The development of paper discs immobilized with luciferase/D-luciferin for the detection of ATP from airborne bacteria
Introduction
Airborne microorganisms including bacteria, fungi, and viruses are a major threat to public health and frequently cause public panic due to the transmission of pathogens through the air without a barrier [1,2]. Our society remains vulnerable and defenseless against unexpected wide-spreading of airborne pathogens. Bacterial infection or viral flus transmission via the air are serious threats worldwide. Due to their small sizes, they can easily be transported long distances (up to several kilometers) by the wind and can enter the lungs via inhalation, which can cause significant public health concerns non-infectious diseases such as hypersensitivity pneumonitis [3], allergies [4], and asthma [5], and infectious diseases such as legionellosis, influenza [6] and sputum-positive tuberculosis [7]. Therefore, rapid detection or identification methods of airborne pathogens are important. Currently, there are a number of conventional methods for the detection and identification of airborne microbes in order to regulate and control the air quality, including culture-based methods [[8], [9], [10]], microscopy [[11], [12], [13], [14]], immunoassay [[15], [16], [17]], and polymerase chain reaction (PCR) [[18], [19], [20]]. However, the real-time detection of airborne bacteria remains challenging due to experimental complexity and costs [1]. Conventionally, the suggested regulation to identifying airborne microbes is the culture based method; however, it takes several days to grow the bacteria in culture medium until colony formation [21,22]. Furthermore, with this method, only a small proportion of microorganisms [2,23], about less than 10%, can be culturable and the remained proportion is not culturable [24]. Conversely, the considerable detection methods of airborne microorganisms to identify the exact species use highly specific antibodies or DNA or RNA gene probes for PCR [25,26]; however, this method requires several hours of preparation, complicated procedures, and operation of experts [1]. Additionally, direct methods have been used that detect the microorganisms based on the adenosine triphosphate (ATP)-driven luciferase/D-luciferin photoemission with high sensitivity and specificity of ATP [21,27]. In luciferase/D-luciferin photoemission, D-luciferin reacted with ATP in the presence of luciferase as a catalyst, molecular oxygen, and Mg2+ ions to produce oxyluciferin, adenosine monophosphate, and light. The relaxation of the electronically excited oxyluciferin to the ground state resulted in the emission of yellow-green light in the wavelength range of 550–570 nm, which was normally measured using a luminometer [28,29]. However, this biochemical method has several limitations owing to the activity of luciferase, which significantly reduced with increasing time in the solution phase, and the requirement of several hours to prepare the luciferase/D-luciferin solution. Numerous bacterial detection methods have been previously suggested; however, real-time airborne bacterial detection and enumeration from the air remains challenging.
In this study, we suggest techniques capable of rapid detection and enumeration of airborne bacteria using paper discs immobilized with a mixture of luciferase/D-luciferin (co-immobilization). The most commercially available ATP assay kits use the liquid-based reaction mixture and the essential enzyme, luciferase, should be kept in cold (−20 °C to 4 °C) before using it for maintenance of enzyme activity. These conditions are the bottleneck to use the ATP assay kit for a field test. Our approach can reduce the detection time using ready-made paper discs for the luciferase/D-luciferin reaction, wherein the activity of luciferase enzyme is well maintained. Following the immobilization process, paper discs are stored stably at room temperature and used directly without any pretreatment rather than solution-based reaction. They are ready-to-use for the heat-lysis of samples collected from the air. We optimized the immobilization procedure for the mixture of luciferase and D-luciferin using paper discs. We also investigated storage stability of the paper discs immobilized and the appropriate lysis methods for the reaction with the paper discs. Using Collison nebulizer, the present study simulated airborne bacteria status of Escherichia coli (E. coli) as a model case, and determined the detectable concentration of E. coli using the paper discs immobilized with luciferase/D-luciferin.
Section snippets
Reagents, instruments, chemicals, and bacterial cells
We used luciferase, D-luciferin, dilution buffer, lysis buffer, and ATP standard directly, which were included in the “Roche ATP Bioluminescence Assay Kit HSII” (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The luciferase/D-luciferin mixture was prepared from the kit components by adding 2.5 mL of supported dilution buffer into a luciferase vial which contains D-luciferin. The luciferase/D-luciferin solution which contains the traces of Mg2+ was then aliquoted to 200 μL each and frozen at
Optimization of lysis method
To prepare the lysates of airborne bacteria, we aimed to identify the most convenient lysis method for the samples captured from the air, considering the automated bioaerosol monitoring system, which is our final goal (not in this study). Lysis is the key contributor for the successful detection of ATP extracted from the bacteria captured from the air, and the method should be simple with a high efficiency. The lysis process starts to break down the cellular membrane of bacterial cells and
Conclusions
We proposed that paper discs immobilized with luciferase/D-luciferin can be used to detect ATP extracted from bacterial samples collected from the air. The paper discs following immobilization could be stored stably for over 30 days at room temperature, and be used to detect bacterial ATP immediately after simple heat lysis of the bacterial samples. The detection time for bacterial samples after collected from the air was less than 7 min included 2 min of heat lysis and 5 min of bioluminescence
Acknowledgments
This work was finally supported by the Korea Institute of Science and Technology (KIST) Institutional Research Program and in part by the Korea Ministry of Environment (MOE) as “Public Technology Program based on Environmental Policy (No. 2016000160008)".
Dung T.Nguyen received her Bc science in Chemistry from Hanoi University of Science in 2009. She is a master student in University Science and Technology, KIST-School. Research interest of her is the development of biosensor to detect airborne microorganism.
References (42)
- et al.
Review of bioaerosols in indoor environment with special reference to sampling, analysis and control mechanisms
Environ. Int.
(2015) - et al.
Non-culturable bioaerosols in indoor settings: impact on health and molecular approaches for detection
Atmos. Environ.
(2015) - et al.
Detection and enumeration of airborne biocontaminants
Curr. Opin. Biotechnol.
(2004) Detection and characterization of biological and other organic-carbon aerosol particles in atmosphere using fluorescence
J. Quant. Spectrosc. Radiat. Transfer.
(2015)- et al.
A new method for the real-time quantification of airborne biological particles using a coupled inertial aerosol system with in situ fluorescence imaging
Sens. Actuators B Chem.
(2017) - et al.
Biosensors for monitoring airborne pathogens
J. Lab. Autom.
(2015) - et al.
Microgravimetric immunosensor for direct detection of aerosolized influenza A virus particles
Sens. Actuators B. Chem.
(2007) - et al.
Application of real-time PCR for total airborne bacterial assessment: comparison with epifluorescence microscopy and culture-dependent methods
Atmos. Environ.
(2008) - et al.
Rapid and specific detection of bacteria using bioluminescence
Anal. Chim. Acta
(2002) - et al.
The assessment of bioaerosols: a critical review
J. Aerosol. Sci.
(1994)
Incorporating polymerase chain reaction-based identification, population characterization, and quantification of microorganisms into aerosol science: a review
Atmos. Environ.
The real-time polymerase chain reaction
Mol. Aspects Med.
Biosensors for pathogen detection: a smart approach towards clinical diagnosis
Sens. Actuators B Chem.
A microfluidic ATP-bioluminescence sensor for the detection of airborne microbes
Sens. Actuators B. Chem.
Firefly luciferase inhibition
J. Photochem. Photobiol. B
A convenient one-step extraction of cellular ATP using boiling water for the luciferin–luciferase assay of ATP
Anal. Biochem.
Quantitating adenylate nucleotides in diverse organisms
J. Biochem. Biophys. Methods
The effects of meteorological factors on atmospheric bioaerosol concentrations-a review
Sci. Total. Environ.
Pathology of hypersensitivity pneumonitis
Curr. Opin. Pulm. Med.
Asthma and respiratory physiology: putting lung function into perspective
Respirology
Characterization of viral agents causing acute respiratory infection in a San Francisco University Medical Center Clinic during the influenza season
Clin. Infect. Dis.
Cited by (20)
Direct and indirect tools for identification and quantification of microbes associated with bioaerosols
2023, Bioaerosols Emission from Anthropogenic Sources: Influencing Factors, Microbial Diversity, Epidemiological Threats, and Control ApproachesAIEgen-based nanoprobe for the ATP sensing and imaging in cancer cells and embryonic stem cells
2021, Analytica Chimica ActaCitation Excerpt :Among them, luminescence-/fluorescence-based sensing systems have gained great attention because of their simple operation process, intuitive signal response, high sensitivity, selectivity, and biocompatibility [14–18]. Currently, several ATP monitoring systems, based on luminescence (luciferin-luciferase) [19–21] and fluorescence (genetically-encoded fluorescent sensors) [22–24], are actively used in biological studies, and some of these are commercially available [25,26]. Although such systems are efficient and useful, developing a small organic fluorescence probe for the ATP is needed in terms of ease-of-use and for a wide range of applications.
Four-stage signal amplification for trace ATP detection using allosteric probe-conjugated strand displacement and CRISPR/Cpf1 trans-cleavage (ASD-Cpf1)
2020, Sensors and Actuators, B: ChemicalNano-lantern on paper for smartphone-based ATP detection
2020, Biosensors and BioelectronicsCitation Excerpt :This is consistent with the threshold for positivity of UTI reported in the literature (105 CFU/mL). When comparing the analytical performance of the ATP sensing paper combined with Oneplus 5 smartphone detection, the LOD (10−14 mol of ATP corresponding to 3.8 × 104 CFU/mL) was about one order of magnitude higher compared to those recently reported by Nguyen et al. (2018) and Santangelo et al. (2018). However, it must be pointed out that our assay contains less amount of reagents than previously reported ATP biosensors relying on D-luciferin/luciferase system and does not require the use of benchtop luminometer for BL acquisition but exploits the smartphone camera as portable optical detector (Nguyen et al., 2018).
Microfluidic paper-based analytical devices for environmental analysis of soil, air, ecology and river water
2019, Sensors and Actuators, B: ChemicalCitation Excerpt :The authors in [137,138] used μPAD-coated AgNPs aggregation and multilayer paper-based device for colorimetric and electrochemical quantification, respectively, to detect particulate matter (PM) pollution and six metals (Cd, Pb, Fe, Cu, Ni, and Cr) in air sample. A special paper-based device is used to detect ATP from airborne bacteria developed by Nguyen et al. [139]. The paper-based device can detect ATP extracted from purified E. coli as low as 1.17 × 103 CFU/mL.
Dung T.Nguyen received her Bc science in Chemistry from Hanoi University of Science in 2009. She is a master student in University Science and Technology, KIST-School. Research interest of her is the development of biosensor to detect airborne microorganism.
Hye Ri Kim received Bc science in Bio-Environmental Chemistry from Chungnam National University in 2017. She is a master student in University Science and Technology, KIST-School. Reasearch interest of her is the development of biosensing recepters to detect airborne microorganism.
Jae Hee Jung has received Ph.D. in mechanical engineering from Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea (2008). He is currently a senior research scientist in the center for environment, health, and welfare research at the Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea. His research has been focused on the detection and control of airborne biological particulate matters.
Kang-Bong Lee is a principal investigator and a professor in Korea Institute of Science and Technology, Seoul, Republic of Korea. His current research includes fabrication and design for various fluorescence sensors and colorimetric nanoparticle sensors.
Byoung Chan Kim has joined Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea as a principal research scientist and serves also as an associate professor in Department of Energy and Environmental Engineering at KIST-school, University of Science and Technology (UST) in Korea. He received Ph.D. (2005) and master degree (2001) in Environmental Engineering from Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea, and bachelor degree (1999) of Chemical Engineering from Sogang University, Seoul, Republic of Korea. Before joining KIST, he worked as a Post-Doc researcher at Pacific Northwest National Laboratory (PNNL), Richland, USA and as a senior research scientist at Institute Pasteur Korea, Seongnam, Republic of Korea. His research interests cover aptamer development against environmental hazardous substances including pathogens and chemicals, and application of aptamer to biosensors. He has published more than 65 peer reviewed papers.