The development of paper discs immobilized with luciferase/D-luciferin for the detection of ATP from airborne bacteria

https://doi.org/10.1016/j.snb.2018.01.009Get rights and content

Highlights

  • The paper discs immobilized with luciferase/D-luciferin were prepared to detect ATP extracted from airborne bacteria.

  • The immobilization of luciferase/D-luciferin in paper discs was prepared by repeats of adsorption/dry loops process.

  • The paper discs prepared maintained storage stability over 30 days after initial preparation.

  • The ATP extracted from bacteria by heat lysis method could be measured within 7 min using paper discs.

Abstract

The presence of various microbes in the air is one of the main causes of respiratory diseases or ailments affecting the health of humans and livestock. Therefore, early identification and detection of microorganisms in the air are key to preventing the risk associated with microbial infection. In this study, we prepared paper discs (diameter = 0.5 cm) immobilized with luciferase/D-luciferin and found that these discs could be used to determine the adenosine triphosphate (ATP) directly from the heat-lysates of airborne bacteria. The repetition of sequential adsorption/drying of the mixture solution (luciferase and D-luciferin) was performed using paper disc. The storage stability of the paper discs at room temperature was maintained for one month following their preparation while the storage stability of the liquid-based ATP assay not maintained even for one day. The paper discs could detect ATP extracted from aerosolized Escherichia coli (E. coli) as low as 1.17 × 103 CFU/mL in pure bacteria samples or 2.32 × 103 CFU/mL in bacteria samples containing dust (1 mg/mL). ATP evaluation using the paper discs for detection of aerosolized bacteria may reduce the detection time to be less than 7 min after sampling. Novel paper discs immobilized with luciferase/D-luciferin will be valuable for the development of fast and sensitive sensors for early detection and enumeration of airborne microorganisms without preparation of enzyme solution for ATP assay.

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.

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    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.

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