Analytica Chimica Acta 998 (2018) 52e59
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Novel immunochromatographic assay based on Eu (III)-doped polystyrene nanoparticle-linker-monoclonal antibody for sensitive detection of Escherichia coli O157:H7 Ke-Yu Xing a, Juan Peng a, Dao-Feng Liu b, Li-Ming Hu a, Chun Wang a, Guo-Qiang Li a, Gang-Gang Zhang a, Zhen Huang a, Song Cheng a, Fang-Fei Zhu a, Na-Mei Liu a, Wei-Hua Lai a, * a b
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China Jiangxi Province Center for Disease Control and Prevention, Nanchang 330047, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Novel ICA based on EuNP-linker-mAb was used to sensitively detect E. coli O157:H7. SA not only conjugated more BiomAb but also provided more active sites of the mAb. EuNP-SA-ICA had advantages over other four ICAs in sensitivity and linear range. EuNP-SA-ICA offers promising application in the detection of milk sample.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2017 Received in revised form 15 October 2017 Accepted 21 October 2017 Available online 27 October 2017
Colloidal gold immunochromatographic assay (ICA) has poor sensitivity when used for Escherichia coli O157:H7 (E. coli O157:H7) detection. Eu (III)-doped polystyrene nanoparticle (EuNP) has a large range of stokes shift, long decay time, and wide excitation spectrum and narrow emission spectra. EuNP has been used as novel probe in ICA to improve sensitivity. In this study, carboxyl-modified EuNPs were prepared with different linkers. ICA based on EuNP, EuNP-6 carbon chain (CC) complex, EuNP-200CC complex, EuNP-1000CC complex, and EuNP-streptavidin (EuNP-SA) complex were systematically compared for the detection of E. coli O157:H7. Under optimized working conditions, the limits of detection (LOD) of EuNPICA, EuNP-6CC-ICA, EuNP-200CC-ICA, EuNP-1000CC-ICA, and EuNP-SA-ICA were 9.54 102, 1.59 102, 3.18 102, 2.98 102, and 1.08 102 colony-forming units (CFU) mL1, respectively. The linear ranges of EuNP-ICA, EuNP-6CC-ICA, EuNP-200CC-ICA, EuNP-1000CC-ICA, and EuNP-SA-ICA were 6.36 102 e1.59 105, 3.18 102e1.59 105, 6.36 102e1.59 105, 6.36 102e1.59 105, and 8.0 101 e1.59 105 CFU mL1, respectively. EuNP-SA-ICA exhibited the highest sensitivity and the widest linear range with good specificity, accuracy, and precision. It could be a promising analytical method for
Keywords: Immunochromatographic assay Escherichia coli O157:H7 Eu (III)-doped polystyrene nanoparticle Streptavidin Carbon chain
Abbreviations: ICA, Immunochromatographic assay; E. coli O157:H7, Escherichia coli O157:H7; EuNP, Eu (III)-doped polystyrene nanoparticle; CC, Carbon chain; SA, Streptavidin; LOD, Limits of detection; CFU, Colony-forming units; ELISA, Enzyme-linked immunosorbent assay; SA-Bio, Streptavidin-biotin; mAb, Monoclonal antibody; pAb, Polyclonal antibody; NC, Nitrocellulose; EDC$HCl, N-(3-dimethylaminopropyl)-N0 -ethylcarbodimide hydrochloride; BSA, Bovine serum albumin; BB, Borate buffer; PBS, Phosphate-buffered saline; TMB, 3,30 ,5,50 -Tetramethylbenzidine; HRP, Horseradish peroxidase; ICTS, Immunochromatographic test strip; Bio-mAb, Biotin-anti-E. coli O157:H7 monoclonal antibody; TEM, Transmission electron microscope; DLS, Dynamic light scattering; PDI, Polydispersity index; CV, Coefficient of variation. * Corresponding author. Tel.: þ86 13879178802; fax: þ86 791 88333708. E-mail address:
[email protected] (W.-H. Lai). https://doi.org/10.1016/j.aca.2017.10.027 0003-2670/© 2017 Elsevier B.V. All rights reserved.
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detecting E. coli O157:H7 in food samples. EuNP-SA-ICA may be a good model for detecting low concentrations of other food-borne pathogens. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Enterohemorrhagic Escherichia coli (E. coli) is a group of E. coli that causes human hemorrhagic diarrhea and enteritis. A particularly dangerous type from this group is referred to as enterohemorrhagic E. coli O157:H7, which is associated with foodborne outbreaks traced to undercooked meats, apple juice or cider, salad, salami, and milk [1]. E. coli O157:H7 was first recognized as a pathogen during a severe hemorrhagic diarrhea in 1982. The US Federal Center for Disease Control and Prevention reported a case on 6 October 2016, which led to the infection of 199 people. The infective strain was E. coli O157:H7. Some methods for detection of pathogens include pure culture [2,3], polymerase chain reaction [4e7], electrochemical strategies [8,9], enzyme-linked immunosorbent assay (ELISA) [10e13], surface plasmon resonance [14,15], gene chip [16], free-flow magnetophoresis [17], and surface enhanced Raman spectroscopy [18,19]. However, these methods are time-consuming, laborious, and not suitable for on-site detection. Colloidal gold immunochromatographic assay (ICA) is convenient and fast but has low sensitivity [20e24]. Eu (III)-doped polystyrene nanoparticle (EuNP) was developed in ICA [25e28]. The nanoparticle has a large stokes shift (>150 nm) and a long fluorescence lifetime and can effectively eliminate the interference of various non-specific fluorescence, thereby leading to high sensitivity. Steric hindrance of label-monoclonal antibody (mAb) is presumed to limit the immunoreaction between target and antibody. Thus, some label complexes conjugating with label, linkers, and antibodies are used in ICA to reduce steric hindrance [27e29]. In this study, novel EuNP was conjugated with antibody via four different linkers and used in ICA for the sensitive detection of E. coli O157:H7 in milk. Linkers enhanced the immunoreaction between target and antibody. Among these linkers, streptavidin-biotin (SABio) system is expected to be more sensitive for an immunoreaction, because the high binding of biotin and streptavidin is a strong combination of multi-level amplification [30e33]. The complex label was first introduced in ICA.
(BSA), borate buffer (BB; 0.05 M, pH 6.0), phosphate-buffered saline (PBS; 0.01 M, pH 7.4), and Tween 20 were purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). 3,30 ,5,50 -Tetramethylbenzidine (TMB) and H2O2 were obtained from Beijing Ke-wei (Beijing, China). Horseradish peroxidase (HRP)econjugated goat anti-mouse IgG was purchased from ZSGB-BIO (Beijing, China). All other chemicals and reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The ELISA reader was purchased from Beijing Pulang New Technology Co., Ltd. (Beijing, China). The portable strip reader for the EuNP-immunochromatographic test strip (ICTS) (excitation wavelength ¼ 365 nm, emission wavelength ¼ 615 nm) was obtained from Fenghang Scientific Instrument Co., Ltd. (Zhejiang, China). 2.2. Bacterial strains culturing E. coli O157:H7 (ATCC 43888) and 14 non-E. coli strains, namely, Staphylococcus aureus (CMCC 26003), Enterobacter sakazakii (CMCC 45407), Shigella flexneri (CMCC 2457), Shigella sonnei (CMCC 51592), Candida albicans (ATCC 10231), Bacillus subtilis (BD 168), Listeria welshimeri (ATCC 35897), Proteusbacillus vulgaris (CMCC 49027), Pseudomonas aeruginosa (CMCC 11997), Micrococcus luteus (CMCC 28001), Salmonella typhimurium (ATCC 13311), Salmonella choleraesuis (ATCC 10708), Salmonella enteritidis (ATCC 13076), and Salmonella paratyphi A (ATCC 9150) were cultured in LuriaeBertani medium (LB, Oxoid, Basing-stoke, UK) at 37 C for 20 h. All these strains were conserved in our laboratory. 2.3. Preparation of biotin-anti-E. coli O157:H7 monoclonal antibody (Bio-mAb) Biotin (10 mM) at 13.3 mL was added to 2 mL of 0.5 mg mL1 anti-E. coli O157:H7 mAb and mixed in a DH-Ⅱ Rotary Mixer from Ningbo Xinzhi Biotechnology Co., Ltd. (Zhejiang, China) at 30 rpm for 45 min at room temperature. Bio-mAb was purified by dialyzing at 4 C for 3 days.
2. Materials and methods
2.4. Preparation of EuNP-SA
2.1. Materials and reagents
EuNP was added to 0.5 mL of 0.05 M BB (pH 6.0) containing 1.75 mg of EDC to activate the carboxyl of surface of EuNP for 15 min. Then, SA was added to form EuNP-SA complexes. The remaining active sites of the complexes were blocked with 50 mL of 0.05 M BB containing 10% (w/v) BSA. Finally, the mixtures were centrifuged at 12,000 rpm at 4 C for 20 min. The pellet was suspended in 1 mL of 0.05 M BB (pH 7.0) containing 0.2% (w/v) BSA and 0.5% (v/v) Tween 20. The residual SA in supernatant was analyzed in a subsequent experiment.
EuNP (excitation wavelength ¼ 365 nm, emission wavelength ¼ 615 nm), EuNP-6 carbon chain (CC), EuNP-200CC, and EuNP-1000CC were purchased from Microdetection Bio-tech Co., Ltd (Nanjing, China). Streptavidin was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Biotin was obtained from Thermo Fisher Scientific (Waltham, MA, USA). AntiE. coli O157:H7 monoclonal antibody (mAb) and rabbit anti-E. coli O157:H7 polyclonal antibody (pAb) were purchased from Meridian Life Science, Inc. (Memphis, TN, USA). Nitrocellulose (NC) membrane, sample pad, conjugate pad, and absorbent pad were purchased from Millipore (Bedford, MA, USA). Polystyrene plates with 96 wells were purchased from Hangzhou Sheng-you Biotech Co., Ltd. (Hangzhou, China). N-(3-dimethylaminopropyl)-N0 -ethylcarbodimide hydrochloride (EDC$HCl), bovine serum albumin
2.5. Detection of coupling rate of EuNP-SA with ELISA The SA solution (100 mL; 1.25 mg mL1) was coated in the ELISA plate and incubated at 4 C for 10 h. After washing thrice with 250 mL of 0.01 M PBS containing 1‰ (v/v) Tween 20, 300 mL of 3% (w/v) BSA solution was added into each well to block active sites
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and then incubated at 37 C for 1 h. After washing thrice, 50 mL of the SA standard solution/supernatant solution and 50 mL 1.25 mg mL1 Bio-mAb were added into the ELISA well and incubated at 37 C for 1 h. After washing five times, 100 mL of HRPconjugated goat anti-mouse IgG complex solution was added into each well and incubated at 37 C for 30 min. After washing five times, 100 mL of the TMB and H2O2 mixed solution was added and incubated at 37 C for 15 min without light. After adding 50 mL of 10% (v/v) H2SO4, the absorbance was measured at 450 nm using a microplate reader. The control sample was 0.01 M PBS. All experiments were performed in triplicate. 2.6. Preparation of EuNP-mAb complexes and EuNP-linker-mAb complexes EuNP, EuNP-6CC, EuNP-200CC, and EuNP-1000CC at 50 mg were added to 0.5 mL of 0.05 M BB containing 1.75 mg of EDC$HCl to activate the carboxyl of surface of the four materials for 15 min. Anti-E. coli O157:H7 mAb was added to form EuNP-mAb, EuNP6CC-mAb, EuNP-200CC-mAb, and EuNP-1000CC-mAb complexes for 2 h. EuNP-SA at 50 mg was added to 0.5 mL of 0.05 M BB. BiomAb was added to form EuNP-SA-Bio-mAb complexes for 2 h. The remaining active sites of EuNP-mAb, EuNP-6CC-mAb, EuNP200CC-mAb, and EuNP-1000CC-mAb, and EuNP-SA-Bio-mAb were blocked with 50 mL of 0.05 M BB containing 10% (w/v) BSA for 2 h. Finally, the mixtures were centrifuged at 12,000 rpm at 4 C for 20 min. The EuNP-mAb and EuNP-linker-mAb complexes were suspended in 1 mL of 0.05 M BB (pH 7.0) with 0.2% (w/v) BSA and 0.5% (v/v) Tween 20. 2.7. Characterization of the EuNP, EuNP-linker, EuNPemAb, and EuNP-linker-mAb complexes The morphology and size of EuNP and EuNP-linkers were characterized using transmission electron microscope (TEM, JEOLJEM 2100, Tokyo, Japan). Dynamic light scattering (DLS), Zeta potential and polydispersity index (PDI) of EuNP, EuNP-linker, EuNPmAb, and EuNP-linker-mAb complexes were analyzed by a particle size analyzer (Nano-ZS, Malvern Instruments Ltd., Worcestershire, U.K.). 2.8. Optimization of key parameters The ICA systems based on EuNP-mAb, EuNP-6CC-mAb, EuNP200CC-mAb, EuNP-1000CC-mAb, and EuNP-SA-Bio-mAb were optimized by varying the following key factors: the concentration of SA (2.5, 5, 10, 20, 40, 80, 160, and 320 mg mg1), the coupling pH (4.0, 5.0, 6.0, 7.0, and 8.0); the concentration of anti-E. coli O157:H7 mAb or Bio-anti-E. coli O157:H7 mAb (25, 50, 100, 150, and 200 mg mL1); the concentration of rabbit anti-E. coli O157:H7 pAb (0.2, 0.4, 0.6, 0.8, and 1.0 mg mL1); and the immunoreaction time. All experiments were conducted in triplicate. 2.9. Preparation of ICTS The rabbit anti-E. coli O157:H7 pAb and goat anti-mouse IgG (0.8 mg mL1) were sprayed by BioDot XYZ platform (Irvine, CA) on the NC membrane and designated as the test and control lines, respectively. Then, the membrane was dried at 37 C for 10 h. The sample pad, conjugate pad, NC membrane, and absorbent pad were assembled as ICTS. 2.10. Detecting E. coli O157:H7 with ICA The spiked E. coli O157:H7 cultures sample solution was diluted
in 10 mM PBS (pH 7.4). Scheme 1 show the negative or positive sample solution (100 mL) was mixed with the label-mAb complexes (2 mL) in an ELISA well for 5 min and then added to ICTS [34]. Fluorescence intensity of the test line (FIT) was recorded after a 200 ms delay and a 400 ms counting window (Scheme 1). All experiments were performed in triplicate. 2.11. Immunological kinetics analysis E. coli O157:H7 (1.59 104 colony-forming units (CFU) mL1) solution at 100 mL was pipetted into a sample well of the strip. The FIT of the five ICTS was recorded every minute and studied for 45 min. The kinetics reaction curve was established by plotting the FIT against the immunoreaction time. 2.12. Specificity analysis The specificity of the five ICAs was assessed by testing the E. coli O157:H7 (104 CFU mL1) target strain and 14 non-E. coli O157:H7 strains (104 CFU mL1). All experiments were repeated in triplicate. 2.13. Detection of real sample Milk sample was spiked with diluted cultures of E. coli O157:H7 with final concentrations of 0, 8.0 101, 1.19 102, 1.59 102, 3.18 102, 6.36 102, 9.54 102, 1.27 103, 1.59 104, 1.59 105, and 1.59 106 CFU mL1. The calibration curve was established by plotting the logarithm of FIT against the logarithm of E. coli O157:H7 concentrations. The limit of detection (LOD) was calculated at negative mean plus three times the standard deviation. The accuracy and precision of the five ICAs were quantitatively analyzed by recovery and coefficient of variation (CV). 3. Results and discussions 3.1. Characterization of the EuNP, EuNP-linker, EuNPemAb, and EuNP-linker-mAb complexes The morphology and size of EuNP and EuNP-linker were characterized by TEM (Fig. S1AeE). They had relative homogeneity in terms of size and had average diameters of 158, 159, 158, 161, and 159 nm, respectively. The hydrodynamic diameters of EuNP, EuNP-6CC, EuNP-200CC, EuNP-1000CC, and EuNP-SA were 213.6 ± 1.8, 201.9 ± 2.1, 209.4 ± 2.7, 201.3 ± 2.2, and 224 ± 3.5 nm, respectively. The hydrodynamic diameters of EuNP-mAb, EuNP-6CC-mAb, EuNP-200CC-mAb, EuNP-1000CCmAb, and EuNP-SA-Bio-mAb were 224.2 ± 3.5, 220.2 ± 3.5, 231.5 ± 6.3, 214.5 ± 3.1, and 230.5 ± 1.7 nm, respectively (Fig. 1AeE). The changes of hydrodynamic diameter showed that five label-mAb complexes were synthesized by coupling the amino group of mAb with the carboxyl group of the labels. The size distribution of the five labels and label-mAb complexes were further investigated by the PDI. Table S1 shows that the five labels and label-mAb complexes had very low PDI (
