Fully Automated Field-Deployable Bioaerosol Monitoring System

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Fully Automated Field-Deployable Bioaerosol Monitoring System using Carbon Nanotube-based Biosensors Junhyup Kim, Joon-Hyung Jin, Hyun Soo Kim, Wonbin Song, SuKyoung Shin, Hana Yi, Daeho Jang, Sehyun Shin, and Byung Yang Lee Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b06361 • Publication Date (Web): 12 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

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Environmental Science & Technology

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Fully Automated Field-Deployable Bioaerosol

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Monitoring System using Carbon Nanotube-based

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Biosensors

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Junhyup Kim,† Joon-Hyung Jin,† Hyun Soo Kim,† Wonbin Song†, Su-Kyoung Shin,‡ Hana

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Yi,‡ Dae-Ho Jang,† Sehyun Shin,† and Byung Yang Lee†*

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†

Department of Mechanical Engineering, Korea University, Seoul 02841, Korea

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‡

BK21PLUS Program in Embodiment: Health-Society Interaction, Department of Public

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Health Sciences, Graduate School, Korea University, Seoul 02841, Korea

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*To whom correspondence should be addressed. E-mail: [email protected], Tel: +82 2 3290

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3365

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KEYWORDS: bioaerosol, fungi, real-time monitoring, carbon nanotube, field-effect

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transistor

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ABSTRACT

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Many progresses have been performed in the field of automated monitoring systems of

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airborne pathogens. However, they still lack of the robustness and stability for field

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deployment. Here, we demonstrate a bioaerosol auto-monitoring instrument (BAMI)

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specifically designed for in-situ capturing and continuous monitoring of airborne fungal

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particles. This was possible by developing highly sensitive and selective fungi sensors based

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on two-channel carbon nanotube field-effect transistors (CNT-FETs), followed by integration

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with a bioaerosol sampler, a Peltier cooler for receptor lifetime enhancement, and pumping

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assembly for fluidic control. These four main components collectively cooperated with each

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other to enable real-time monitoring of fungi. The two-channel CNT-FETs can detect two

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different fungal species simultaneously. The Peltier cooler effectively lowers the working

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temperature of the sensor device, resulting in extended sensor lifetime and receptor stability.

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The system performance was verified in both laboratory conditions and real residential areas.

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The system response was in accordance with reported fungal species distribution in the

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environment. Our system is versatile enough that it can be easily modified for the monitoring

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of other airborne pathogens. We expect that our system will expedite the development of

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hand-held and portable systems for airborne bioaerosol monitoring.


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INTRODUCTION

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Bioaerosols, occupying a large portion of air pollutants, are air suspensions including a

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variety of microbial particles such as bacteria, fungi, viral particles, pollens, and so forth.1

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Among these airborne microbial particles, fungi are known as a one of major causes of

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allergic disorders.2-4 Children, for example, who stay during most of their daily life in public

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facilities such as schools or community centers are even more susceptible to fungal allergens

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and have more probability of suffering from many pulmonary diseases caused by fungi

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group.5 Indeed, fungus-originated allergens have currently attracted many attention due to

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their adverse impacts on public health and indoor air quality.5 Therefore, we need a reliable

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and robust monitoring systems for the early detection of hazardous allergenic fungi in the

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environment. This will enable one to take active measures to reduce or remove those hazards

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as soon as possible by methods such as filtering or ventilation.6, 7 Other laboratory bench-top

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equipments involve techniques such as surface plasmon resonance,8 high performance liquid

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chromatography,9 gas chromatography mass spectrometry10 and so on. However, although

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these methods are highly sensitive and reliable, they are usually time consuming, costly, labor

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intensive, and therefore, not applicable for in-situ real-time detection of allergenic

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bioaerosols in the field. Meanwhile, new biosensors using nanoscale materials such as

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nanoparticle,11 nanotube (NT),12 nanowire (NW),13 nanosheets and graphene14 as sensor

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transducers have been demonstrated to have remarkable sensitivity, enabling sometimes

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single molecule detection.15 In particular, nanobiosensors based on NT or NW-based field-

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effect transistors (FETs) are suitable for miniaturization, mass production, and real time

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monitoring, due to their compatibility with conventional microfabrication process and

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versatile functionalization methods.16 However, these techniques are still in their

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development stage and usually need further system integration for field deployment. A recent 3 ACS Paragon Plus Environment


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work using nanomaterials demonstrated that airborne influenza virus particles can be

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collected from air and detected by antibody immobilized Si NW FETs integrated with

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microfluidic systems.17 However, their system was aimed to virus and limited in the long-

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time stability and operation time needed for the continuous real-time monitoring of airborne

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fungal allergens in real field situations.

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In this work, we demonstrate a Bioaerosol Auto-Monitoring Instrument (BAMI) for real-time

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detection of two kinds of allergenic fungal species, Aspergillus niger (A. niger) and

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Alternaria alternata (A. alternata), which are related to various allergenic and pulmonary

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diseases.18,

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several days are demonstrated using the same sensor device. This was possible by developing

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highly sensitive and selective nanomaterial-based biosensors for A. niger and A. alternata

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based on carbon nanotube (CNT) FETs, combined with sensor cooling capabilities and a

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microfluidic system that enables extensive washing of the sensor device for sensor

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regeneration. The system was tested in real field conditions. The response in the field was in

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accordance with reported fungal species distribution in the environment. Our system is

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versatile enough and its use can be easily extended to detect microbial particles other than

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fungi in the future. We expect that our system will expedite the development of hand-held

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and portable systems for airborne allergenic fungi monitoring.

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Sensor regeneration capabilities and extended system operation time up to


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MATERIALS AND METHODS

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A. Preparation of Target Fungal Solution

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To perform quantitative experiments, we first prepared A. alternata solutions with known

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concentration (in weight per volume) by carefully weighing the fungi amount using a ultra-

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microbalance (XF2U, Mettler Toledo).20 The Alt a 1 mAb 121-immobilized CNT channel

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was recorded as a function of time with continuous injection of 2 µL of different

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concentrations of A. alternata suspended in 1 mM PBS solution at every 50 sec.

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B. Activity Verification of A. alternata Antibody

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The effect of temperature on antibody activity was determined using enzyme-linked

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immunosorbent assay (ELISA). Alt a 1 monoclonal antibody (mAb, 1.83 mg/mL, Indoor

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Biotechnologies, Inc.) was diluted in 50 mM carbonate-bicarbonate buffer (pH9.6) and added

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in each well of microplate (NUNC Maxisorp, Nalgene). The plate was incubated overnight at

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different temperatures (4, 25, 37 °C), then washed three times with 200 µL PBS washing

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buffer with 0.05% Tween-20 (PBST) and blocked with 100 µL PBST solution with 1% BSA

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(BSA/PBST) at various temperatures (4, 25, 37 °C) for 30 min. After washing three times

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with 200 µL PBST, 100 µL aliquots of different concentrations of A. alternata cells (0.1 ~

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100 µg/mL) in 1% BSA/PBST were then applied to each well and the plates were incubated

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at different temperatures (4, 25, 37 °C) for 1 h. After washing three times with 200 µL PBST,

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100 µL aliquots of 1000 times diluted biotinylated anti-Alt a 1 mAb (1 mg/mL) in 1%

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BSA/PBST were added to each well of plate. Then, after washing the plates three times with

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200 µL PBST, 100 µL aliquots of 1000 times diluted Streptavidin-peroxidase (Aldrich, Inc.,

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0.25 mg/mL) in PBST was added and the plate was incubated at room temperature for 30 5 ACS Paragon Plus Environment


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min. Finally, after washing three times with PBST, 100 µL aliquots of 1 mM ABTS in 70

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mM citrate phosphate buffer (pH 4.2) and 1000 times diluted hydrogen peroxide were added

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to each well and the plate was incubated for 15 min at room temperature for blue-green color

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development. The enzyme reaction was stopped by adding 100 µL 2% oxalic acid (Sigma).

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The optical density (O. D.) at 405 nm (Figure 2) was read using a microplate reader Victor™

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X3 (Perkin Elmer Inc., USA). In field tests, ELISA was carried out by coating 500 times

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diluted Alt a 1 mAb to double the antibody concentration. Then, sampled solutions were

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reacted for 2 h to double the reaction time. Finally, 100 µL aliquots of 1000 times diluted

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hydrogen peroxide in 70 mM citrate phosphate buffer (pH 4.2) and 1 mM ABTS were added

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to each well and left to react for 30 min.

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C. Alexa Fluor Dye Labeling of Alt a 1 mAb

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We prepared 1 mL of Alt a 1 mAb antibody solution of concentration 1 mg/mL, and then

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added 100 µL of 1 M sodium bicarbonate solution. 100 µL of the Alt a 1 mAb antibody

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solution was added to Alexa Fluor® dye tube and gently inverted a few times to mix and then

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incubated for 1 h at room temperature. The purification column was assembled and 1.5 mL of

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purification resin was applied to the column. The purification was then centrifuged for 3 min

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at 1100 G. G represents gravitational constant. Afterwards, 100 µL of the dye-conjugated

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antibody solution was applied to the purification column and centrifuged for 5 min at 1100 G

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to collect the dye-labeled antibody solution. The fluorescence image was taken with Nikon

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eclipse LV100ND optical microscope with excitation wavelength at 510–560 nm and

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exposure time = 60 sec.

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D. CNT-FET Preparation


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We prepared the CNT-FET following a previously reported process.20 Briefly, the CNT-FETs

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were fabricated on a highly doped p-type silicon wafer with 300 nm thick thermal oxide

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(SiO2). Carboxylic acid functionalized single-walled CNTs (> 90%, Aldrich, Inc.) were

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patterned to form a channel of 3 µm width and 10 µm length utilizing directed assembly on a

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molecularly patterned substrate with octadecyltrichlorosilane (> 90%, Aldrich, Inc.). Source

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and drain electrodes consisting of 30 nm-thick Au films over 5 nm-thick Cr adhesion layer

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were fabricated by conventional metal evaporation and lift-off process.

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E. Functionalization of CNT-FETs with Primary Antibody

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For antibody immobilization, the carboxyl-functionalized CNT surface was treated with 1-

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ethyl-3-(3-dimethylaminopropyl)-carbodiimide

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(Aldrich, Inc.) chemistry to anchor the antibodies to the carboxyl-functionalized CNT

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surface.21 Antibodies for selective detection of A. niger and A. alternata were purchased from

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Bio-Rad Laboratories, Inc. and Indoor Biotechnologies, Ltd., respectively. As described

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above, our sensor device was comprised of two CNT channels, each one immobilized with

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different kind of antibodies. In channel 1, antibodies (Alt a 1 monoclonal antibody (mAb))

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that specifically binds to the A. alternata coat protein (Alt a 1 Immunogen) were immobilized

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on the CNT-FET using EDC/NHS chemistry. For this purpose, the CNT-FET were immersed

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in 0.2 M/0.1 M EDC/NHS in 10 mM PBS for 4 h at room temperature. The EDC/NHS

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process enables substitution of the hydroxyl group of the carboxylic acid group with a more

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facile leaving group, i.e., NHS group. The mAb solution was prepared by diluting the as-

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received antibody solution by 1000 times in 1% BSA, 50% glycerol, 49% 1 mM pH 7.4 PBS

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buffer mixture, filtered by a 0.2 µm pore size filter. This antibody solution was dropped on

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the EDC/NHS-treated CNT-FET and kept for 4 h. In this process, the N-terminal of the

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antibody formed a strong amide bond with the carbonyl carbon of the CNT-FET. The

(EDC)/N-hydroxysuccinimide

(NHS)



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resulting antibody-immobilized CNT-FET devices were stored at 4 ºC before use. In channel

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2, an antibody (WF-AF-1) that specifically binds to the A. niger coat protein (galactomannan)

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was immobilized on the CNT-FET using EDC/NHS chemistry. We followed a previously

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reported process.20 The WF-AF-1 antibody solution was prepared by diluting the as-received

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antibody solution by 10 times in 1 mM PBS buffer solution. Phosphate-buffered saline (PBS,

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pH 7.4) solution was purchased from Aldrich, Inc.

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F. Liquid Gating Experiments

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The liquid gating experiments on A. alternata sensor was performed by first preparing a

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CNT-FET immobilized with an A. alternata antibody (Alt a 1 mAb). Then, 10 µL of 1 mM

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PBS buffer was dropped on the sensor region. The source-drain bias was maintained at 0.1 V.

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A cyclic liquid gate voltage from -0.5 to 0.5 V was applied using a Pt pseudo-reference

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electrode. All the application of voltage and current monitoring was performed using a

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semiconductor parameter analyzer (4200-SCS, Keithley, USA). To observe the effect of

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nonspecific target, 5 µL of 10 pg/mL A. niger solution was applied to the PBS buffer droplet.

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The gating scan did not show any significant shift in the gating curve (Figure 4C). However,

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when we applied another 5 µL of 10 pg/mL A. alternata solution, the current increased and

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the gating curve shifted upwards, which is due to the gating effect of the negatively charged

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fungi fragments to the p-type CNT-FET. In case of A. niger sensors, the liquid gating

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experiments were performed in the same method as above, only the sample being

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immobilized with A. niger antibody (WF-AF 1), as shown in Figure S4.

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G. BAMI System Configuration

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A photo image of the BAMI is shown in SI Figure S5. The base vessel of the bioaerosol

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sampler was modified from its original form by using an acryl vessel attached with four 8 ACS Paragon Plus Environment

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fluidic channels at the vessel bottom using silicon tubing (inner diameter = 1 mm), each one

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connected to four peristaltic pumps (P1, P2, P3, P4). Note that this modification enabled

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washing the vessel with PBS buffer solution and resampling of the fungal solution. The

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pumping actions of the four peristaltic pumps were completely controlled by built-in micro

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controller unit (MSP430, Texas Instruments, Inc.) communicating through RS-232 cable with

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a PC running a serial communication program. Each peristaltic pump can be separately

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operated by a switching relay. P1 supplies fresh PBS solution from the buffer reservoir to

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bioaerosol sampler base with a flow rate of 10 mL/min. P2 can drain the buffer from

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bioaerosol sampler base with a flow rate of 10 mL/min. P3 and P4 supply fungi-suspended

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buffer solution to the channels 1 and 2 of the CNT-FET sensor module (SI Figure S8),

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respectively, through polyether ether ketone-based microfluidic channels with flow rates of 1

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mL/min, corresponding to a flow velocity of 13 mm/s. The sensor response of the CNT-FETs

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was measured by monitoring the current change of each device under a DC bias of 0.1 V.

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Current-time characteristics of the CNT-FETs were recorded using a Keithley 2636A dual-

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channel sourcemeter driven by LabVIEW software. The Peltier cooler is in contact with the

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CNT-FET holder from the bottom side. A heat sink is in close contact with the cooler.

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Proportional-integral-derivative controller sets up a temperature, receives feedback

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temperature from the heat sensor installed in the CNT-FET sensor holder, and eventually

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controls the cooling temperature by turning on/off the power supply on the basis of the pulse

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width modulation.

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RESULTS AND DISCUSSION

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Figure 1 shows the functional diagram of the BAMI composed of a bioaerosol sampler based

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on a commercial air-to-liquid impactor (BioSampler®, SKC Inc), a microfluidic assembly

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including four peristaltic pumps for injection and withdrawal of the sampled fungal solution,

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two-channel CNT-FET-based detection circuitry for the detection of both A. niger and A.

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alternata, Peltier cooling for receptor lifetime elongation, and PC-based electronic control

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system. Here, we developed a novel biosensor for A. alternata and combined it with a

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previously reported sensor for A. niger.20 The CNT-FETs were combined with Peltier cooler,

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and then integrated with microfluidics and modified the bioaerosol sampler. The Peltier

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cooler has two effects: that of precooling the fungal solution before being injected to the

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CNT-FETs, and that of lowering the operation temperature of the immobilized antibodies.

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The sampled solution reaches the sensing surface through the cold side of the Peltier cooler to

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cool down the solution in advance to a temperature compatible to that of the CNT-FET

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sensor. To verify the activity of the Alt a 1 mAb and identify the optimum operation

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temperature, we performed sandwich ELISA experiments in three different temperature

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conditions (Figure 2). The A. alternata antibody showed enhanced binding activity as we

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lowered the temperature from 37 ºC to 4 ºC. Since the antibody lifetime in general is known

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to elongate as temperature is lowered,22 and the binding activity for A. alternata was

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enhanced at lower temperatures of 4 ºC, this justifies the usage of a Peltier cooler in our

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BAMI system.

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The CNT-FETs were prepared by previously reported methods.20 The CNT-FETs were

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produced in wafer-scale (SI Figure S1). Each device had two parallel channels, each channel

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composed of single-walled CNT network channels of with 10 µm length and 3 µm width (SI

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Figure S1 (A-B)). The IV characteristics showed a current of ~3.2 µA at 1 V (SI Figure S1 10 ACS Paragon Plus Environment

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(C)). The resistance distribution was in the range of 3.6 ± 2.5 MΩ (SI Figure S1 (D)). The

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gate characteristics showed a current on-off ratio of 2~3 in air and liquid gating conditions

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(SI Figure S1 (E-F)). Afterwards, the antibodies were immobilized onto CNT-FETs using

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EDC/NHS chemistry to prepare the A. alternata biosensors. The antibody immobilization

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was verified with atomic force microscopy and fluorescence imaging (Figure 3). As shown in

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Figure 3 (A-C), the topography showed increase of height by ~5 nm after antibody

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immobilization, which is similar to the reported size of the immunoglobulin G (IgG) such as

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Alt a 1 mAb.23 The binding activity of immobilized antibodies was confirmed by exposing

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the antibody-immobilized CNT to A. alternata solution and then conjugating it with Alexa-

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Fluor labeled secondary antibody. Figure 3 (D) shows that the fluorescence is maximum at

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the CNT-channel region. The non-specific binding was confirmed by performing a negative

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control experiment where no primary antibody was immobilized on the CNT-FET. When

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there was no primary antibodies immobilized, we could not observe any significant

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fluorescence intensity on the CNT regions (Figure S2). It should be noted that the images in

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Figure 3 reflects the binding of the antibody with fungi fragments and not with whole spores.

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It is known that airborne particles of A. niger and A. alternata fungi are composed of both

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spores/hyphae and fragments of them.24 And these fragments, in contrast to viruses which are

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infectious only as whole viruses, play the role as pathogens that cause atopic dermatitis,

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allergy and pulmonary diseases such as asthma and pneumonia.25 Also, the size of A. niger

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and A. alternata spores are in the range of 3 µm and 15 µm, respectively,26, 27 which makes

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the direct binding event between antibody and spores difficult. Therefore, the primary target

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material of our sensors were fungal fragments and not spores or hyphae.

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The sensitivity and selectivity of the A. alternata biosensor was first tested (Figure 4). Figure

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4 (A) shows that the sensor response was linearly proportional to the A. alternata 11 ACS Paragon Plus Environment


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concentration in the range from 10 pg/mL to 1 µg/mL. The limit of detection, defined here as

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the minimum target concentration resulting in a detectable sensor response, was 10 pg/mL.

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Furthermore, the detection limit was two-fold lower, compared with new immunoassay

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method.28 Here, we maximized the sensitivity by increasing the Debye length around the

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CNTs using diluted PBS buffer at 1 mM ionic concentration. This results in extended sensing

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region around the CNTs at liquid conditions.29 The Debye length at 1 mM PBS buffer can be

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estimated to be about 9.7 nm.30 Since Alt 1 mAb is an IgG type antibody which has a planar

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structure with diameter of ~8.5 nm and thickness of ~4 nm, we can expect IgG antibodies to

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be immobilized around the CNTs with random direction while providing multiple binding

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sites within the Debye length.23, 29, 30 The current increase with increasing concentration can

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be explained from electrostatic gating effect from the A. alternata spores and fragments.

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Since our sensors show quasi-linear response to log-concentration (inset of Figure 4 (A)), and

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both spores and hyphae contribute to the concentration, the signal reflects the total amount of

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allergens in air. In neutral pH, the surface of A. alternata is negatively charged because the

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isoelectric point of the A. alternata is around 4.2.31 The negatively charged A. alternata

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spores and fragments apply a negative gating effect to the p-type CNT channel (SI Figure S1

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(E)), and therefore, the current is increased in the CNT-FET channel. The sensor showed a

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large dynamic range of fungi concentration in the range of 10 pg/mL to 1 µg/mL with 65%

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total signal change (Figure 4 (A)). The conversion between mass concentration to actual

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spore number per volume needs additional parameters such as the ratio of spore to fragment

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that gets sampled into the PBS buffer during aerosol sampling for each fungal species and the

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fraction of fragments bound to the immobilized antibody that contribute to signal change,

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which is left for further study. As shown in Figure 4 (B), the selectivity of A. alternata sensor

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was tested by consequently injecting a set of predesigned mixture solutions of four different

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kinds of fungi: A. alternata, A. niger, Cladosporium cladosporioides (C. cladosporioides), 12 ACS Paragon Plus Environment

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and Penicillium chrysogenum (P. chrysogenum). The sensor showed no response to the first

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two mixtures containing no A. alternata. However, the electrical signal showed increasing

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sensor response to the last two mixtures that contained A. alternata of concentrations of 10

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and 100 pg/mL. Also, the selectivity was observed by liquid gating experiments (Figure 4

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(C)). The liquid gate characteristics showed a typical p-type channel behavior. When exposed

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to A. niger, the gate characteristics showed almost no change. However, when the sensor was

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exposed to the target fungi A. alternata, the sensor showed a rightward shift of the threshold

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voltage due to the hole enhancement effect by the negatively charged A. alternata

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fragments.32 For A. alternata sensor, sensor regeneration experiment was performed up to

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several times (Figure 4 (D)). However, the sensor showed reduced sensitivity with repeated

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number of washing. We expect that the number of repetitive regeneration can be increased

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with the utilization of more robust synthetic receptors in the future.

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The A. alternata sensor developed above was combined with A. niger sensor previously

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developed20 on each channel of the CNT-FET devices. The sensor for the A. niger also

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showed increasing current with the addition of A. niger solution in the concentration range of

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10 pg/mL to 1 µg/mL in the same way as A. alternata sensor (Figure S3). The liquid gating

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characteristics of A. niger sensors showed similar sensitivity and selectivity as reported

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before (SI Figure S4).20 These devices were combined with a bioaerosol sampler, Peltier

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cooler and microfluidic systems to build BAMI for the real-time monitoring of airborne fungi

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(SI Figure S5). The Peltier cooler provides the BAMI with control of operation temperature,

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allowing an optimized condition for detection and receptor lifetime elongation. This function

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is expected to be particularly useful for biosensing applications using protein-based receptors,

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because biomacromolecules such as enzymes and antibodies frequently demand a particular

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temperature

and

a

pH

for

best

performance

of

the

immobilized

functional 13

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biomacromolecules.33 Actual surface temperature of the Peltier cooler was confirmed with a

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thermocouple, resulting at temperatures of 6±0.5 °C, 25±0.2 °C, and 35±0.5 °C for set

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temperatures of 4, 25, and 37 °C, respectively. The Alt a 1 mAb showed its maximum

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activity at 4 °C (Figure 2), meaning that a low temperature operation enabled by the Peltier

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cooler allows enhanced antibody lifetime and more sensitive binding activity of the receptors.

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The operation of the microfluidics system was verified by observing the direct response to

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ammonium hydroxide solutions of concentrations in the range of 100 nM to 10 mM

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concentration with devices with bare CNTs (SI Figure S6). Decrease of CNT conductivity

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with increased ammonium hydroxide solution is known to be due to electron donating

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property of ammonium to the CNTs.34 The stability of the A. alternata antibody-

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functionalized sensor was verified by exposing the sensor to target fungi and comparing the

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sensor response at two different temperatures of 4 and 25 ºC for more than 5 days (Figure 5).

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When the sensor was kept at 25 ºC, the sensor response drooped after ~1 day, presumably

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due to denaturation of the antibodies. However, when the sensor was kept at 4 ºC, the sensor

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response maintained constant for up to 9 days (data only shown up to 5 days), showing the

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effectiveness of lowering the sensor operation temperature using a cooler.

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The sensitivity and selectivity of the sensors were verified by injecting solutions of fungi

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solutions with known concentrations to the sampler vessel and transported to the CNT-FET

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by the microfluidic system (Figure 6). First, the sensor response of the A. niger sensor in the

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range of 10 pg/mL to 1 ng/mL concentration is shown in Figure 6 (A). In contrast, when we

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introduced just PBS buffer, the A. niger sensor showed no response. Also, the selectivity of

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the A. niger sensor was verified by injecting solutions of other fungal species. The sensor

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showed no response to other fungal species such as A. alternata or C. cladosporioides (SI

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Figure S7). Next, the operation of the A. alternata was observed in the range of 10 pg/mL to 14 ACS Paragon Plus Environment

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100 ng/mL concentration (Figure 6 (B)). Note that the signal change is in accordance with

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Figure 4 (B). The sensor response showed a fast average rising time of ~20 sec. When the

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sensor was washed with PBS using the microfluidic system, the sensor response returned to

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its initial value with an average falling time of ~2 min. Here, the rising and falling time has

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been defined as the time required to change between 10% and 90% of the sensor response.

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This sensor recovery is probably due to moderate binding affinity and washing.35 Note that

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the sensor recovery characteristics is an important function that enables regeneration and

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lifetime elongation of the sensor device. The simultaneous operation of A. niger and A.

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alternata sensors are shown in Figure 6 (C). Here, we subsequently introduced 10 pg/mL A.

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niger and 10 pg/mL A. alternata, followed by PBS in intervals of 5 min. We can observe the

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sequential selective response of each sensor to their target and simultaneous recovery to their

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initial values when washed with PBS. We tested the selective sensor response in the presence

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of two additional interferent fungal species: C. cladosporioides and P. chrysogenum (Figure 6

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(D)). The four mixtures in Figure 6 (D) were applied additively. It shows that the sensor

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response only depends on the concentration of the specific target. These results show that our

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BAMI is effective in detecting fungal allergenic particles with extended sensor lifetime

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enabled by sensor cooling and washing system.

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The overall performance of the BAMI system including the sampler was tested in laboratory

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conditions by placing the BAMI system inside a homemade acryl chamber (size 300 × 300 ×

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500 mm) and exposing it to dish-cultured fungi to avoid contamination from other

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interferents (SI Figure S8 (A)). The bioaerosol sampler vessel contained 40 mL of D.I. water

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at the beginning of the experiment, and the volumetric flow rate from the bioaerosol sampler

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to the CNT-FET was 1 mL/min. To apply a fungal bioaerosol input to the bioaerosol sampler

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in a consistent way, we sampled the dish-cultured fungi by setting colony size criteria. For A. 15 ACS Paragon Plus Environment


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niger, a drop (1 mL) of A. niger suspension was spread on a agar plate (DifcoTM malt extract),

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and left to grow for 7 days. Among the A. niger colonies, those 10 colonies with a size

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smaller than 1 mm in diameter were carefully sampled with cotton ball and placed into a

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specifically-designed glass pre-chamber connected in front of the bioaerosol sampler. For A.

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alternata, we followed the same procedure as A. niger. However, the sampled colonies had

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diameter around 1 cm, considering the size difference between A. niger and A. alternata

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spores, the latter being ~5 times bigger in diameter compared to the former.26, 27 After placing

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the sampled cotton ball in front of the bioaerosol sampler, we turned on the bioaerosol

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sampler. After operating the bioaerosol sampler for 5 min, the sampled fungal solution in

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PBS was transported to the CNT-FET device by the microfluidic system. For initial 5 min,

339

the BAMI was operated to sample A. niger (SI Figure S8 (B), red) or A. alternata (SI Figure

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S8 (B), blue) fungi species in 30 sec intervals. Each curve reached its maximum in around 5

341

min. This implies the system requires at least 5 mL of a sample solution considering the fluid

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flow rate. After detection, the pumping system was operated to supply pure buffer solution to

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the fungi sensor for sensor regeneration. As control experiment, no current change was

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observed when we supplied clean buffer to the fungi sensor (SI Figure S8 (B), black). These

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results imply that our BAMI is effective in detecting fungal spore/hyphae fragments with

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extended sensor lifetime enabled by sensor cooling and washing system.

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Finally, we field-tested the BAMI system in real residential areas to verify the field

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deployment capability of the system. We chose two different locations for comparison (SI

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Figure S9). One location was at a basement level of a building with considerable fungi

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contamination, while the other was in upper level of a building with no observable fungal

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growth. The sampler was operated for 5 min at 12.5 L/min aspiration rate to concentrate

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environmental fungi in the PBS. Afterwards, the sampling was stopped and the sampled 16 ACS Paragon Plus Environment

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solution was injected to the antibody-immobilized CNT-FETs by the microfluidic channels.

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This sequence was repeated on already existing fungal solution to gradually increase the

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fungal concentration in the PBS solution. It should be noted that the system can in principle

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sample and detect at the same time (Figure S8). Here, the system was not operated in that

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way to ensure sufficient sampling of fungal fragments. Figure 7 (A) shows the BAMI

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response at different environmental conditions. In case of A. niger, the signal increased faster

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than that of A. alternata at both locations. The signal saturated after the signal changed by

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~80%. This relatively higher response compared to A. alternata is expected since A. niger is

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known to be an indoor-originated fungi. In contrast, the A. alternata response was lower than

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A. niger in both locations, and the A. alternata response was lowest at upper level residence.

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This is expected since A. alternata is outdoor-originated fungal species.33 The fungal solution

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collected by the sampler was verified to contain the target fungal species by ELISA and

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morphological analysis. The sampled solution were analyzed with ELISA and the O. D. was

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observed to increase at extended sampling time and therefore at higher fungal concentrations

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(Figure 7 (B)). Also, the fungal species after 5-day incubation at 25 ºC showed to contain

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both Aspergillus and Alternaria species when their morphology was inspected by SEM and

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optical image (SI Figure S10).36-39 Finally, a control experiment was performed to observe

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the effect of ambient pollutants such as gases and dust particles to the sensor response (SI

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Figure S11). Ambient indoor air was sampled using the BiosamplerTM at 12.5 mL/min

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aspiration rate for 5 min to prepare a bioaerosol solution in PBS buffer in the same way as

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Figure 7. The solution was then filtered with 0.02 µm pore size filter (Anodisc 47, Whatman,

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USA) to eliminate any particles larger than 0.02 µm including fungal spores and their

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fragments. The sensor was an A. niger sensor with antibody WF-AF-1 immobilized on the

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CNT-FET. The sensor showed no response to the filtered solution. This shows that the sensor

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response is not due to ambient gases or particles smaller than 0.02 µm in size. 17 ACS Paragon Plus Environment


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A practical fungi biosensor should be able to monitor fungi concentration in real-time, while

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enabling in-situ and real-time bioaerosol sampling, high sensitivity towards target fungi,

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selectivity towards target, and acceptable sensor life-time. The protein-based receptors such

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as antibodies can provide high selectivity when combined with highly sensitive nanomaterials

382

such as CNTs. However, their proper binding will happen only at adequate working

383

temperatures and pH values. Therefore, we developed the BAMI by combining bioaerosol

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sampler, Peltier cooler, microfluidic system, and CNT-FET to enable air-to-liquid sampling

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of airborne fungi particles, sensor life-time elongation, fungi solution transport, sensor

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regeneration and nanomaterial-based sensitive detection of fungal particles. The BAMI

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system additionally supports device regeneration option, and is perfectly compatible with any

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micro/nano bio devices. Future work will involve the introduction of miniaturized sampling

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system and pre-filtering process to eliminate dust particles and other interferents from fungal

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particles. Our system is versatile enough that it can be easily expanded for the monitoring of

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other airborne pathogens. We expect that our system will expedite the development of hand-

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held and portable systems for airborne bioaerosol monitoring.

393 394

Acknowledgments

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This project was supported by the Center for Integrated Smart Sensors funded by the Ministry

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of Science, ICT & Future Planning as Global Frontier Project (CISS-2011-0031866) and by

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National Research Foundation (2015R1A2A2A04002733).


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398

Supporting Information

399 400 401 402 403 404 405 406

Figures showing the basic electrical characteristics of CNT-FET devices. Fluorescence image of a CNT-FET with no primary antibody immobilized. Real-time response of A. nigerspecific sensor. Liquid gate characteristics of A. niger-specific sensor. Photo image showing the BAMI system. Figures showing selectivity results of A. niger sensor, chamber test using BAMI systemc, and optical images of field test location for BAMI system. Optical and SEM images for morphological analysis of Aspergillus and Alternaria species for samples collected from the field test locations using conventional impactor. This information is available free of charge via the Internet at http://pubs.acs.org/.

407 408

Notes

409

The authors declare no competing financial interest.



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410 411

412 413

Figure 1. Schematic block diagram of bioaerosol auto-monitoring instrument (BAMI)

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composed of bioaerosol sampler, pumping assembly, Peltier cooler, and carbon nanotube

415

field-effect transistor (CNT-FET) biosensor module. The bioaerosol sampler preconcentrates

416

the airborne fungi into PBS buffer. Peristaltic pumps P1 and P2 enable the washing of the

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collecting vessel. Peristaltic pumps P3 and P4 inject the preconcentrated fungi solution to the

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two-channel sensing module, composed of two-channel CNT-FETs, each immobilized with

419

selective receptor (antibody) to fungi Aspergillus niger (A. niger) and Alternaria alternata (A.

420

alternata), respectively. A Peltier cooler maintains the sensing module to a low temperature

421

(~4 ºC) to enhance the binding efficiency and extend the lifetime of the antibodies.

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423 424

Figure 2. Enzyme-linked immunosorbent assay (ELISA) measurements for testing antibody

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activity of A. alternata antibody (Alt a 1 monoclonal antibody (mAb)) at three different

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temperatures of 4 ºC, 25 ºC, and 37 ºC. The optical density (O. D.) of each reaction well was

427

read at 405 nm wavelength. The antibody showed enhanced binding activity at lower

428

temperatures.

429



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430 431

Figure 3. Verification of antibody immobilization to CNT-FET and target-receptor binding.

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(A) Atomic force microscopy (AFM) image of the CNT-FET channel. (B) AFM image of the

433

CNT-FET channel immobilized with A. alternata antibody (Alt a 1 mAb). (C) Comparison of

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height profiles before and after target binding. (D) Fluorescence microscopy image of CNT-

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FET sensor after fungal fragment binding followed by conjugation with Alexa Fluor®-568

436

labeled secondary antibody.

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Figure 4. Sensitivity and selectivity of A. alternata sensor. (A) Real-time response of A.

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alternata antibody (Alt a1 mAb)-immobilized CNT-FET device to consecutive injection of A.

441

alternata suspension in PBS solution of various concentrations (each injection volume = 2

442

µL). Note that the initial volume of the 1 mM pH 7.4 PBS solution on the device is 2 µL.

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(Inset) Corresponding calibration curve. The x-axis values are the injected A. alternata

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concentrations from 10 pg/mL to 1 µg/mL differing by ten times. (B) Sequential injection of

445

four different mixtures of fungal solutions. The first two mixtures contain no A. alternata,

446

while the last two mixtures contain A. alternata of concentrations differing by 10 times (10

447

and 100 pg/mL, respectively). Here, A: A. alternata, B: A. niger, C: P. chrysogenum, D: C.

448

cladosporioides. (C) Liquid gate characteristics change of our sensor to target and non-target 23 ACS Paragon Plus Environment


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fungi. Injection volume and concentration were always 5 µL and 10 pg/mL, respectively.

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Here, Vds = 0.1 V, and initial volume of PBS was 10 µL. (D) A. alternata sensor regeneration

451

test.

452

Afterwards, 2 µL of 10 pg/mL A. alternata solution was applied. After 50 sec, the sensor was

453

extensively washed with PBS buffer to recover the initial sensor signal level. This process

454

can be repeated for several times before the sensor sensitivity is reduced.

Initially, a 2 µL of PBS droplet was applied on the antibody-immobilized CNT-FET.

455


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Figure 5. Dependence of stability of antibody-immobilized CNT-FET sensor on temperature.

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The sensor was exposed to A. alternata and kept at 4 ºC and 25 ºC, respectively, for 5 days.



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Figure 6. Response of BAMI system to pure and mixtured fungal solutions. (A) Normalized

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conductance change time of the A. niger sensor to target concentrations from 10 pg/mL to 1

462

ng/mL, followed by sensor regeneration process composed of PBS washing. (B) Sensitivity

463

of A. alternata sensor to various target concentrations followed by sensor regeneration. (C)

464

Simultaneous response of the two-channel A. niger and A. alternata sensor to sequential

465

injection of A. niger and A. alternata followed by sensor regeneration with PBS washing. (D)

466

Two-channel sensor response to sequential injection of mixtures of four fungal species. The

467

mixtures were applied additively. In case of A. niger antibody-immobilized CNT-FET, it

468

responded to A. niger only. Meanwhile, the A. alternata antibody-immobilized CNT-FET

469

responded to A. alternata only. (A: A. niger; B: A. alternata; C: C. cladosporioides; D: P.

470

chrysogenum)


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471 472

Figure 7. Field testing of BAMI system in indoor residential areas. (A) Operation of BAMI

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system at indoor residential areas. The response at basement residential area (green/red) and

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upper level residential area (magenta/blue) of A. niger/A. alternata sensor were compared to

475

chamber conditions (black) with no fungi. (B) ELISA measurements on A. alternata content

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of the sampled solution in (A). The fungal solution collected by the sampler was verified to

477

contain A. alternata fungal species with increased concentrations with sampling time.



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Fully Automated Field-Deployable Bioaerosol Monitoring System

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