanced, the beads based

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microfluidic preconcentrator increased the detection sensitivity of immunoassay (by at least 500

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fold). In presented studies, each unit was equipped with a reaction channel (100 µm wide × 30 µm

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high) packed with antibody-modified microspheres in a single layer. The microspheres were

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immobilized by a weir structure which formed at the junction between the reaction channel of 30

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µm in height and the outlet channel of 10 µm in height (Figure 1E). It could intercept

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microspheres (20.38-µm diameter) and let liquid flow through during the processes of


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microsphere packing and sample loading. Compared with direct modification of antibodies on the

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surface of channels, the microspheres in the channel could provide a larger surface area to modify

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antibodies for capturing target spores. Meanwhile, the single spore cells in sample would have

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more chances to touch immobilized antibodies easily when they flow through the tiny gaps

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between microspheres, which also could improve the capture efficiency. Figure 2A illustrated the

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process of A. niger capture and detection on the microspheres. The monoclonal antibody was used

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to identify A. niger spores with high specificity. After the spores were labeled with FITC, the

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FITC could profile spore cell by emitting green fluorescence under 490 nm excitation.46,47 The

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amount of target spores was calculated according to the intensity of fluorescence signal.

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Validation of the enrichment chip. Like other approaches based on fluid mechanics and air

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dynamics (most of them belong to the impingement method),48,49 its collection efficiency for

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particles with different sizes and qualities might be different. We firstly testified the feasibility of

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the enrichment chip for A. niger spores collection. As shown in Figure 3A, the spore-aerosols

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were generated in the closed cube and monitored by APS to test its stability. The data of six

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repeated trials was shown in Figure 3B. The figure only showed the numbers of particles from 3.5

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µm to 12 µm measured by APS due to A. niger spores as the detection objects would form aerosol

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particles with diameters larger than 3.5 µm50 and particles with diameter larger than 10 µm are

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easier to combine with water to fall down. This result showed that (1) approximately 75% of

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particles that detected by APS distributed in the range of 3.5 to 5 µm of aerodynamic diameters

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which matched with the size of A. niger spore; (2) other particles that smaller than 10 µm might be

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the particles containing two spores or enwrapped with a thin water film;51 (3) the sampling volume

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of APS was ~1.67 L each trial (sampling time: 20 s) which was similar to ~1.5 L of the


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enrichment chip (working time: 1 min). The result indicated that the concentration of mimic

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spore-aerosol in the generation cube were stable and it could provide an effective aerosol for the

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feasibility tests. Subsequently, the enrichment chip was placed in the cube for sampling with 30

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min and 2 h, respectively. As shown in Figure 4, the spores adhered to the inside wall of channels

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were observed under the 20× objective. The number of spores collected for 2 h was significantly

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more than that of 30 min collection. This result directly proved that the enrichment chip could be

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used for spore-aerosol collection.

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Table 1. Collection efficiency of enrichment chip with different experimental times. Experimental time (min) 0 10 20 30 40 50 60

Total no. of enriched A. niger spores (cells) 0 10500 23300 31700 45900 62800 91500

Total no. of unenriched A. niger spores (cells) 0 0 600 1700 4600 8900 16000

Enrichment efficiency 0 100% 97.4% 94.6% 90.0% 85.8% 82.5%

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Figure 4. Images of enrichment channel on the chip for spore-aerosol capture with different


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sampling time. (A) sampling time: 0 min, (B) sampling time: 30 min, (C) sampling time: 2 h.

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In order to investigate the collection efficiency changing with the time, 6 different

293

experimental times were selected to be 10, 20, 30, 40, 50, and 60 min, as shown in Table 1. The

294

cube in testing system (Figure S2) was charged with the spore suspension of 1.1 × 107 cells·ml−1

295

to generate spore-aerosol droplets. During experiments, the spore-aerosol droplets were kept in a

296

saturated state to provide channels of the enrichment chip enough spores for absorption.

297

Experimental data about A. niger spore collection efficiency was shown in Table 1. The results

298

showed that the enrichment chip was well capable of capturing A. niger spores from aerosol. The

299

total numbers of collected spores increased as experimental time prolonged. As the sampling time

300

prolonged from 20 to 60 min, the collection efficiency slightly declined from 97.4% to 82.5%.

301

However, its efficiency was much better than that of the traditional sedimentation method. When

302

the sampling time was 20 min, the collection efficiency of the enrichment chip was 35 times

303

higher than that of the traditional sedimentation method. When sampling time was 60 min, the

304

collection efficiency of the enrichment chip was still 5 times higher than that of the traditional

305

sedimentation method. It is clear that the designed microfluidic chip with SHM structure can

306

capture A. niger spores at a very good efficiency from the aerosol. In addition, we listed some

307

typical existing methods for spore-aerosol collection, shown in Table S1. Compared with these

308

methods, our enrichment chip provided a relatively shorter sampling time.

309

Validation of the spore-aerosol detection system. Our system provided quantitative analyses of

310

spores by using a detection chip to evaluate collected spores from the upstream enrichment chip.

311

The feasibility of the detection chip was validated by testing A. niger spore suspension containing

312

~500 cells. Figure 5A-a showed the image of reaction channel under bright field. The


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microspheres formed a monolayer in the 100-µm wide reaction channel, which facilitated the

314

result reading due to all microspheres could be seen under the same microscopic vision field.

315

Figure 5A-b and Figure 5A-c showed the positive result and negative result, respectively, under

316

490 nm extraction. The green fluorescence presenting in the positive result demonstrated that the

317

immunoassay could analyze the amount of spores in the reaction channel. The negative result

318

indicated the monolayer of microspheres could provide a clean background for the immunoassay.

319

To realize quantitative analyses of A. niger spores, the standard curve was established between

320

spore count and fluorescence density. The spore suspensions containing 1000, 750, 500, 200, 100,

321

50, 20, and 10 cells, respectively, were tested on the detection chip as samples. The fluorescence

322

image of each sample was shown in Figure 6A. The fluorescence density of sample containing

323

1000 spores was largest among all samples and it decreased as spore number decreased in samples

324

(as shown in Figure 6B). The detection limit of the chip was 20 spores, equivalent to ~300

325

spores·m−3 that could be detected by 60-min sampling using the enrichment chip. A fitting formula

326

with good R-squared (> 0.90) was established from the standard curve, which would be

327

convenient to the quantitative analysis. In addition, we found that when the number of spores in

328

sample was larger than 500, the spores could be easily intercepted and deposited at the front of

329

microsphere channel, which facilitate the chip to capture large amounts of spores avoiding the

330

escape of target spores. Generally, the concentration of spores in the indoor air was at the range of

331

50 to × 103 CFU·m−3 changing with seasons3. As reported in some researches, the outbreak growth

332

of fungi could release a high concentration of spores, approximately 104 CFU·m−3 10,25,52. These

333

results indicated that our system is feasible to handle the samples from common indoor air.


ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

334 335

Figure 5. (A) Validation of the feasibility of the detection chip, (a) Bright-field image of reaction

336

channel packed with microspheres, (b) Fluorescence image of reaction channel for detection of

337

500 spores, (c) Fluorescence image of reaction channel for detection of 0 spores as a blank. (B)

338

Optical density comparison of immunofluorescence detections of 500 spores (b) and 0 spores as a

339

blank (c). Values are the mean ± s.d. for n = 3, **p < 0.01.


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340 341

Figure 6. (A) Fluorescence images of reaction channels for detection of 10, 20, 50, 100, 200, 500,

342

750, and 1000 of A. niger spores, respectively. (B) Standard curve for evaluation of spore count in

343

reaction channel via optical density of fluorescence. Standard deviations for n = 3.

344

To further test specificity of the proposed system, A. oryzae spore was used as interferent

345

component mixed with A. niger samples in spore-aerosol generations. The A. oryzae spore was

346

chosen as the interferent because its diameter is similar to that of A. niger spore which makes it

347

can be physically captured by the enrichment chip. In addition, these two kinds of Aspergillus

348

spores are hard to be distinguished under the bright-field microscope. It is a good example to test

349

the specificity of the proposed system. The spore mixtures containing 25 %, 75%, and 100% of A.

350

niger spores were aerosolized into the generation cube following the aerosol generation

351

procedures above, respectively. For 5 min sampling by the enrichment chip, the collected spores


ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

352

were eluted with PBS and then analyzed on the detection chip. The fluorescence images of each

353

sample was shown in Figure 7A. The fluorescence density of sample containing 75% A. niger

354

spores was 2.31 times higher than that of sample containing 25% A. niger spores (as shown in

355

Figure 7B). The results demonstrated our detection chip could distinguish the specific pathogenic

356

spores from other species with good specificity. Furthermore, we calculated the number of A.

357

niger spores in each sample (as Table S3-5 shown) by converting fluorescence density to spore

358

quantity using the fitting formula. The data of estimated spore-aerosol concentration could match

359

with the data from APS. The actual samples with unknown concentrations also be detected

360

through the proposed system and traditional sedimentation method (shown in Table S6). Therefore,

361

the proposed system has the capability for quantitative analysis of specific pathogenic spores

362

directly from the air. Furthermore, the spore analysis in this system took relative less time

363

compared with other methods in Table S2. Its detection limit might be not as low as those highly

364

sensitive method such as biosensors30,31 and qPCR28, however, the sensitivity of our system used

365

for the forewarning of airborne spores in air could fulfill practical detection, since the fungal

366

spores usually threaten human health under a relative high concentration.


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367 368

Figure 7. (A) Fluorescence images of reaction channels for detection different mixed samples

369

containing A. niger spores and A. oryzae spores, (a) 100% A. niger spores, (b) 75% A. niger spores

370

and 25% A. oryzae spores, (c) 25% A. niger spores and 75% A. oryzae spores. (Total number:

371

~500 spores). (B) Optical density comparison of immunofluorescence detections of 100% A. niger

372

spores (a), 75% A. niger spores and 25% A. oryzae spores (b), 25% A. niger spores and 75% A.

373

oryzae spores (c). Values are the mean ± s.d. for n = 3, **p < 0.01.

374

CONCLUSIONS

375

Herein, we presented a microfluidic platform for direct and precise analysis of specific pathogenic

376

spores from the air. Experimental results demonstrated that the system can be used for enrichment

377

and semi-quantitative detection of airborne Aspergillus niger spores. The elution volume of the

378

upstream enrichment chip is small and can be directly used by the downstream detection chip

379

avoiding secondary condensation. The whole process could be completed within 3 h from spores

380

enrichment to fluorescence analysis on the chip. Moreover, the detection limit of the analysis chip


ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

381

is ~20 spores, equivalent to ~300 spores·m-3 of the concerned targets in air. In addition, the

382

detection chip could be read by a mini fluorescence detector, suitable for point-of-care application.

383

Overall, we believe that the presented system can help to monitor airborne pathogenic spores in

384

the public buildings, public transportations, as well as private places. It also has potential to enable

385

automatic assessment of other specific spore levels in indoor air.

386

ASSOCIATED CONTENT

387

Supporting Information

388

Additional data including the schematic diagram of spore-aerosol generation system, illustration

389

of the system to test the efficiency of the enrichment chip, the comparison of different methods for

390

fungi sampling and detection, the number calculation of A. niger spores in different mixture

391

samples by converting fluorescence density to spore quantity using the fitting formula and actual

392

samples detection by the microfluidic system are given in the Supporting Information.

393

AUTHOR INFORMATION

394

Corresponding Author

395

* E-mail: [email protected] (G.S.)

396

Notes

397

The authors declare no competing financial interest.

398

Acknowledgments

399

This work was supported by National Natural Science Foundation of China (21577019, 21527814,


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21377026), the National Key Research and Development Program of China (2016YFD0500604),

401

the

402

(No.17JC1401001).

Program

of

Science

and

Technology

Commission

403 404 405 406 407 408 409 410


of

Shanghai

Municipality

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A microfluidic system for rapid detection of airborne pathogenic fungal

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