Article pubs.acs.org/acssensors
Dual Functional Core−Shell Fluorescent Ag2S@Carbon Nanostructure for Selective Assay of E. coli O157:H7 and Bactericidal Treatment Ning Wang, Xing Wei, An-Qi Zheng, Ting Yang, Ming-Li Chen,* and Jian-Hua Wang* Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Shenyang, China, 110819 S Supporting Information *
ABSTRACT: A dual functional fluorescent core−shell Ag2S@ Carbon nanostructure is prepared by a hydrothermally assisted multi-amino synthesis approach with folic acid (FA), polyethylenimine (PEI), and mannoses (Mans) as carbon and nitrogen sources (FA-PEI-Mans-Ag2S nanocomposite shortly as Ag2S@C). The nanostructure exhibits strong fluorescent emission at λex/λem = 340/450 nm with a quantum yield of 12.57 ± 0.52%. Ag2S@C is bound to E. coli O157:H7 via strong interaction with the Mans moiety in Ag2S@C with FimH proteins on the fimbriae tip in E. coli O157:H7. Fluorescence emission from Ag2S@C/E. coli conjugate is closely related to the content of E. coli O157:H7. Thus, a novel procedure for fluorescence assay of E. coli O157:H7 is developed, offering a detection limit of 330 cfu mL−1. Meanwhile, the Ag2S@C nanostructure exhibits excellent antibacterial performance against E. coli O157:H7. A 99.9% sterilization rate can be readily achieved for E. coli O157:H7 at a concentration of 106−107 cfu mL−1 with 3.3 or 10 μg mL−1 of Ag2S@C with an interaction time of 5 or 0.5 min, respectively. KEYWORDS: core−shell nanostructure, Ag2S@C, dual-function, fluorescence, E. coli assay, antibacterial effect
A
s an indicator pathogen, Escherichia coli (E. coli) O157:H7 can cause a series of diseases.1,2 As one of the most dangerous pathogen types, E. coli O157:H7 can also cause other serious illnesses especially in young and immunocompromised individuals.3 Therefore, the development of sensitive and selective approaches for E. coli O157:H7 assay is highly required. Various methods for the determination of bacteria have been investigated, including culture and colony counting,4 microscopy,5 electrochemistry,6,7 enzyme-linked immune sorbent assay,8 and spectrometry.9−11 Among those analysis schemes, fluorescence based procedures play a significant role in biosensing applications.12 Meanwhile, fluorescent methods are capable of elucidating biological processes, investigating structure and dynamics of biological macromolecules, as well as determining the trace level of biological macromolecules.13,14 For this purpose, quite a lot fluorescent materials have been exploited, e.g., magnetic nanoparticles,12 quantum dots,15 organic materials,16 and natural carbohydrate.17 Recently, antibacterial agents and their applications have received extensive attention due to the fact that more and more problems were caused by bacteria. In practice, the commonly used antibacterial agents, i.e., ozone, hypochlorite, and antibiotics,18,19 tend to cause contamination or salting of freshwater sources20 and bacterial antibiotic resistance.21 In the © 2017 American Chemical Society
past decade, inorganic nanoparticles have held promise for serving as a probe for the biological analysis as well as bacterial and mammalian cell killing.22−24 Meanwhile, many effective antimicrobial materials were developed.25,26 Silver-based nanomaterials27,28 have shown excellent potential for bactericidal applications. In addition, silver nanoparticles exhibit significant toxicity when their size becomes smaller.29 For further applications, these antibacterial agents were handicapped due to their considerable cytotoxicity.30,31 Furthermore, a lot of attention was devoted to the preparation of different hybrid heteronanostructures of the core−shell structure.32,33 Various specific functional groups34 and semiconductors35 as shell materials were fabricated on the surface of the core, which can prevent the core from agglomeration and oxidation. Monodisperse and stable core−shell type nanoparticles could obviously reduce toxicity of the materials. These observations provide obviously promising potentials in biosensing and antibacterial applications. However, so far there is no report on dual-functional nanostructured material that serves as both a fluorescence sensing probe for bacteria and an Received: October 31, 2016 Accepted: February 8, 2017 Published: February 8, 2017 371
DOI: 10.1021/acssensors.6b00688 ACS Sens. 2017, 2, 371−378
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ACS Sensors antibacterial agent. In this work, a novel environmentally benign mannose-modified dual-functional core−shell fluorescent nanostructure is described, i.e., Ag2S@C core−shell nanostructure. Ag2S@C is bound on E. coli O157:H7 and fluorescence emission by Ag2S@C/E. coli conjugate is closely related to the content of E. coli O157:H7. This observation provides a sensitive and selective procedure for the assay of E. coli O157:H7. Meanwhile, the nanocomposite also offers a favorable bactericidal effect to E. coli O157:H7. Furthermore, the Ag2S@C core−shell nanostructure exhibits low toxicity and extraordinary photostability.
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Figure 1. HRTEM images (a) and AFM images (b) of Ag2S@C core− shell nanostructure.
EXPERIMENTAL SECTION
Preparation of Ag2S@C Core−Shell Nanostructure. 16 mL FA suspension (0.2 g of FA) and 4 mL PEI solution (100 mg mL−1) were successively added into a 150 mL conical flask and sonicated for 30 min. 0.02 g of Mans was then added followed by 10 min sonication, and afterward injection of 2 mL of 0.1 mol L−1 AgNO3 solution. The reaction mixture was continuously stirred for 12 h followed by addition of 20 mL Na2S solution (0.02 mol L−1) and 8 mL deionized water. After stirring the reaction mixture for 8 h, it was transferred into a 100 mL Teflon-lined pressure vessel, and the mixture was heated at 160 °C for 5 h. The product, i.e., Ag2S@C core−shell nanostructure with a brown color, was obtained. The agglomerated particles were removed by centrifugation at 10 000 rpm for 10 min for twice. The final product was achieved after purification by Sephadex G-25 gel column with deionized water as eluent. For the purpose of comparison, the following nanostructures containing part of the components in Ag2S@C, e.g., FA-Man, FAMan-Ag2S, FA-PEI-Man, and FA-PEI-Ag2S, were prepared by following the procedures described here. Fluorescent Sensing of E. coli O157:H7 with Ag2S@C. The fluorescence nature of the Ag2S@C core−shell nanostructure was found to depend on ionic strength. This was studied by introducing various amounts of NaCl. In addition, the influences of other frequently encountered species in biological sample matrixes, e.g., cationic and anionic species, amino acids, and proteins, were also evaluated in the presence of each individual species. In all the studies, the excitation of Ag2S@C solution was performed at 340 nm, and fluorescence at 450 nm was recorded. E. coli O157:H7 suspension of ca. 108 cfu mL−1 was cleaned for twice at pH 7 by PBS buffer at 0.2 mol L−1 for the purpose of removing the culture medium, followed by diluting to 1.0 × 103 to 5.0 × 107 cfu mL−1 with PBS buffer. 900 μL suspension of E. coli O157:H7 and 450 μL Ag2S@C solution (4.0 mg mL−1) were mixed in a tube, followed by incubating at 37 °C for 1 h. Afterward, the bacteria were separated by centrifuging at 8000 rpm for 5 min, and they were then cleaned by using PBS buffer for twice for future use. The experimental procedures for antibacterial activity and cytotoxicity of Ag2S@C were given in the Supporting Information.
The full scan spectrum indicates the typical peaks for C 1s (284.7 eV), N 1s (398.3 eV), O 1s (531.3 eV), and S 2p (167.6 eV). Elemental analysis for the Ag2S@C core−shell nanostructure revealed the composition of C (52.89 wt %), N (25.52 wt %), O (14.25 wt %), and S (4.03 wt %). In Figure 2a, the high-resolution C 1s XPS spectra are deconvoluted into the following peaks, e.g., 284.2, 284.7, 285.2, 286.0, 286.5, and 288.2 eV, corresponding to the chemical bonds of C−C, CC, C−S, C−N, C−O, and CO/C N.36,37 The N 1s spectrum (Figure 2b) gives rise to the peaks at 398.3 and 399.6 eV, attributed to pyridine-like N and amino N,38 respectively. The O 1s spectrum (Figure 2c) exhibits four main bands at 530.3, 531.2, 531.7, and 532.7 eV, which can be identified as CO, C−N−O, OC−O, and C−OH/C−O− C.39,40 The S 2p spectrum (Figure 2d) illustrates two major peaks at 163.4 and 167.6 eV, ascribed to sulfur in the C−S and S−O groups, respectively. The 163.4 eV peak is further resolved to 163.4 and 164.2 eV, which can be safely assigned to covalent bonds of S 2p3/2 and S 2p1/2.36,41,42 FT-IR spectra of FA, Mans, and Ag2S@C core−shell nanostructure were shown in Figure S2. The broad absorptions in the range of 3200−3500 cm−1 related to the O−H stretching and N−H stretching vibrations for amine groups.43 Absorptions of 2930 and 2840 cm−1 were contributed respectively by the stretching vibration of −CH2− in PEI.44 The broad absorption within 1600−1740 cm−1 was identified as amide carbonyl, while that appearing at 1500 cm−1 was contributed by CC.43,45 The characteristic absorption bands at 1385, 1330, 1090−1200, and 937 cm−1 could be attributed by O−H bending vibration, C−N stretching vibration, C−O/C−S stretching vibration, and γC−O group in the Ag2S@C core−shell nanostructure.46,47 The identification of −OH, −NH, C−O/C−S, and −CONH− groups in the Ag2S@C core−shell nanostructure ensures its solubility in the aqueous medium. The surface charge analysis for the Ag2S@C core−shell nanostructure revealed that they are positively charged with a zeta potential of 3.07 ± 0.14 mV at pH 9.2. This might be because of the fact that there are amine groups in the structure of Ag2S@C. XRD patterns in Figure S3 illustrated a significant difference for Ag2S@C and FA-PEI-Mans. The only difference for the structures of Ag2S@C and FA-PEI-Mans is the presence of Ag2S in the former, while there is none in the latter. Three strong peaks at 31.70°, 38.44°, and 44.83°(marked with ■) are identified in the XRD pattern for Ag2S@C, corresponding to the (−311), (−330), and (171) planes of C6H13−AgS (PDF card No.: 00-047-2093). While the diffraction peaks at 34.20°, 36.50°, and 40.80° (marked with •) are identified for the (150), (−310), and (141) C6H5−AgS (PDF card No.: 00-048-1976). Furthermore, diffraction peaks at 2θ 40.80°, 64.68°, 77.55°, and
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RESULTS AND DISCUSSION Characterization of Ag2S@C Core−Shell Nanostructure. HRTEM images of the Ag2S@C core−shell nanostructure are given in Figure 1a. They are well dispersed in aqueous medium and no obvious aggregation is observed showing ca. 30−40 nm in size. The inset illustrated the encapsulation of Ag2S@C core−shell nanostructure into a gray carbon shell of ca. 8.5 nm in size. AFM images (Figure 1b) indicate the monodispersed Ag2S@ C core−shell nanostructure, and they are mostly distributed within a range of 25−45 nm, giving a diameter of 32.90 nm on average (Figure S1a). This result is consistent with that observed by HRTEM images. XPS spectrometry was performed to investigate the surface state of the Ag2S@C core−shell nanostructure (Figure S1b). 372
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Figure 2. XPS spectra for Ag2S@C core−shell nanostructure. C 1s (a), N 1s (b), O 1s (c), and S 2p (d).
Figure 3. Absorption and fluorescence spectra for the Ag2S@C core−shell nanostructure. Inset: photographs of the Ag2S@C core−shell nanostructure at natural light (left) and 365 nm (right) irradiation in aqueous medium (a). Fluorescence emission spectra for Ag2S@C core−shell nanostructure at λex 300−440 nm (b). The concentration of Ag2S@C: 1.3 mg mL−1.
81.51° (marked with ★) indicate the (031), (302), (125), and (251) planes of Ag2S (PDF card No.: 00-003-0844). These observations illustrated the presence of −AgS moiety (or Ag−S bond) in the Ag2S@C core−shell nanostructure. TGA analytical results for FA-PEI-Mans and Ag2S@C core− shell nanostructure were given in Figure S4. It indicated that a slight weight loss at 80−105 °C was the result of water depletion. Thereafter, a significant weight loss was observed within 200−400 °C because of stripping of the functional groups of FA-PEI-Mans and Ag2S@C core−shell nanostructure, respectively.48 After 400 °C, the TGA curves show a slight drop in both cases, corresponding to residues of ca.18.28% (wt
%) for FA-PEI-Mans and ca. 25.61% (wt %) for Ag2S@C. A difference of ca.7.33% (wt %) was due to the presence of Ag2S in the Ag2S@C, corresponding to a mass ratio of 6.38% (wt %) for silver in Ag2S@C. Meanwhile, the content of Ag was also investigated by ICP-MS, and the results showed that the mass fraction of Ag is 7.64% (wt %) in the Ag2S@C core−shell nanostructure. This result is consistent with that achieved from TGA analysis. The fluorescence emission and UV−vis adsorption spectra of Ag2S@C core−shell nanostructure were illustrated in Figure 3a. The obvious absorption at 270 nm could be assigned to σ−π* transition for the CO bond, and that at 350 nm is assigned to 373
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Figure 4. Variation of fluorescence intensities (λex/λem = 340/450 nm) of Ag2S@C core−shell nanostructure at 1.3 mg mL−1 in the presence of various commonly encountered ions at 10 mmol L−1 (a), amino acids at 10 mmol L−1, along with albumin at 5 μmol L−1 (b).
π−π* transition for the conjugated CC bond. On the other hand, Ag2S@C displays an intense fluorescence at λex/λem = 340 nm/450 nm. Further studies gave rise to a quantum yield (QY) of 12.57 ± 0.52% for the Ag2S@C core−shell nanostructure. The inset in Figure 3a illustrated the photographs of Ag2S@C solution by exciting at 365 nm and daylight, respectively. The bright blue color with excitation by UV light further confirms that the Ag2S@C core−shell nanostructure is an excellent fluorescence probe. The fluorescence spectra in Figure 3b illustrated that by excitation at 300−440 nm, a red shift of the fluorescence wavelength is observed from 450 to 490 nm. The excitationdependent nature of emission for luminescent carbon nanostructures is widely reported, which might be due to different surface defects of the band gap in the Ag2S@C core− shell nanostructure.49 Fluorescence Behavior of Ag2S@C Core−Shell Nanostructure. The dependence of the fluorescence behavior of the Ag2S@C core−shell nanostructure on the ionic strength was investigated by incorporating 0−1.0 mol L−1 NaCl (Figure S5a) in a Ag2S@C solution of 1.3 mg mL−1. It indicated that when varying the ionic strength within a certain range, no obvious variation was observed for the fluorescence of Ag2S@C. Moreover, it is obvious that the Ag2S@C solution shows a favorable stability, illustrated by the fact that fluorescence change is very limited by illumination for 2 h (Figure S5b). Further investigations indicated that some commonly encountered species in biological samples, e.g., 10 mmol L−1 Na+, K+, Ca2+, Mg2+, CO32−, HPO42−, Cl−, and CH3COO−, pose a very small effect on the assay as demonstrated by the virtually constant fluorescence for Ag2S@C (Figure 4a). In addition, 10 mmol L−1 of the following amino acids, e.g., glycine, isoleucine, alanine, threonine, lysine, valine, arginine, proline, serine, and leucine, and 5 μmol L−1 of albumin including cytochrome c, transferring, ovalbumin, horseradish peroxidase, bovine serum albumin, and whey protein result in limited variation of fluorescence (Figure 4b). Detection of E. coli O157:H7. The sensing of E. coli O157:H7 with Ag2S@C core−shell nanostructure as probe was evaluated in a PBS buffer medium. Figure S6 illustrated a linear calibration as obtained within the range of 1.0 × 103 to 5.0 × 107 cfu mL−1 by plotting the bacteria concentration versus fluorescence intensity of the Ag2S@C/E. coli conjugate. A detection limit of 330 cfu mL−1 was obtained, which is
obviously more sensitive than those of the recently reported procedures for E. coli detection.15,17,50−52 The literature illustrated that type 1 fimbriae on the surface of Enterobacteriaceae,53 e.g., E. coli, are responsible for their activities for Mans and mannoside-binding, which produces a strong interaction between Mans and FimH.15,17 In this study, the employment of Mans containing a precursor for the preparation of Ag2S@C core−shell nanostructure tends to exhibit a strong interaction with FimH proteins generally found on fimbriae tips in E. coli O157:H7. This provides the basis for its selectivity toward E. coli O157:H7. For the purpose of comparison with Ag2S@C (a FA-PEIMans-Ag2S core−shell nanostructure), FA-PEI-Ag2S nanocomposite was fabricated, where no Mans precursor was involved with respect to that in the preparation of Ag2S@C. The interaction of FA-PEI-Ag2S with the bacteria was investigated. The experiments showed that, for FA-PEI-Ag2S, a fluorescence emission at λex/λem = 340/450 nm was observed. After performing similar operations as those for Ag2S@C, i.e., allowing E. coli O157:H7 to incubate/interact with FA-PEIAg2S and afterward isolated the bacteria by centrifugation, no obvious fluorescence increase was observed, which is opposite to what is observed for Ag2S@C and E. coli interaction. This observation clearly indicated that for the preparation of the Ag2S@C core−shell nanostructure, the precursor Mans plays a crucial role in making Ag2S@C a suitable probe to perform the E. coli O157:H7 assay, and the interaction of mannose with FimH proteins provides special selectivity toward E. coli O157:H7. Selectivity to E. coli O157:H7 against Coexisting Bacteria. E. coli O157:H7 is known as a Gram-negative (G−) bacterium. Further studies were carried out for evaluating the selectivity of the fluorescent Ag 2 S@C core−shell nanostructure to E. coli O157:H7. For this purpose, four other bacteria species at 1.0 × 105 cfu mL−1 were chosen to investigate their effect on the fluorescence of Ag2S@C. These bacteria include Staphylococcus aureus (S. aureus, G+), Saccharomycetes, Bacillus subtilis (B. subtilis, G + ), and Pseudomonas aeruginosa (P. aeruginosa, G−), and the results were given in Figure 5. For E. coli O157:H7 assay at three concentration levels, i.e., 1.0 × 103, 1.0 × 105, and 1.0 × 107 cfu mL−1, the fluorescence of Ag2S@C was investigated when there was 1.0 × 105 cfu mL−1 S. aureus, Saccharomycetes, B. subtilis, or P. aeruginosa. The results indicated that at 1.0 × 105 and 1.0 × 107 cfu mL−1, the coexisting bacteria cause a fluorescence 374
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of the bacteria. It is observed that a 99.9% sterilizing rate was recorded for E. coli O157:H7 with 3.3 μg mL−1 Ag2S@C and an interaction time of 5 min. When a higher concentration of Ag2S@C, i.e., 10 μg mL−1, was employed, a same sterilization rate could be readily obtained with a much shorter interaction time, i.e., 0.5 min. These observations were further demonstrated by the photographs for E. coli O157:H7 colonies grown in broth agar plates with 3.3 or 10 μg mL−1 Ag2S@C core−shell nanostructure (Figure S7). In comparison with those obtained in the control plate (without Ag2S@C), where a large number of E. coli O157:H7 colonies were observed, however, very few colonies were found by treatment with Ag2S@C. Before and after incubation by 3.3 μg mL−1 of the Ag2S@C core−shell nanostructure for 10 min, SEM images for E. coli O157:H7 were given in Figure S8. Native E. coli O157:H7 cells have smooth surfaces, while they are obviously destroyed after incubation with Ag2S@C with very rough appearance. For the purpose of further elucidating the antibacterial mechanisms, E. coli O157:H7 viability was thoroughly evaluated by treating with FA-Mans, FA-Mans-Ag2S, FA-PEI-Mans, FAPEI-Ag2S, and Ag2S@C at three concentration levels (1.0, 2.0, and 3.3 μg mL−1). With a fixed interaction time of 10 min, the results were shown in Figure 6b. In the absence of PEI and Ag2S, a very limited antibacterial effect was observed for FAMans nanocomposite. When treated by the same concentration of FA-Mans-Ag2S and FA-PEI-Mans, E. coli O157:H7 viability significantly declined to ca. 27.8% and 25.5%, respectively. This indicated that the antibacterial effect might be associated with the synergetic effect of PEI and Ag2S. Similarly, after treatment with Ag2S@C an obvious improvement to the antibacterial effect is observed with respect to FA-PEI-Ag2S, giving rise to a viability of 0.01% for E. coli O157:H7 with 3.3 μg mL−1 Ag2S@ C. This clearly demonstrated that Mans play a vital role in the excellent bactericidal effect of Ag2S@C. The cytotoxicity of the Ag2S@C core−shell nanostructure was investigated by incubating HeLa cells with Ag2S@C (0.001−10.0 mg mL−1, Figure S9). It was seen that Ag2S@C exhibits low cytotoxicity to HeLa cells, providing a viability of >90% at 5 mg mL−1. In addition, at 10 mg mL−1 of Ag2S@C a >85% viability could still be achieved. This might be because in the core−shell structure the innocuous carbon shell alleviated the toxicity of Ag2S moiety. The observations herein demonstrated that the low cytotoxicity of the Ag2S@C core− shell nanostructure facilitates its application in biological systems.
Figure 5. Effect of coexisting bacteria, e.g., 1.0 × 105 cfu mL−1 of B. subtilis, S. aureus, P. aeruginosa, or Saccharomycetes, on the fluorescence (λex/λem = 340/450 nm) of 1.3 mg mL−1 Ag2S@C core−shell nanostructure, in the presence of 1.0 × 103 (***), 1.0 × 105 (**), or 1.0 × 107 (*) cfu mL−1 E. coli O157:H7.
variation of
