Article pubs.acs.org/JAFC
Selective Detection of Shiga-like Toxin 1 from Complex Samples Using Pigeon Ovalbumin Functionalized Gold Nanoparticles as Affinity Probes Chun-Hsien Li, Yi-Ling Bai, Karuppuchamy Selvaprakash, Kwok-Kong Tony Mong, and Yu-Chie Chen* Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan S Supporting Information *
ABSTRACT: Escherichia coli O157:H7 is a foodborne pathogen. This bacterial strain can generate Shiga-like toxins (SLTs), which can cause serious sickness and even death. Thus, it is important to develop effective and sensitive methods that can be used to rapidly identify the presence of SLTs from complex samples. Pigeon egg white (PEW) contains abundant glycoproteins, including pigeon ovalbumin (POA) (∼60%). POA possesses Gal-α(1→4)-Gal-β(1→4)-GlcNAc termini, which can recognize the B subunits in SLT type 1 (SLT-1B). Thus, POA is a suitable probe for trapping SLT-1B. In this work, we used PEW proteins as starting materials to react with aqueous tetrachloroauric acid for generation of PEW-protein-immobilized gold nanoparticles (AuNPs@PEW) via one-pot reactions. We demonstrated that the generated AuNPs@PEW were mainly dominated by POAimmobilized Au NPs. The as-prepared AuNPs@PEW were used as affinity probes to selectively probe SLT-1B from complex cell lysates derived from E. coli O157:H7. The selective trapping step can be completed within ∼90 s under microwave heating (power = 450 W) to enrich sufficient SLT-1B for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. Furthermore, this approach can be used to detect SLT-1B at a concentration as low as ∼40 pM. The feasibility of using the proposed method to selectively detect SLT-1B from ham contaminated by E. coli O157:H7 was also demonstrated. KEYWORDS: Shiga-like toxin, Escherichia coli O157:H7, pigeon egg whites, pigeon ovalbumin, gold nanoparticles, MALDI-MS, foodborne pathogens
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complex cell lysate samples.24,28 One-pot reactions have been used to generate protein/peptide functionalized gold nanoprobes by reacting proteins with tetrachloroaurate in aqueous solution.32−34 Proteins/peptides containing specific amino acids, such as cysteines, are suitable reducing agents that can be used to generate protein/peptide-immobilized Au NPs.32−35 PEW contains abundant proteins, and POA contains three cysteines in its sequence.36 Thus, we believed that PEWimmobilized Au NPs (AuNPs@PEW) can be readily generated via one-pot reactions by simply reacting PEW proteins with tetrachloroauric acid for a given time. Furthermore, the generated AuNPs@PEW should be dominated by the Au NPs immobilized with the abundant POA (AuNPs@POA). Therefore, the purification steps of POA from PEW can be eliminated. The generated AuNPs@PEW can be used as affinity probes for SLT-1 based on Gb3 ligands on POA. In the current study, we initially explored the synthesis method for generation of AuNPs@PEW by using PEW proteins as starting materials. To shorten the analysis time, microwave heating24,37,38 was used for selective enrichment of SLT-1B from complex samples. Furthermore, the feasibility of using the as-prepared AuNPs@PEW as affinity probes for SLT-1B from complex samples was demonstrated.
INTRODUCTION Foodborne disease outbreaks resulting from Escherichia coli O157:H7 1−4 have elicited considerable attention. The pathogenicity of E. coli O157:H7 mainly results from the generation of Shiga-like toxins (SLTs), including SLT-1 and SLT-2.5,6 STL-1 can induce cell apoptosis at a concentration as low as ∼10 ng/mL.7 SLTs may be present in undercooked meat,8−11 contaminated drinking water,12,13 and even human milk.14 Thus, sensitive and reliable analytical methods that can be used to rapidly screen for the presence of SLTs in complex samples should be developed. SLTs are AB5 proteins.15−17 The single A subunit can inhibit ribosome activity for protein synthesis, and the B subunit possesses three binding sites toward globotriaosylceramide (Gb3), which contains Gal-α(1→ 4)-Gal-β(1→4)-Glc-β-O-ceramide on the outer membrane of host cells.18 Therefore, Gb3 analogues have been used as probes to interact with SLT1-B.19−22 To enhance the binding affinity, Gb3 analogue-immobilized nanoparticles (NPs)23−25 have been demonstrated as effective probes for selectively targeting SLT1B from complex samples. Gb3-containing glycoproteins such as pigeon ovalbumin (POA) are available in nature.26 Thus, POA can be used as a suitable probe to interact with SLT-1B. Moreover, POA is abundant (∼60%) in pigeon egg white (PEW) proteins.27 Thus, inexpensive PEW proteins can be used as starting materials to generate suitable probes for targeting SLT-1B. Several studies have demonstrated the possibility of selectively enriching or immobilizing POA on Sepharose gels28 or magnetic NPs.29−31 POA-immobilized adsorbents were successfully used to trap SLT-1B from © 2017 American Chemical Society
Received: Revised: Accepted: Published: 4359
February 24, 2017 May 9, 2017 May 11, 2017 May 11, 2017 DOI: 10.1021/acs.jafc.7b00863 J. Agric. Food Chem. 2017, 65, 4359−4365
Article
Journal of Agricultural and Food Chemistry
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followed by the addition of aqueous sodium hydroxide (2 M, 3 μL). To prevent aggregation of the generated Au NPs during reaction, 2 μL of aqueous sodium hydroxide was added first under stirring for 1 min followed by the addition of another 1 μL of aqueous sodium hydroxide solution. The reaction was moved to a water bath at 60 °C and stirred at 400 rpm for ∼4 h. The resultant solution was centrifuged at 26,810g (4 °C) for 30 min. The supernatant was discarded to remove small particles and excess reagents. The remaining AuNPs@PEW were redispersed in deionized water (1 mL) followed by centrifugation at 3750g for 10 min to remove large particles. The procedures were repeated three times. Preparation of SLT-1-Containing Cell Lysate. One colony of E. coli O157:H7 was cultured in the broth prepared from tryptic soy broth and yeast extract broth (TSBY) at 37 °C for 12 h to generate SLT-1. TSBY was prepared by mixing tryptic soy broth (12 g) and yeast extract (2 g) in deionized water (400 mL). The resultant bacterial cells were centrifuged at 3750g for 10 min to remove the supernatant. The remaining bacterial cells were rinsed by Tris buffer at pH 7.4 (25 mM, 1 mL × 2) under centrifugation at 3750g for 10 min. The resultant bacterial suspension (1 mL) with the concentration measured at optical density at the wavelength of 600 nm (OD600) equal to 1 was mixed with aqueous urea (8 M, 1 mL). The mixture was incubated at 37 °C for 2 h to lyse the cells. The resultant solution was filtered by a syringe filter (pore size = 0.2 μm) to collect the filtrate. The filtrate was desalted using a ZipTip. The desalted filtrate (100 μL) containing SLTs was dissolved in Tris buffer at pH 7.4 (25 mM, 100 μL) before use. Selective Enrichment of SLT-1B. AuNPs@PEW (2.65 mg mL−1, 10 μL) were mixed with the cell lysate sample derived from E. coli O157:H7 prepared above (40 μL) under microwave heating (power = 450 W) for ∼90 s. Subsequently, the sample was centrifuged at 26,810g at 4 °C for 10 min, and the supernatant was discarded. The remaining AuNP@PEW−target species conjugates were rinsed by Tris buffer at pH 7.4 (25 mM, 50 μL) by pipetting. The resultant sample was centrifuged at 26,810g for 10 min to remove the supernatant. The remaining conjugates were treated with Galα(1→4)Gal (50 nM, 10 μL) solution, which was prepared in Tris buffer to release target species from the AuNPs@PEW. The resultant solution was centrifuged again at 26,810g for 10 min. The supernatant (2 μL) containing the released target species was mixed with CHCA (20 mg mL−1, 2 μL). The mixture (1 μL) was deposited on a MALDI sample plate, followed by solvent evaporation, and subsequent introduction to a MALDI mass spectrometer for MALDI mass spectrometric analysis. The other sample (40 μL) containing ricin B (20 μL, 10−6 M) mixed with the same lysate (20 μL) was also used as the model sample for conducting the same experiments stated above. Tryptic Digestion of the Conjugates of AuNP@PEW-Target Species. After using AuNPs@PEW as affinity probes to enrich their target species from cell lysate, the resultant conjugates were digested by trypsin to characterize the target species trapped by the nanoprobes. The trapped target species were released by Galα(1→ 4)Gal glycoside (50 nM, 10 μL) prepared in Tris buffer (25 mM, pH 7.4) from the conjugates of AuNP@PEW−target species. After 60 s of pipetting, the resultant solution was centrifuged at 26,810g for 10 min. The released species (10 μL) was mixed with aqueous ammonia bicarbonate buffer (10 μL, 100 mM, pH 8). The resultant solution (20 μL) was digested by trypsin (2 μL, 42.4 μg mL−1) under microwave heating (power = 900 W) for 1 min. The digested product (2 μL) was mixed with CHCA (25 mg mL−1, 2 μL) containing 0.1% TFA followed by depositing the mixture (1 μL) on the MALDI plate. After solvent evaporation, the sample was ready for MALDI mass spectrometric analysis. Preparation of SLT-1-Contaminated Food Samples. Food samples contaminated by SLTs were prepared by culturing two colonies of E. coli O157:H7 on a small piece of ham (∼1 g) placed in TSBY broth (25 mg mL−1, 3 mL) at 37 °C for 12 h. The cell concentration of E. coli O157:H7 in the prepared sample was estimated to be ∼1.7 × 105 cfu mL−1. The resultant sample was centrifuged at 3750g for 10 min to remove the supernatant. The remaining ham was vortex-mixed in Tris buffer at pH 7.4 (25 mM, 3
EXPERIMENTAL PROCEDURES
Reagents and Materials. α-Cyano-4-hydroxycinnamic acid (CHCA), 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), dithiothreitol, iodoacetic acid, sodium pyruvate, and trypsin (from bovine pancreas) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid (TFA) (99%) was obtained from Riedel-de Haen (Buchs, St. Gallen, Switzerland). Acetonitrile (99%) and streptomycin sulfate were purchased from Merck (Darmstadt, Germany). Ammonium bicarbonate, tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid (36.5%), and urea were acquired from J. T. Baker (Phillipsburg, NJ, USA). Tetrachloroauric acid was obtained from Showa (Tokyo, Japan). Luria−Bertani broth, tryptic soy broth, and yeast extract were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). 10-Mecaptodecyl 4-O-(α-Dgalactopyranosyl)-1-D-galactopyranoside (Gal-α(1→4)-Gal) was synthesized according to the method reported previously.24 Ricin B subunit was obtained from Vector Laboratories (Burlingame, CA, USA). Deionized water was obtained from a Merck-Millipore water purification system (Darmstadt, Germany). ZipTips were obtained from Millipore (Bedford, MA, USA). Amicon Ultra 4 centrifugal filters were purchased from Millipore (Billerica, MA, USA). Pall acrodisc syringe filters with Supor membrane (PN-4612; pore size, = 0.2 μm) were obtained from Voigt Global Distribution (Lawrence, KS, USA). Pigeon (Columba livia) eggs were obtained from a local pigeon farm. E. coli O157:H7 (BCRC 13085) was acquired from Bioresource Collection and Research Center (Hsinchu, Taiwan). Ham was obtained from a local supermarket. Instrumentation. Absorption spectra were obtained from a Varian Cary 50 ultraviolet−visible (UV−vis) absorption spectrophotometer (Palo Alto, CA, USA) or a NanoVue plus absorption spectrophotometer (Buckinghamshire, UK). Transmission electron microscopic images were obtained using a JEM2000 FXII transmission electron microscope (TEM) from JEOL (Tokyo, Japan). Mass spectra were obtained using an AutoFlex III matrix-assisted laser desorption/ ionization (MALDI) mass spectrometer (Bruker Daltonics, Breman, Germany). When the analytes had molecular weights
