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ATP Bioluminescence-Based Bacteria Detection Using Targeted Photothermal Lysis by Gold Nanorods Seong U Kim, Eun-Jung Jo, Yuseon Noh, Hyoyoung Mun, Young-Deok Ahn, and Min-Gon Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00254 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018
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Analytical Chemistry
ATP Bioluminescence-Based Bacteria Detection Using Targeted Photothermal Lysis by Gold Nanorods Seong U Kim†, Eun-Jung Jo†, Yuseon Noh, Hyoyoung Mun, Young-Deok Ahn and Min-Gon Kim*
Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology (GIST), 123 Cheomdangwagi-ro, Buk-gu, Gwangju, 61005, Republic of Korea
†
*
These authors contributed equally to the work
Corresponding author. M.-G. Kim: Tel: +82-62-715-3330; Fax: +82-62-715-3419; E-mail address: mkim@gist.ac.kr
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ABSTRACT: Bacterial infections are common causes of morbidity and mortality worldwide; therefore, environmental contamination by bacterial pathogens represents a global public health concern. Consequently, a selective, rapid, sensitive, and in-field detection platform for detecting significant bacterial contamination is required to ensure hygiene and protect public health. Here, we developed a fast and simple platform for the selective and sensitive detection of bacteria by measuring adenosine triphosphate (ATP) bioluminescence following targeted photothermal lysis mediated by antibody conjugated gold nanorods. This method employed both targeted photothermal lysis of bacteria by near-infrared irradiation and highly selective detection of the lysed bacteria via ATP bioluminescence within 36 min (incubation, 30 min; NIR-irradiation, 6 min). Using the proposed method allowed limits of detection in pure solution of 12.7 CFU, 70.7 CFU, and 5.9 CFU for Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes, respectively. Additionally, bacteria were successfully detected on artificially inoculated plastic cutting boards. Furthermore, this method was highly specific, without cross-reaction among pathogenic bacteria. We believe that the proposed method has significant potential as an on-site diagnostic tool for applications associated with public health and environmental-pollution monitoring.
Keywords Bacteria, Photothermal lysis, Gold nanorods, Adenosine triphosphate, Bioluminescence
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INTRODUCTION Pathogenic bacteria are recognized as a major global concern due to the significant risk posed by their causing infectious diseases, such as foodborne illnesses, pneumonia, tetanus, typhoid fever, diphtheria, syphilis, and leprosy. These infectious diseases are a significant source of morbidity and mortality, causing hospitalization and/or possibly death. Therefore, infection control and microbialcontamination management are important tasks in both the healthcare and food industries for hygiene monitoring. Due to the increased demand for adequate hygiene, consumers and patients have become aware of public health, food safety, security, and medicine issues caused by pathogenic bacteria. Therefore, the rapid, convenient, and sensitive detection of pathogenic microorganisms is of great importance for the monitoring of environmental quality and food safety, as well as in clinical diagnostic applications to address health and safety problems. 1-10 Adenosine triphosphate (ATP)-based bioluminescence methods are applied in environmental examinations, the food industry, and healthcare facilities for assessing the efficacy of sanitation standard operating procedures for various environmental surfaces to enable cleanliness and hygiene monitoring. The measurement of ATP, a molecule found in living cells, via bioluminescent firefly luciferin/luciferase reaction has been widely employed for the detection of pathogenic bacteria. This method is based on luciferase-based detection of ATP, which results in the production and emission of bioluminescent light via enzymatic reaction. The bioluminescent signal is directly proportional to the amount of ATP released from lysed bacteria. 11-13 Compared with traditional methods, such as culturebased microbiological testing, this method is simple and rapid, and has, therefore, been widely been applied for detection of bacterial contamination on food and environmental surfaces. This method is used on various hospital surfaces (floors, tables, kitchens, sinks, utensils, door handles and panel, and bathrooms) and in operating rooms, wards, and intensive care units for the prevention of bacterial epidemics and to assess the overall cleanliness of healthcare institutions.7, 9 Additionally, surfaces in contact with food, such as the kitchen sink, drains, faucet handles, cutting boards, and canteen tables, in the food industry are tested to ensure product safety and quality.8, 10 However, the method has limited applications for the selective detection and quantification of specific bacteria, because it 3
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cannot distinguish between different sources of ATP from live/dead or beneficial/harmful bacteria to correctly assess microbial risk; therefore, current methods used to measure ATP levels do not provide detailed information for the identification of target bacteria.13 Given these limitations, recently developed biosensors14 that rely on the use of immunomagnetic filtration,15 fluorescence,3, 6, 16 surface plasmon resonance (SPR), 2, 16, 17 quartz crystal microbalance, 6, 16, 18
and electrochemistry2, 16, 19 have been explored in the context of their use for the detection of
specific bacteria and based on interactions between bacterial antibody and target. However, these methods require laborious washing steps, which limit their use for on-site testing due to their low sensitivity. Because of these issues, the development of a biosensor exhibiting high sensitivity and selectivity for on-site bacterial detection is needed. Our efforts to this end have led to new bacterial detection platform based on ATP-specific bioluminescence following targeted photothermal lysis of bacteria mediated by antibody conjugated gold nanorods (GNRs). The photothermal effect is a phenomenon associated with electromagnetic radiation,20 which is produced by converting the photoexcitation of materials, such as polymers,21 graphene oxide, 22 carbon nanotubes,23 and GNRs,24-26 into thermal energy (heat). These nanomaterials conjugated with bioreceptors have promising applications for the killing and/or treatment of infectious bacterial pathogens through targeted photothermal lysis.21-25 Although these methods are reasonably selective for targeted bacteria, quantitative detection of specific bacteria remains difficult. Here, we employed direct detection of targeted pathogenic bacteria using antibody conjugated GNRs to selectively target and lyse pathogenic bacteria via localized heating caused by near-infrared (NIR) irradiation. This system, which combines the advantages of ATP bioluminescence, including high sensitivity, simplicity, and reliability, with targeted photothermal lysis, allowed the highly sensitive and selective detection of targeted bacteria.
EXPERIMENTAL SECTION Materials and sampling. Cetyltrimethyl ammonium bromide (CTAB), gold (III) chloride trihydrate (HAuCl4·3H2O), sodium borohydride (NaBH4), silver nitrate (AgNO3), L-ascorbic acid, 4
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sodium chloride (NaCl), potassium phosphate (KH2PO4), potassium chloride (KCl), sodium phosphate (Na2HPO4), and thiol-PEG-carboxyl (HS-PEG-COOH; average Mn: 7500) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). An antiEscherichia coli O157:H7 polyclonal antibody was obtained from KPL (Gaithersburg, MD, USA). An anti-Salmonella polyclonal antibody was purchased from Bioss (Woburn, MA, USA). An anti-Listeria polyclonal antibody was obtained from Thermo Fisher Scientific. E. coli O157:H7 (ATCC 35150), Salmonella typhimurium (ATCC 13311), and Listeria monocytogenes (ATCC 19116) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Tryptic soy agar (TSA), tryptic soy broth (TSB), brain heart infusion (BHI) agar and broth, brilliant green agar (BGA), eosin methylene blue (EMB) agar, sorbitol- MacConkey (SMAC) agar, and rappaport-vassiliadis R10 broth were purchased from BD Bioscience (San Jose, CA, USA). An intracellular ATP kit (266-111) was purchased from BioThema (Handen, Sweden). UltraSnap ATP surface test was obtained from Hygiena (Camarillo, CA, USA) for luciferin-luciferase solution. Ready-made, insulated K-type thermocouple with a subminiature connector and spool cap was purchased from Omega Engineering, Inc. (Stamford, CT, USA). Portable luminometer (Clean-Q; TBD1000) was obtained from Teltron Inc. (Daejeon, Republic of Korea).
Instrumentation. Absorbance spectra and bioluminescence intensity were recorded using an Infinite 200 PRO and a microplate reader from TECAN (Mannedorf, Switzerland). The size, shape, and uniformity of synthesized GNRs and bacterial morphology were measured by transmission electron microscopy (TEM; Tecnai G2 F30 S-Twin; FEI, Hillsboro, OR., USA). Potentiometry was performed using an electrochemical interface analyzer (CompactStat; Ivium technologies, Netherlands).
Bacterial culture. E. coli O157:H7 strains were streaked on EMB agar and incubated at 37°C for 16 h. Blue-black colonies exhibiting a green metallic sheen were identified, and picked colonies were dispersed in 0.85% NaCl solution. The solution was streaked on SMAC agar and incubated at 37°C for 16 h. The white and transparent colonies were selected and grown in TSB at 37°C with 5
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shaking at 180 rpm for 16 h. S. typhimurium strains were streaked on BGA and incubated at 35°C for 18 h. The colonies were then selected and grown in Rappaport-vassiliadis R10 broth at 41°C for 22 h. L. monocytogenes strains were streaked on BHI agar and incubated at 37°C for 18 h, and the resulting colonies were grown in BHI broth at 37°C under aerobic conditions for 18 h. Bacterial concentrations were determined by colony counting and real-time polymerase chain reaction. Cultured bacteria solution in phosphate-buffered saline (PBS) was mixed with ATP-eliminating reagents (BioThema) to eliminate extracellular ATP, and the mixture was incubated at room temperature for 20 min. After incubation, the bacteria were collected by centrifugation at 6000 rpm for 10 min. The supernatant was discarded, and the pellet was re-suspended in 5.7 mM PBS. The centrifugation and washing steps were repeated twice, followed by resuspension of the pellets in 5.7 mM PBS.
GNR synthesis. GNR synthesis was conducted according to a previously reported seedmediated method, with modifications.27 A seed solution was prepared by adding cold NaBH4 (0.01 M; 0.3 mL) to a mixture containing CTAB (0.1 M; 3.75 mL), HAuCl4·3H2O (0.01 M; 0.125 mL), and deionized water (0.825 mL). The solution was stirred vigorously for 2 min and left to stand at 37°C for at least 2 h. The growth solution was obtained by adding HAuCl4·3H2O (0.1 M; 0.25 mL) and AgNO3 (0.01 M; 0.5 mL) to CTAB (0.1 M; 50 mL), followed by the addition of ascorbic acid (0.1 M; 0.3 mL) and observation of a change in the solution from light yellow-brown to colorless. Subsequently, the resulting solution was mixed with seed solution (0.65 mL) and incubated overnight at 37°C. The synthesized GNRs underwent two rounds of centrifugation at 10,000 rpm for 120 min and washing, with the pellets re-suspended in deionized water, followed by storage at 25°C until use.
GNR functionalization and preparation of antibody conjugated GNRs. GNR functionalization using HS-PEG-COOH was conducted according to a previously reported method, with modifications.28 The GNR pellet obtained by centrifugation was mixed with 25 µL HS-PEGCOOH (2 mM) and 400 µL Tris buffer (50 mM; pH 3) and stirred at room temperature for 30 min by rotation. The solution was then centrifuged and re-dispersed in 1 mL phosphate buffer (10 mM; pH 7.2). The concentration of the prepared carboxylate-modified GNRs solution was denoted as “1×”. To obtain consistent data using the conjugates at a constant concentration, the carboxylate-modified 6
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GNRs, stored at a high concentration, were diluted (0.048×) with phosphate buffer before conjugation. Using a microplate reader, the optical density of the 0.048× carboxylate-modified GNRs was determined as 0.41 and 1.57 at 510 nm (OD510; transverse absorption wavelength) and 830 nm (OD830; longitudinal absorption wavelength), respectively. Each bacteria-specific antibody was covalently conjugated to the carboxylated GNRs using EDC coupling. EDC solution (100 mg/mL; 10 µL) was added to the carboxylated GNR solution (OD510 = 0.41 and OD830 = 1.57; 0.5 mL), and the carboxylated GNRs were activated by slow shaking at room temperature for 10 min. The resulting solution was centrifuged at 10,000 rpm and 22°C for 56 min and washed three times with deionized water. After discarding the supernatant, the anti-bacteria antibody (5 µg) and 250 µL deionized water were added to the activated GNRs, followed by incubation at room temperature for 2 h with shaking. The solution was subjected to a second round of centrifugation and pellet resuspension in PB buffer (10 mM; pH 7.4) using the same methods, and the antibody conjugated GNRs were then re-suspended in 50 µL PBS (pH 7.2). The concentration of the prepared GNR-antibody conjugate solution was denoted as “10×” and stored at 4°C until use.
ATP-bioluminescence-based bacterial detection following photothermal lysis. We optimized key parameters, such as conjugate concentration and light-emitting diode (LED) irradiation times, associated with the bioluminescence-based detection of bacteria following photothermal lysis mediated by GNRs. Bacteria (100 µL) prepared in PBS were mixed with conjugates (0.01–80×; 1 µL) and incubated at room temperature for 30 min. Thereafter, solutions were irradiated for various time periods (0–12 min) using an 845-nm LED (300 mW/cm2) at a distance of 15 mm from the top of the solution. A 10-µL aliquot of the resulting solution was added to a luciferinluciferase solution (Hygiena), and bioluminescence was immediately measured using a microplate reader; the bioluminescent signal was measured in less than 30 s. Additionally, bacteria were mixed with conjugates as described and diluted to a ratio of 1:1000, followed by irradiation.
Detection of E. coli O157:H7 on the surfaces of plastic cutting boards. Plastic cutting boards were washed with 70% ethanol and sterile ultrapure water. After washing, the plastic cutting boards were soaked in a solution containing 20% ethanol and 1% lactic acid for 30 min to 7
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sterilize. Then, the resulting plastic cutting boards were washed ten times with sterile ultrapure water and artificially inoculated with bacteria (5 × 105 CFU) on small sections (0.5 cm × 0.5 cm) of equal size, and the inoculated samples were maintained at room temperature for 3 h to absorb the bacteria. PBS (10 µL) was added to the plastic cutting boards and left at room temperature for at least 5 min. The resulting solution was pipetted 15 times back and forth. Extracted bacteria (5 × 105 CFU) from the plastic cutting boards were also tested as described in method section. For the detection of bacteria using the proposed method integrated with existing commercial products, the absorbed bacteria on the plastic cutting boards were incubated with PBS including 20× GNR-antibody conjugates for 30 min. Thereafter, solutions were irradiated for 6 min using an 845-nm LED at a distance of 15 mm from the top of the solutions on the plastic cutting boards. The resulting solution was swabbed by the sampling device (UltraSnap ATP surface test; Hygiena). Next, the activated sampling device was inserted into the portable luminometer (Clean-Q; Teltron Inc.) to measure the bioluminescence intensity, in accordance with the product manual.
RESULTS AND DISCUSSION System schematic. A schematic describing the platform is illustrated in Figure 1(a). GNRs exhibiting suitable aspect ratios can absorb NIR radiation and transfer light energy into the surrounding environmental as heat. 20, 24-26 Antibody conjugated GNRs are capable of identifying specific bacteria via antibody antigen recognition. The localized heating of the GNRs via NIR irradiation causes photothermal lysis of targeted bacteria, and, consequently, the ATP released from the lysed bacteria reacts with luciferin to produce a bioluminescent signal proportional to the ATP concentration (Figure 1b). Therefore, this method allows the detection of pathogenic bacteria using ATP bioluminescence following photothermal lysis and the selective destruction of bacteria targeted by antibody conjugated GNRs.
Characterization of GNRs and conjugates. A representative TEM image (Figure S1a) of the GNRs synthesized using the seed-mediated method shows that the GNRs were uniformly sized, with a length and width of ~33.11 ± 2.75 nm and ~8.02 ± 0.92 nm (average aspect ratio: 4.13), 8
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respectively. The UV–vis absorption spectra of the colloidal bare GNRs showed two distinct maxima corresponding to a transverse-absorption wavelength at 510 nm and a longitudinal-absorption wavelength at 819 nm. The surface of the GNRs was modified with 7.5-kDa HS-PEG-COOH, which exhibited a strong, localized SPR (LSPR) peak at 830 nm. Following antibody conjugation, a red shift in the LSPR peak of the carboxylate-functionalized GNRs from 830 nm to 845 nm was observed (Figure S1b). These results demonstrated successful conjugation of the bacteria-specific antibody and the carboxyl-functionalized GNRs. The maximum-absorbance wavelength (λmax = 845 nm) of the GNR-antibody conjugates matched the 845-nm NIR LED required for effective photothermal effects (Figure S2). Figure S3 (a) shows the absorption spectra of the GNR-antibody conjugates (using the carboxylate-modified GNRs at concentrations ranging from 0.25-fold to 4-fold) when 0.048× carboxylate-modified GNRs was used “1-fold”. When the concentration of the carboxylate-modified GNRs was greater than 2-fold, the wavelength was reduced (Figure S3b). The maximum absorption wavelength did not reach 845 nm because the antibody could not completely cover the surface of the GNR when the concentration of the carboxylate-modified GNRs was too high (> 2-fold). Because the maximum absorption wavelength of the GNR-antibody conjugates, using concentrations higher than 2-fold, were not consistent with those of the 845-nm NIR LED, they were not suitable for the effective photothermal effect. Therefore, we selected 0.048× as the optimal carboxylate-modified GNRs concentration for optimum conjugation of antibodies.
System optimization. In the presence of target bacteria (5 × 105 CFU E. coli O157:H7), we found that the bioluminescent intensity reached a maximum upon addition of 20× (initial concentration) GNR-antibody conjugates as compared with results observed with carboxylatefunctionalized GNRs. This result suggested that specific photothermal lysis occurred between the functionalized antibacterial GNR-antibody conjugates and the bacteria (Figure 2a). We determined time courses specific for relative bioluminescence intensity observed following ATP release and using the GNR-antibody conjugates and antigen recognition. Figure 2(b) shows that the degree of bioluminescence rose gradually and stabilized after a maximum incubation time of 30 min. Because the photothermal effect initiated by the GNR-antibody conjugates was dependent upon NIR LED9
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irradiation time, we tested various irradiation times (0-12 min) to select an appropriate length according to the plate-counting method (Figure 2c and Figure S3) and turbidity measurement (Figure S4) for photothermal lysis of target bacteria (E. coli O157:H7). After NIR LED-irradiation, the 1000fold diluted samples were grown in liquid and solid bacterial culture. For the solid culture, the diluted samples were spread onto TSA plates and incubated at 37°C for 18 h, followed by quantification of bacterial viability (Figure 2c and Figure S3). The percentage of bacterial viability at various NIRirradiation times was calculated from the ratio between colony counts in the presence and absence of NIR irradiation using the following equation: Viability (%) =
Colony counts after NIR irradiation × 100 Colony counts before NIR irradiation
Liquid culture was performed in TSB at 37°C overnight with shaking at 180 rpm, and turbidity of the resulting solution was measured at OD600 (Figure S4). Our results (Figures 2c, S3, and S4) indicated activity within a 6-min period, whereas extended irradiation (>8 min) caused ATP release via nontargeted photothermal lysis by carboxylate-functionalized GNRs. After 10 min, ATP release also occurred in bacterial samples alone due to the heat generated by NIR irradiation. Additionally, heat treatment of samples above a certain temperature and for a suitable period of time results in bacterial lysis29; however, simple heat treatment is a limiting factor for selective detection and quantification of specific bacteria, because it cannot distinguish between different sources of ATP from target/nontarget bacteria in the heat-treated sample. On the other hand, we were capable of detecting targeted pathogenic bacteria using antibody conjugated GNRs to selectively target and lyse pathogenic bacteria via localized heating caused by NIR irradiation within a 6-min period (Figure 2c, S3, and S4). Therefore, 6 min was chosen as an optimal irradiation time for targeted photothermal lysis and antigen recognition. The overall optimized conditions for photothermal lysis meditated by the GNR-antibody conjugates for bacterial detection included the use of 20× GNR-antibody conjugates at an incubation time of 30 min, with a 6-min NIR-irradiation time.
Confirmation of system efficacy. Following system optimization, we examined its 10
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efficacy using various sample combinations (bacteria alone, bacteria in the presence of carboxylated GNRs, or bacteria in the presence of GNR-antibody conjugates) in the presence and absence of NIR irradiation. In the presence of target bacteria, strong bioluminescence was observed following targeted photothermal lysis of GNR-antibody conjugates by NIR irradiation under the optimized conditions. By contrast, the bioluminescence of other samples (bacteria only and in the presence of carboxylated GNRs) was very weak in the presence or absence of NIR irradiation. These observations suggested that the GNR-antibody conjugates bound target bacteria specifically, resulting in high levels of bioluminescence associated with ATP release by targeted photothermal lysis (Figure 2d). TEM images subsequently confirmed this activity in the presence and absence of NIR irradiation (Figure 3), showing that GNR-antibody conjugates attached to the bacterial cell surface, thereby promoting targeted photothermal lysis in the presence of NIR irradiation (Figure 3b) under the optimized conditions. Our results suggested that targeted bacteria in the presence of GNR-antibody conjugates were destroyed by exposure to NIR irradiation, although bacteria alone were undamaged, regardless of exposure to NIR irradiation (Figure 3a). We measured the temperature applied to the solution in the proposed method under 845-nm LED irradiation for 6 min. The bulk temperature of the solution was obtained from the measured potentiometry data by soaking the thermocouple in the solution under 845-nm LED irradiation for 6 min. Figure S6 (a) shows the bulk temperature of the various sample types in the presence or absence of NIR irradiation. Although the temperature of the solutions was increased by the NIR irradiation, the maximum temperature reached by all the solutions after 6 min irradiation was similar and only ~50 °C. The temperature reached in this method was lower than that previously reported to be used for bacterial lysis.29,
30
Additionally, strong bioluminescence was
observed only in the presence of target (E. coli O157:H7) bacteria (Figure S6b). Therefore, the proposed method induces the photothermal lysis of targeted bacteria by localized heat, based on antibody-antigen recognition, and not by the bulk temperature of the solution. These data (Figure 3 and S6) demonstrated successful targeted photothermal lysis by GNR-antibody conjugates.
Method sensitivity and specificity. This system was validated by determining sensitivity levels in the presence of different bacterial concentrations. The most common foodborne pathogens, 11
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including E. coli O157:H7, S. typhimurium, and L. monocytogenes, were tested to determine method sensitivity and specificity. Figure 4(a) shows the normalized bioluminescence intensities for each target bacteria at concentrations ranging from 5 × 100 to 5 × 106 CFU. We observed linear increases in the bioluminescence intensities along with increases in bacterial concentration according to a logarithmic plot (Figure 4a), resulting in linear relationships for E. coli O157:H7, S. typhimurium, and L. monocytogenes of R2 = 0.988, R2 = 0.976, and R2 = 0.9415, respectively, and LODs of 12.7 CFU, 70.7 CFU, and 5.9 CFU, respectively. The LODs for this method were comparable with other bacteria-detection platforms, and the specificity was also evaluated in the presence of interference and the coexistence of other bacteria. Each target bacteria showed strong bioluminescence intensity in the presence of each target-specific GNR-antibody conjugate, whereas non-targeted bacteria (5 × 105 cells) showed weak bioluminescence intensity (Figure 4b). Additionally, coexistence of target bacteria with other non-targeted bacteria did not affect bioluminescent intensity (Figure 4c). These results indicated that the proposed method showed high degrees of specificity toward the target bacteria, regardless of the presence of or potential cross-reactivity with other bacteria.
Detection of E. coli O157:H7 inoculated onto the surface of plastic cutting boards. To further demonstrate the efficacy of this method, we detected E. coli O157:H7 inoculated onto the surface of plastic cutting boards (Figure 5). Commercially available products used for bacterial detection are based on measuring the release of ATP by bacterial lysis using lysis buffer containing detergents capable of disrupting hydrophobic-hydrophilic interactions. The ATP released from the bacterial sample can be quantified by measuring the generated bioluminescence from the enzymatic reaction via luciferin-luciferase solution. Although this conventional method is rapid and simple, complete lysis is impossible. Additionally, conventional methods involving detergent lysis make it impossible to detect specific E. coli strains due to their inability to distinguish between different ATP sources from the target/non-target bacteria (Figure 5a). Moreover, these detergents may interfere with the enzymatic reaction via luciferin-luciferase.31, 32 On the other hand, we detected targeted E. coli O157:H7 using antibody conjugated GNRs to selectively target and lyse pathogenic bacteria via localized heating caused by NIR irradiation. Our findings showed that this method was 12
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capable of detecting E. coli O157:H7 on these surfaces, even in the presence of interference by and coexistence with other bacteria (Figure 5b). The localized heat used for the proposed method caused the complete and specific disruption of targeted bacteria (Figures 2c, 3, and S3); therefore, this higher efficiency targeted photothermal lysis was capable of detecting target bacteria with higher selectivity and sensitivity. Additionally, the detection time and specificity of our proposed method were comparable to other methods for bacterial detection on the contaminated surfaces (Table S1). Additionally, to further demonstrate the possibility of using this method for in-field or on-site bacteria detection, we integrated this method with commercial products. These commercial products have been widely used for real time hygiene on-site monitoring of surfaces of processing equipment and environments in a wide range of industries, healthcare institutions, and canteens.33-36 The proposed method, integrated with the commercial products, showed specificity (Figure S7) toward the target bacteria (E. coli O157:H7), regardless of the presence of or potential cross-reactivity with non-target bacteria (S. typhimurium). The relative light unit (RLU) value of target bacteria was higher than that of the non-target bacteria due to the more presence of ATP by the localized heating of the GNRs via NIR irradiation causes photothermal lysis of targeted bacteria. Therefore, with GNR-antibody conjugates and 845-nm LED irradiation, the proposed method can be applied to in-field or on-site bacteria detection because it can easily be integrated with existing commercial products. Our method offers a novel capability in this regard and can be used for the selective detection of target bacteria on various contaminated food and environmental surfaces as a tool to monitor surface cleanliness in both the food industry and healthcare institutions.
CONCLUSIONS In this study, we described the development of a fast and simple method for the selective detection of bacteria using ATP bioluminescence based on photothermal lysis mediated by antibody conjugated GNRs. Our results showed that bioluminescent efficiency increased along with increasing concentrations of bacteria selectively lysed by localized heating due to the GNR-antibody conjugates. This detection was successfully performed within 36 min (incubation, 30 min; NIR-irradiation, 6 min) 13
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and highly specific to pathogenic bacteria. These results indicated that the proposed method can be used to rapidly detect pathogenic bacteria associated with food contact/environmental-surface contamination and has significant potential as an on-site evaluation and monitoring tool for cleanliness and safety.
AUTHOR INFORMATION Corresponding Authors * E-mail:
mkim@gist.ac.kr (Min-Gon Kim)
ORCID Min-Gon Kim: 0000-0002-3525-0048 Author Contributions †
S. U. Kim and E.-J. Jo contributed equally to this work.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was financially supported by grants from the Global Research Laboratory (GRL) Program (NRF-2013K1A1A2A02050616) and the Mid-career Researcher Program (NRF-2017R1A2B3010816) through a National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT. This research was supported by a grant from the World Institute of Kimchi funded by the Ministry of Science and ICT (KE1701-5), Republic of Korea.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterization of GNRs and conjugates; Absorption spectrum of GNR-antibody conjugates and 845-nm NIR LED; Absorption spectra and the maximum absorption wavelength of the GNR-antibody conjugates; Photographs of E. coli O157:H7 colonies grown on the TSA plates; Bacteria-viability test according to turbidity measurements; Comparison of the methods developed for detecting bacteria on surfaces; Solution temperature and bioluminescence by 14
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NIR irradiation; Detection of bacteria using the proposed method integrated with existing commercial products (PDF)
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Figure 1. Detection of bacteria using GNR-mediated photothermal lysis and measurement of ATP bioluminescence. (a) Schematic illustration of this system involving NIR irradiation. ATP is released from the lysed bacterial targets, followed by (b) chemical reactions associated with ATP bioluminescence catalyzed by luciferase. AMP, adenosine monophosphate; PPi, pyrophosphate.
Figure 2. Optimization and confirmation of the proposed method for bacterial detection (E. coli O157:H7; 5 × 105 CFU) via GNR-mediated photothermal lysis and bioluminescence measurement. (a) Bioluminescence intensity from various initial concentrations (1×, 1.25×, 2.5×, 5×, 10×, 20×, 40×, and 80×) of GNRs in the presence of bacteria. (b) Bioluminescence intensity from various incubation times (5–30 min). (c) Bacterial-viability according to a plate-counting method using various samples (bacteria alone, bacteria in the presence of carboxylated GNRs, and bacteria in the presence of GNRantibody conjugates) at various NIR-irradiation times (0–12 min). (d) Bioluminescence intensity of various samples (bacteria alone, bacterial in the presence of carboxylated GNRs, and bacterial in the presence of GNR-antibody conjugates) in the presence and absence of NIR irradiation under optimized conditions. ILum, bioluminescence intensity; IAbs, absorbance intensity.
Figure 3. TEM images of bacteria following NIR irradiation. (a) Bacteria in the absence of GNRantibody conjugates. (b) Targeted photothermal lysis of bacteria by GNR-antibody conjugates in the presence and absence of NIR irradiation.
Figure 4. Sensitivity and specificity of the proposed method for the detection of bacteria in pure samples by GNR-mediated photothermal lysis and bioluminescence measurement. (a) Normalized bioluminescence intensity for each target bacteria (E. coli O157:H7, S. typhimurium, and L. monocytogenes) ranging from 5 × 100 CFU to 5 × 106 CFU. (b) Specificity of this method in the presence of interferential bacteria (5 × 105 CFU). (c) Specificity of this method by coexistence of 19
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target bacteria and non-target bacteria (5 × 105 CFU). ILum, bioluminescence intensity.
Figure 5. Comparison of the proposed method with other methods for detection of E. coli O157:H7 inoculated onto the surfaces of plastic cutting boards. Specificity by the (a) conventional ATP methods and (b) proposed method in the presence of interference and co-existence with other non-target bacteria. E, E. coli O157:H7; L, L. monocytogenes; S, S. typhimurium.
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Figure 1.
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Figure 3.
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Figure 5.
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