A microfluidic ATP-bioluminescence sensor for the detection of airborne microbesIntroductionMaterials and methodsMicrobes and reagentsAerosol generation and condensation systemMicrofluidic systemDetection of ATP bioluminescenceResults and discussionAnalysis of bioaerosolsOptimization of flow rate in the microfluidic channelCondensation systemCalibration of electric circuit and measurement of pure ATPATP-bioluminescence vs. concentrations of airborne microbesConclusionsAcknowledgementsReferencesAvailable online at www.sciencedirect.comSensors and Actuators B 132 (2008) 443–448A microfluidic ATP-bioluminescence sensor for the detectionof airborne microbesSeung Jae Lee, Jae Sung Park, Hee Taek Im, Hyo-Il Jung∗Laboratory of Biochip Technology, School of Mechanical Engineering, Yonsei University, Seoul, South KoreaAvailable online 18 December 2007AbstractAirborne pathogenic microorganisms are hazardous bioaerosols which often cause serious respiratory diseases. To prevent airborne infectiousdisease, real-time detection and monitoring systems of airborne pathogens are needed. Since ATP (adenosine triphosphate) is a major biologicalenergy source, the detection of ATP from aerosol reflects the existence of living microbes. Therefore, we developed a new biosensor to detect ATPfrom aerosols in real-time using an aerosol condensation system, a microfluidic channel, and an ATP-bioluminescence transducer. The condensationsystem enabled aerosol microbes (4 L) to be hydrosolized (0.2 ml) in 2 min. The bacterial intracellular ATP was then extracted in the passage throughthe microfluidic channel. The concentration of ATP could be determined by a bioluminescence sensor integrated in the channel. In this study, weused B. subtilis and E. coli JM110 as model airborne microbes. Our system can determine the existence of airborne microbes within 10 min. In thefuture, the application of our device will extend to the detection of fungi and consequently contribute to improving indoor air quality.© 2007 Elsevier B.V. All rights reserved.Keywords: Airborne microbes; Aerosol condensation; Microfluidic channel; ATP-bioluminescence sensor1. IntroductionThe real-time detection of microorganisms such as virus,bacteria, and fungi is an emerging and rapidly evolving fieldof research. The spread of airborne pathogens like measles,anthrax, Legionella, influenza, smallpox, and rhinovirus is oftenregarded as major threats of public health since they cause severeairborne infectious diseases with high mortality rates [1]. Fur-thermore, most bacterial and viral pathogens can be used forbiological weapons capable of immense destruction [2]. Theseairborne diseases can spread rapidly by means of airborne trans-mission from person to person via the respiration of pathogenicbioaerosols. In order to prevent the transmission of such airborneinfectious diseases and control dangerous biological particles inpublic places and dwellings, efficient real-time detection sys-tems are required.Conventionally, the detection of airborne bacteria has beenachieved by collecting and culturing, a method which is veryeffective but requires a long incubation time (at least 24 h). Ingeneral, the collection of living organisms is achieved by com-∗Corresponding author. Tel.: +82 2 2123 5814; fax: +82 2 312 2159.E-mail address: [email protected] (H.-I. Jung).monly used sampling methods in aerobiology, e.g. filtration, airwashing, impingment, and impaction [3].In recent years, new techniques have been developed toreplace the standard sampling method in order to reduce theduration of testing. Mainelis et al. [4] developed a new bioae-orosol sampler, called as electrostatic precipitator, which utilizedan electric field to deposit charges on bacterial samples anda solid agar as a bacterial growth media. After the develop-ment of the sedimentation method, Vadrot et al. [5] adaptedthe polymerase chain reaction (PCR) for direct detection ofMycobacterium tuberculosis. Deloge-Abarkan et al. [6] testedand compared the main principles for bioaerosol collectionmethods, i.e. solid impaction, liquid impingement and filtration,and they performed fluorescent in situ hybridization (FISH) forairborne Legionella bacteria detection. Most recently, Senguptaet al. [7] reported a detection method based on Raman spec-troscopy which relies on inelastic scattering, or Raman scatteringof monochromatic light, usually from a laser in the visible range.They utilized a silver coated bioanalyte suspension to obtain anenhanced Raman spectrum, and then could detect and character-ize airborne bacteria rapidly by injecting the suspension througha light scattering chamber.ATP (adenosine triphosphate) is the most important biolog-ical fuel in living organisms. Detecting ATP originating in air0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.snb.2007.10.035444 S.J. Lee et al. / Sensors and Actuators B 132 (2008) 443–448Fig. 1. Schematic diagram of our experimental set-up. The system consists of PAGS (Pneumatic Aerosol Generation System), a high-efficient condenser and amicrofluidic chip for ATP extraction and detection of bioluminescence.could thus be an important method for detecting living organ-isms like airborne pathogens, although the critical biohazardconcentration is yet unknown. Quantitative measurements ofATP have been applied to biological and environmental systemsfor years. For example, the growth of bacteria was monitoredin real time by the measurement of bioluminescence [8,9].However, most of the applications in the area of ATP detec-tion were limited to food and hygienic systems, for instance,determining the surface cleanliness of kitchen using traditionalhygiene swabbing method plus ATP-bioluminescence [10] andevaluating the microbial load on hands or domestic surfacesby ATP-bioluminescence monitoring as a surrogate marker[11].In this paper, we demonstrate a new real-time detection sys-tem to measure ATP extracted from airborne microbes using (1)a condensation system to concentrate aerosol, (2) a microfluidicchannel to extract bacterial ATP, and (3) a bioluminescence sen-sor to measure ATP content. Our technique will help improveenvironmental monitoring methods to prevent airborne infec-tious diseases.2. Materials and methods2.1. Microbes and reagentsBacteria were obtained from Korean Culture Center ofMicroorganisms (KCCM). E. coli JM110 (ATCC 47013) andB. subtilis (ATCC 6633) were grown in nutrient culture mediaat a