Ultrasensitive detection of Escherichia coli O157:H7 by

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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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Journal of Agricultural and Food Chemistry

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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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ACS Paragon Plus Environment


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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

263

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

11


using

the

biotinylated


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

272

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

281

(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

283

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

284

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

286

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

287

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,

289

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

290

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

292

environmental samples.

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

294

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

295

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

303

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

304

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

305

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

306

1

307

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

308

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

309

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

310

ABBREVIATIONS USED

311

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

312

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

313

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

314

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

315

AUTHOR INFORMATION

316

Corresponding Author

317

* E-mail:

318

ORCID

319

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

320

Author Contributions

321

, 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.

322

Notes

323

The authors declare no competing financial interest.

324 325

ACKNOWLEDGMENT 13



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

327

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

328

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

329

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

330

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

331

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

332

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

333 334

ASSOCIATED CONTENT

335

Supporting Information

336

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

337

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

338

nitrocellulose membrane filters; hydrodynamic diameters of various MNP conjugates;

339

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

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colloidal gold immunochromatographic signal-amplifying system for ultrasensitive detection of

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Figure legends

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

414

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

415

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

416

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

417

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

419

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

420

AP/MNP conjugates surfaces (lower).

421 422

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

423

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

424

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

425

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

426

CFUmL-1)-conjugates complexes.

427 428

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

429

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

430

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

431

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

432 17



433

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

434

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

435

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

436 437

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

438

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

439

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

440

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

441

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

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Ultrasensitive detection of Escherichia coli O157:H7 by

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