Ultrasensitive detection of Escherichia coli O157:H7 by

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Food Safety and Toxicology

Ultrasensitive detection of Escherichia coli O157:H7 by immunomagnetic separation and selective filtration with NBT/BCIP signal amplification Seong U Kim, Eun-Jung Jo, Hyoyoung Mun, Yuseon Noh, and Min-Gon Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00973 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 1, 2018

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Ultrasensitive detection of Escherichia coli O157:H7 by

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immunomagnetic separation and selective filtration with NBT/BCIP

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signal amplification

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Seong U Kim†, Eun-Jung Jo†, Hyoyoung Mun, Yuseon Noh, and Min-Gon Kim*

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Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and

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Technology (GIST), 123 Cheomdangwagi-ro, Buk-gu, Gwangju 61005, Republic of Korea

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These authors contributed equally to the work.

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Corresponding author M.-G. Kim: Tel: +82-62-715-3330; Fax: +82-62-715-3419; E-mail address: [email protected]

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Keywords

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Immunomagnetic separation, Selective filtration, Enzyme-catalyzed precipitation, Colorimetry,

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Escherichia coli O157:H7

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ABSTRACT: Here, we report an enhanced colorimetric method using enzymatic amplification with

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nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) precipitation for the

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ultrasensitive detection of Escherichia coli O157:H7 through immunomagnetic separation-selective

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filtration. Biotinylated anti-E. coli O157:H7 antibody and streptavidin−alkaline phosphatase were

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conjugated to the surface of magnetic nanoparticles, and the large complexes remained on the

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membrane filter surface. The resultant light brown spots on the membrane filter were amplified with

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NBT/BCIP solution to yield enzyme-catalyzed precipitation, which increased with increasing E. coli

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O157:H7 concentration. E. coli O157:H7 was detected in pure samples with LODs of 10 and 6.998

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colony-forming units per milliliter through visual observation and measurement of optical density,

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respectively. The proposed method was applied to a lettuce sample inoculated with selective E. coli

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O157:H7, which was detected within 55 min without cross-reactivity to non-target bacteria. This

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enhanced colorimetric method has potential for on-site detection of food contaminants and

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environmental pollutants.

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INTRODUCTION

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Pathogenic bacteria are a major global concern as they represent a significant source of morbidity and

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mortality leading to hospitalization and/or possibly death.1,

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Organization, there are 76 million cases of foodborne disease resulting in 5000 deaths each year.3 One

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of the most important pathogens is Escherichia coli O157:H7, which produces Shiga toxin and causes

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infections that often lead to severe, acute hemorrhagic diarrhea and abdominal cramps. Infection with

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E. coli O157:H7 has a high mortality rate for elderly patients, children younger than 5 years of age,

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and patients whose immune systems are otherwise compromised. Since the majority of E. coli

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O157:H7 infections are caused by the consumption of raw and contaminated foods, including

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vegetables, fruits, milk, and meat, a method that can achieve the earlier and highly sensitive detection

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of pathogenic bacteria is an essential component for improving the monitoring of E. coli O157:H7-

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related food poisoning for public health considerations and for clinical diagnostics.2, 4, 5

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According to the World Health

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The current gold-standard technique for detecting E. coli O157:H7 is the conventional culture

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method, which is considered to be reliable and accurate. However, this technique is unsuitable for on-

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site testing because it is laborious and time-consuming, requiring a few days to obtain a result.6, 7 To

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overcome these limitations, other methods are commonly used for E. coli O157:H7 detection,

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including enzyme-linked immunosorbent assays (ELISAs)8, 9 and polymerase chain reaction.7, 10, 11

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Although these detection methods are reasonably sensitive and more rapid than the culture method,

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they still involve laborious steps and specifically trained personnel. In addition, these methods

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generally require an additional step for removing the inhibitors from the complex sample matrices for

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application to retail and field samples.12, 13 For this purpose, immunomagnetic separation (IMS) using

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magnetic nanoparticles (MNPs) or microbeads that are conjugated with antibodies specific to the

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target bacteria are widely used to achieve efficient and simple isolation or to concentrate the target

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bacteria from complex samples. 14, 15

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Indeed, several detection methods coupled with IMS have been developed for E. coli O157:H7.

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After isolating and concentrating the bacteria through IMS, the bacteria–MNP complexes can be 3

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applied to various detection methods such as surface enhanced Raman spectroscopy,16 surface

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plasmon resonance,17 Fourier-transform infrared spectroscopy,18 quartz crystal microbalance,19

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electrochemistry,20 fluorescence,21 and luminescence.22 Although these methods are capable of highly

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sensitive and selective E. coli O157:H7 detection, they require expensive equipment, and are therefore,

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not widely applicable for on-site detection. Given these limitations, naked-eye biosensors that rely on

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the use of a homogeneous colorimetric assay23 and lateral flow immunoassay24, 25 have been explored

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for their potential suitability for the low-cost and convenient detection of E. coli O157:H7. However,

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the relatively low sensitivity of these methods currently limits their use for on-site testing. Therefore,

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development of a biosensor exhibiting high sensitivity based on naked-eye observation is needed.

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Toward this end, we propose a highly sensitive, selective, and enhanced colorimetric technique that

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was designed to detect E. coli O157:H7 using IMS-selective filtration combined with enzymatic

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amplification based on nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP)

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precipitation. After IMS-selective filtration via recognition between the MNP conjugates and E. coli

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O157:H7, the enzyme [alkaline phosphatase (AP)]-catalyzed precipitation reaction on the conjugates–

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E. coli O157:H7 complex was expected to effectively amplify the signal of colored spots on the

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membrane to allow for visual detection. We tested the application of this distinctive enhanced

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colorimetric design for its ability to detect E. coli O157:H7 on artificially inoculated vegetables.

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MATERIALS AND METHODS

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Materials and Sampling. ampling. An anti-E. coli O157:H7 polyclonal antibody was obtained from KPL

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(Gaithersburg, MD, USA). Carboxymethyldextran-coated MNPs were purchased from Chemicell

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(fluidMAG-CMX 100 nm; Eresburgstrasse, Berlin, Germany). 1-Ethyl-3-(3-dimethylaminopropyl)

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carbodiimide hydrochloride (EDC), EZ-Link™ Hydrazide-LC-Biotin, Zeba Spin Desalting Columns

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(MWCO: 7 K), and PYREX® Fritted Glass Support Base (47 mm) for graduated funnel assembly

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were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Bovine serum albumin (BSA)

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was purchased from Fitzgerald Industries International (Acton, MA, USA). Streptavidin−alkaline 4

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phosphatase (STA-AP), sodium chloride (NaCl), sodium tetraborate (Na2B4O7), boric acid (H3BO3),

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Tris, sodium (meta)periodate (NaIO4), sodium acetate (CH3COONa), biotin, sodium, Tween-20, and

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sodium azide (NaN3) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Amicon

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Ultra Centrifugal Filter Units (MWCO: 3 kDa, 100 kDa) and nitrocellulose membrane filters with a

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pore size of 1.2 µm were obtained from Merck Millipore (Burlington, MA, USA). A DynalTM magnet

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was purchased from Invitrogen (Grand Island, NY, USA). AP Conjugate Substrate Kit was purchased

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from Bio-Rad Laboratories (Hercules, CA, USA).

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Instrumentation. Instrumentation. The shape, pore size, and uniformity of the membrane filter paper were

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measured using a scanning electron microscope (Electron S-4700 EMAX System; Hitachi High-

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Technologies, Japan). The hydrodynamic sizes of nanoparticles and conjugates were measured by a

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dynamic light scattering size analyzer (ELSZ-1000; Otsuka Electronics, Japan). The color intensity on

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the membrane was quantified using a ChemiDocTM MP System (Bio-Rad, CA, USA), which

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converted the color signal to optical density.

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Bacterial Strains and Culture. ulture. Escherichia coli (ATCC 25922), Escherichia coli O157:H7

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(ATCC 35150), Salmonella Typhimurium (ATCC 13311), Listeria monocytogenes (ATCC 19116),

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Staphylococcus aureus (ATCC 23235), and Vibrio vulnificus (ATCC 27562) were purchased from

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American Type Culture Collection (Manassas, VA, USA). Bacillus cereus (KCTC 1092) was

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purchased from the Korean Collection for Type Cultures (Korea Research Institute of Bioscience and

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Biotechnology, South Korea), and Salmonella Mbandaka (NCCP 13695) was purchased from the

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National Culture Collection for Pathogens (South Korea). E. coli O157:H7 strains were streaked on

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eosin methylene blue agar (BD Bioscience, San Jose, CA, USA) and incubated at 37°C for 16 h.

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Blue-black colonies exhibiting a green metallic sheen were identified, and picked colonies were

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dispersed in a 0.85% NaCl solution. The solution was streaked on sorbitol-MacConkey agar (BD

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Bioscience) and incubated at 37°C for 16 h. The white and transparent colonies were selected and

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grown in tryptic soy broth (TSB; BD Bioscience) at 37°C with shaking at 180 rpm for 16 h. L.

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monocytogenes strains were streaked on brain heart infusion (BHI; BD Bioscience) agar and 5

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incubated at 37°C for 18 h, and the resulting colonies were grown in BHI broth at 37°C under aerobic

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conditions for 18 h. B. cereus, E. coli, and S. aureus strains were grown in TSB at 37°C with shaking

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at 180 rpm for 16 h. Salmonella Typhimurium and Mbandaka strains were streaked on brilliant green

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agar (BD Bioscience) and incubated at 35°C for 18 h. The colonies were then selected and grown in

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nutrient broth (BD Biosciences) at 37°C under aerobic conditions for 22 h. V. vulnificus strains were

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grown in marine broth 2216 (BD Biosciences) at 30°C under aerobic conditions for 18 h. The cultured

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bacteria were collected by centrifugation at 3300 ×g for 15 min. The supernatant was discarded and

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the pellet was re-suspended in phosphate-buffered saline (PBS, pH 7.4; Biosesang, Seongnam, South

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Korea). The centrifugation and washing steps were repeated twice, followed by resuspension of the

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pellets in PBS.

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Antibody ntibody Biotinylation. iotinylation. Biotinylated anti-E. coli O157:H7 antibody was prepared by hydrazine

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linkage using hydrazide-biotin reagents. Anti-E. coli O157:H7 antibody (1 mg/mL, 20 µL) was

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dialyzed with sodium acetate buffer (0.1 M, pH 5.5) at 4°C overnight. Cold sodium meta-periodate

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solution (50 mM, 0.4 µL) was prepared in sodium acetate buffer added to the cold antibody solution

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by oxidizing the sialic acid groups of the antibody to form aldehyde groups. After incubation in the

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dark for 30 min on ice, the oxidized sample was subjected to desalting columns (MWCO: 7 K) to

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remove the excess periodate. The sample was then washed three times with PBS using the 3-kDa

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centrifugal filter device. Hydrazide-biotin solution (50 mM, 2 µL) was added to the oxidized and

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buffer-exchanged antibody sample. After incubation for 2 h at room temperature, the biotinylated anti-

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E. coli O157:H7 antibody was washed three times with PBS using the 100-kDa centrifugal filter

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device, followed by storage at 4°C until use.

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Preparation of Biotinylated Antibody/STAntibody/STA-AP/MNP Conjugates. onjugates. STA-AP was covalently

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conjugated to carboxymethyldextran-coated MNPs using EDC coupling. In brief, the EDC solution in

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deionized water (6.67 mg/mL, 3 µL) and STA-AP solution (1 mg/mL, 2 µL) were added to the

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solution of MNPs (25 mg/mL, 8 µL). The mixture was incubated with slow shaking at room

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temperature for 2 h. The STA-AP/MNP conjugate was isolated using a magnet and washed three times 6

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with PBS, to which the biotinylated anti-E. coli O157:H7 antibody was added and incubated at room

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temperature for 30 min. The sample was washed three times with PBS and recovered using a magnet.

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Finally, the biotinylated antibody/STA-AP/MNP conjugates were re-suspended and stored in 40 µL of

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PBS containing 0.1% BSA and 0.05% sodium azide. The concentration of the as-prepared

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biotinylated antibody/STA-AP/MNP conjugates solution was denoted as “1×”, which was stored at

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4°C until use.

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Immunological Magnetic Capture for E. coli O157:H7 Detection. etection. To determine the capture

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efficiency of biotinylated antibody/STA-AP/MNP conjugates for E. coli O157:H7 (100 µL),

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biotinylated antibody/STA-AP/MNP conjugates (1×, 1 µL) were incubated together for 30 min at

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room temperature. After exposure to a magnet for 15 min, the E. coli O157:H7-conjugates complexes

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were washed with PBS and spread onto plates containing tryptic soy agar medium, and incubated for

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18 h at 37°C. The resultant E. coli O157:H7 colonies on the agar dish were counted.

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To determine the optimal exposure time to the magnet, E. coli O157:H7 (100 µL) and biotinylated

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antibody/STA-AP/MNP conjugates (1×, 1 µL) were incubated for 30 min at room temperature. After

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various exposure times, the sample was washed with PBS (1 mL) and re-suspended with 1% BSA in

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PBS (100 µL). After filtration of the sample, colorimetric images of the membrane were detected

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using the ChemiDocTM imaging system (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed by

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the ChemiDoc analyzing program (Image Lab Software version 4.0).

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The capture efficiency of the biotinylated antibody/STA-AP/MNP conjugates for E. coli O157:H7 was calculated using the following formula:

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Capture efficiency (%) = (Csep/Ctotal) × 100

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where Ctotal is the number of colonies based on the initial number of cells (2 × 102 cells in 100 µL

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PBS) and Csep is the number of colonies on the agar dish using the cells captured by E. coli O157:H7-

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conjugates complexes through IMS.

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Enhanced Colorimetric Method based on IMSIMS-selective Filtration for E. coli O157:H7

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Detection. etection. For enzymatic amplification with NBT/BCIP precipitation based on IMS-selective

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filtration, the biotinylated antibody/STA-AP/MNP conjugates (1×, 1 µL) were added to the bacteria

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suspension (1 mL), and the complexes were incubated for 30 min at room temperature followed by 15

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min of magnetic separation. The supernatant was removed, and the resulting pellet was washed with

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PBS (1 mL) and re-suspended with 1% BSA in PBS (100 µL). An aluminum foil tape with 1-mm-

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diameter holes was attached to the fritted glass support base for assembly of the Büchner flask. The

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nitrocellulose membrane filter was then placed on the aluminum foil and moistened with PBS before

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being placed on a 500-mL Büchner flask including the fritted glass support base insert to allow for

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assembly under vacuum. Subsequently, 100 µL of the re-suspended bacteria-conjugates complexes

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were filtered through the membrane under vacuum pressure. The membrane was washed away by

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filtering with PBS containing 0.05% Tween 20. The NBT/BCIP solution (1% NBT and 1% BCIP in

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1× AP color development buffer) was spread onto the resultant membrane, and the membrane was

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dried and incubated at room temperature for 10 min. Finally, the colored spots that developed on the

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membrane were quantified and analyzed by imaging as described above.

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Specificity of the Enhanced Colorimetric Method based on IMSIMS-selective Filtration. iltration. The

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specificity of the enhanced colorimetric method based on IMS-selective filtration described above

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was determined using the target strain E. coli O157:H7 and other bacteria such as E. coli, B. cereus, L.

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monocytogenes, Salmonella Typhimurium, Salmonella Mbandaka, S. aureus, and V. vulnificus. The

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IMS-selective filtration and enzymatic amplification with NBT/BCIP precipitation were performed as

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described above for all bacteria.

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Detection of E. coli O157:H7 on Artificially Inoculated Lettuce. ettuce. The proposed method was

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tested in lettuce inoculated with various E. coli O157:H7 concentrations. The lettuce (purchased from

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a local supermarket in Gwangju, Republic of Korea) was washed with sterile water and soaked in 20%

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ethanol for 10 min for the preparation of “E. coli O157:H7-free” lettuce by sterilization. The cut

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lettuce (1 g) was then placed onto a petri dish and artificially inoculated with E. coli O157:H7 [25, 50, 8

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500, 5000, and 50,000 colony forming units (CFU)/g]. The sample was left at room temperature for 2

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h so that E. coli O157:H7 would be absorbed onto the surface. The lettuce sample was then mixed

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with 5 mL of autoclaved PBS and shaken at room temperature for 5 min. The extracts (1 mL) of

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lettuce were centrifuged at 3300 ×g for 10 min. The supernatant was discarded, and the pellet was re-

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suspended in autoclaved PBS (1 mL). The resultant infected “E. coli O157:H7-positive” lettuce

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sample was subjected to the proposed enhanced colorimetric method based on IMS-selective filtration.

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RESULTS AND DISCUSSION

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Principles of the Enhanced Colorimetric Method based on IMSIMS-selective Filtration. iltration. The

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basic principle of the proposed enhanced colorimetric method using IMS-selective filtration combined

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with enzymatic amplification based on NBT/BCIP precipitation for the ultrasensitive detection of E.

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coli O157:H7 is schematically displayed in Figure 1. The biotinylated antibody/STA-AP/MNP

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conjugates can bind to the target bacteria due to recognition between the antibody and target. The

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target bacteria-conjugates complexes are magnetically isolated from the unbound non-target bacteria

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(Figure 1A). The re-suspended target bacteria-conjugates complexes are selectively filtered using the

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Büchner flask containing a fitted glass support insert base under vacuum pressure (Figure S1).

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Unbound biotinylated antibody/STA-AP/MNP conjugates, which have a much smaller diameter than

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the pore size (1.2 µm) of the nitrocellulose membrane filter (Figure S2), may then easily pass through

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the membrane filter via the holes of the aluminum foil. However, the larger target bacteria-conjugates

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complexes cannot penetrate the pores and thus remain on the membrane filter surface (Figure 1B).

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The resultant spots that form on the membrane filter are light brown in color (Figure 1C upper). The

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remaining AP of the target bacteria-conjugates complexes on the membrane surface then reacts with

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the NBT/BCIP solution to yield enzyme (AP)-catalyzed precipitation. This reaction intensifies the

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color of the spots through the dark-blue indigo dye formed by the oxidation of BCIP (an AP substrate)

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and the blue-purple precipitate formed by the reduction of NBT. Ultimately, the reaction of AP with

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the NBT/BCIP substrate leads to the formation of grayish-purple color signal spots on the membrane 9

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surfaces for selective visual detection of the presence of the bacteria (Figure 1C lower).

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Characterization and Immunological Magnetic Capture of Conjugates. To confirm the

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formation of biotinylated antibody/STA-AP/MNP conjugates, the hydrodynamic diameters of the

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conjugates were analyzed using dynamic light scattering. The hydrodynamic diameters of the

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conjugates gradually increased in the order of carboxymethyldextran-coated MNPs (115.6 ± 1.5 nm),

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STA-AP/MNP conjugates (146.5 ± 3.6 nm), and biotinylated antibody/STA-AP/MNP conjugates

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(172.1 ± 1.2 nm) due to sequential conjugation (Figure S3). These results confirmed that the

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conjugation between the biotinylated antibody/STA-AP and carboxymethyldextran-coated MNP had

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indeed occurred, and the biotinylated antibody/STA-AP/MNP conjugates would easily pass through a

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membrane with a pore size of 1.2 µm.

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Next, we verified whether the biotinylated antibody/STA-AP/MNP conjugates could separate the

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target bacteria via immunological magnetic capture. As shown in Figure 2A, less than one colony was

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observed to be formed by the MNP and STA-AP/MNP conjugates, whereas the average number of

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colonies formed by the biotinylated antibody/STA-AP/MNP conjugates was 185.3 ± 4.2,

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corresponding to a capture efficiency of the biotinylated antibody/STA-AP/MNP conjugates for E.

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coli O157:H7 of 92.0 ± 0.6%. This demonstrated that the synthesized biotinylated antibody/STA-

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AP/MNP conjugate was useful for separating the target E. coli O157:H7 and shows potential as an

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effective enhanced colorimetric method based on IMS-selective filtration.

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In addition, to evaluate the optimal exposure time with a magnet, E. coli O157:H7 and biotinylated

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antibody/STA-AP/MNP conjugates were processed with various exposure times (5–20 min) to a

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magnet (Figure 2B). The intensities of the colored spots on the membrane gradually increased along

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with increasing magnet exposure time and reached a maximum at 15 min. For all concentrations of E.

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coli O157:H7 tested (102, 107, and 108 CFUmL-1), the color intensities observed at 20 min were

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similar to those observed at 15 min. Consequently, 15 min was chosen as the optimal magnet

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exposure time for IMS.

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Sensitivity and Specificity of the Enhanced Colorimetric Method based on IMSIMS-selective 10

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Filtration. iltration. The proposed method was then validated by determining the sensitivity in the presence of

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different E. coli O157:H7 concentrations in a pure sample (PBS). Figure 3 shows the normalized color

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intensities on the membrane for the target bacteria at concentrations ranging from 5 to 104 CFUmL-1.

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We observed a linear increase in the color spot intensities along with an increase in E. coli O157:H7

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concentration according to a logarithmic plot. The light brown color of the resultant spots on the

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membrane was intensified due to enzymatic amplification with NBT/BCIP precipitation (Figure 3). In

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addition, the linear correlations (from 101 to 104 CFUmL-1) before and after application of the

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enhanced colorimetric method via enzymatic amplification were high, at R2 = 0.9811 and R2 = 0.9777,

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respectively, with a limit of detection (LOD) of 27.515 CFUmL-1 and 6.998 CFUmL-1, respectively.

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These LOD result of optical density values indicated that the enhanced colorimetric method by

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enzymatic amplification improved the LOD for E. coli O157:H7 detection. Although the detection

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time of the proposed method may not be substantially faster than that of previous methods developed

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for visual colorimetry-based bacteria detection, it has the advantage of being able to detect the

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bacteria at very high sensitivity (visual LOD: 10 CFUmL-1) by visual observation alone without

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requiring additional equipment (Table S1). The ELISA has a total assay time of 3–5 h, which is much

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longer than the whole procedure (incubation, washing, filtration, and enzymatic amplification) of the

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proposed method, which takes only about 55 min to generate the results. By contrast, the colorimetry

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and lateral flow immunoassay methods only require approximately 20 min to produce results.

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However, these rapid methods have a poor detection limit, which is significantly higher than that

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demonstrated by our detection method. In addition, the method developed in the present study is

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similar to the IMS and selective filtration technique described in our previous reports except for the

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additional

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antibody/STA-AP/MNP conjugates. Although the proposed method takes about 10–20 min longer to

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produce results due to the additional signal amplification step, it is nevertheless capable of the highest

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sensitivity and clearly enables color discrimination based on the bacterial concentration. These clear

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differences between various concentrations of bacteria conferred by the enhanced colorimetric method

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allow for the highly sensitive detection of the target bacteria with naked-eye observation without

enzymatic

amplification

with

NBT/BCIP precipitation

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the

biotinylated

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requiring expensive instrumentation, making it a suitable on-site-testing tool.

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To evaluate the specificity of this method, different concentrations of the bacteria were evaluated in

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the presence of the target bacteria (E. coli O157:H7) and non-target bacteria (B. cereus, E. coli, L.

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monocytogenes, Salmonella Mbandaka, Salmonella Typhimurium, S. aureus, and V. vulnificus). As

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shown in Figure 4, E. coli O157:H7 exhibited strong colorimetric intensity, whereas the non-targeted

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bacteria showed a considerably lower color intensity. These results demonstrated that the proposed

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method has a high degree of specificity toward the target E. coli O157:H7, regardless of the presence

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of or potential cross-reactivity with other bacteria.

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Detection of E. coli O157:H7 Inoculated on Lettuce Samples. amples. To further demonstrate the

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efficacy of this method, we detected E. coli O157:H7 that was artificially inoculated on lettuce sample

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(from 5 to 104 CFUmL-1) before (Figure 5A) and after (Figure 5B) enzymatic signal amplification

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with NBT/BCIP precipitation. Although the color intensity level on the lettuce sample (Figure 5) was

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lower than that detected in PBS (Figure 3), linear increases in the intensities were observed along with

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an increase in the E. coli O157:H7 concentration according to a logarithmic plot. The light brown

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color of the resultant spots on the membrane was clearly intensified by enzymatic amplification with

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NBT/BCIP precipitation (Figure 5). In addition, the linear relationships (from 101 to 104 CFUmL-1)

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before and after application of the enhanced colorimetric method using enzymatic amplification were

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R2 = 0.9876 and R2 = 0.9397, respectively, with an LOD of 91.896 CFUmL-1 and 21.216 CFUmL-1,

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respectively. The visual LOD of 10 CFUmL-1 by naked-eye observation was obtained using the

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enhanced colorimetric method. Therefore, this platform offers novel capability in this regard and can

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be useful as a tool for the sensitive and selective detection of target bacteria in various foods and real

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environmental samples.

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In conclusion, we have developed an enhanced colorimetric method using enzymatic amplification

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with NBT/BCIP precipitation based on IMS-selective filtration for the ultrasensitive detection of E.

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coli O157:H7. The AP of the target bacteria-conjugates complexes on the membrane surface reacted

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with the NBT/BCIP solution to intensify the colored spots formed on the membrane via enzyme-

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catalyzed precipitation. In the presence of the target bacteria, the colorimetric intensities increased 12

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along with an increase in the E. coli O157:H7 concentration. Qualitative and quantitative detection of

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E. coli O157:H7 in pure samples were successfully realized with LODs of 10 and 6.998 CFUmL-1 by

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visual observation and measurements of optical density values, respectively. This detection was

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successfully performed within 55 min and was highly specific to the target bacteria for naked-eye

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observation. In addition, the method was successfully applied to detect E. coli O157:H7 in lettuce

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samples. The LODs by visual observation and measurements of optical density values for E. coli

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O157:H7 inoculated in lettuce samples were 10 and 21.216 CFUmL-1, respectively. Despite inhibition

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of the lettuce sample, the visual LOD for both the pure sample and the lettuce sample was 10 CFUmL-

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1

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enhanced colorimetric method using an enzymatic amplification system can be employed to detect

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target bacteria in diverse food contaminants and environmental pollutants, and can be used for

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biosafety assays in an on-site manner without requiring expensive additional equipment.

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ABBREVIATIONS USED

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AP, alkaline phosphatase; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; BHI, brain heart infusion;

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BSA, bovine serum albumin; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride;

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ELISA, enzyme-linked immunosorbent assay; IMS, immunomagnetic separation; MNP, magnetic

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nanoparticle; NBT, nitroblue tetrazolium; PBS, phosphate buffered saline; TSB, tryptic soy broth

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AUTHOR INFORMATION

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Corresponding Author

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* E-mail:

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ORCID

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Min-Gon Kim: 0000-0002-3525-0048

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Author Contributions

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, which was effectively able to detect the target bacteria using the proposed method. As a result, the



[email protected] (Min-Gon Kim)

S. U. Kim and E.-J. Jo contributed equally to this work.

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENT 13

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The authors would like to acknowledge financial support from the Gwangju Institute of Science and

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Technology (GIST), Korea, under the Practical Research and Development support program

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supervised by the the GIST Technology Institute (GTI). This research was supported by a grant from

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the World Institute of Kimchi funded by the Ministry of Science, ICT and Future Planning (KE1701-

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5), Republic of Korea, and by grants from the Mid-career Researcher Program (NRF-

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2017R1A2B3010816) through a National Research Foundation grant funded by the Ministry of

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Science, ICT, and Future Planning, Republic of Korea.

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ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website.

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Photograph of the selective filtration system; scanning electron microscopy images of

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nitrocellulose membrane filters; hydrodynamic diameters of various MNP conjugates;

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comparison of methods developed for naked-eye colorimetric detection (PDF)

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Figure legends

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Figure 1. Enhanced colorimetric method using enzymatic amplification with NBT/BCIP

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precipitation based on immunomagnetic separation (IMS)-selective filtration for the

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ultrasensitive detection of E. coli O157:H7. (A) IMS for target bacteria using biotinylated

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antibody/STA-AP/MNP conjugates. (B) Selective filtration of target bacteria–conjugates

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complexes. (C) Colored spots on the filter membrane by target bacteria-bound biotinylated

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antibody/STA-AP/MNP conjugates (upper), and enhanced colorimetric spots by enzymatic

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amplification with NBT/BCIP precipitation on the bacteria-bound biotinylated antibody/STA-

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AP/MNP conjugates surfaces (lower).

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Figure 2. Immunological magnetic capture and separation of E. coli O157:H7. (A) Capture

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efficiencies of various MNP conjugates (a: MNP, b: antibody/MNP conjugates, c: STA-

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AP/MNP, d: biotinylated antibody/STA-AP/MNP conjugates) for E. coli O157:H7 (2 × 102

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CFUmL-1). (B) Optimized magnet exposure time of E. coli O157:H7 (102, 107, and 108

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CFUmL-1)-conjugates complexes.

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Figure 3. Sensitivity of the proposed method for the detection of E. coli O157:H7 in PBS

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before (A) and after (B) enzymatic signal amplification with NBT/BCIP precipitation based

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on IMS-selective filtration. Data represent the normalized color intensity for E. coli O157:H7

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at concentrations ranging from 5 × 100 CFUmL-1 to 104 CFUmL-1.

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Figure 4. Specificity of the proposed method in the presence of the target bacteria (E. coli

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O157:H7) and non-target bacteria (B. cereus, E. coli, L. monocytogenes, Salmonella

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Mbandaka, Salmonella Typhimurium, S. aureus, and V. vulnificus).

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Figure 5. Sensitivity of the proposed method for the detection of E. coli O157:H7 in a lettuce

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sample artificially inoculated with E. coli O157:H7 (from 5 × 100 to 104 CFUmL-1) before (A)

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and after (B) enzymatic signal amplification with NBT/BCIP precipitation based on IMS-

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selective filtration. Data represent the normalized color intensity for E. coli O157:H7 at

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concentrations ranging from 5 × 100 CFUmL-1 to 104 CFUmL-1.

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