Surface-Engineered Starch Magnetic ... - ACS Publications

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Surface engineered starch magnetic microparticles for highly effective separation of broad range of bacteria Ke Luo, Ki-Baek Jeong, Sang-Mook You, Da-Hee Lee, Jong-Yun Jung, and Young-Rok Kim ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b03611 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on September 3, 2018

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ACS Sustainable Chemistry & Engineering

Surface engineered starch magnetic microparticles for highly effective separation of broad range of bacteria

Ke Luo,† Ki-Baek Jeong,† Sang-Mook You, Da-Hee Lee, Jong-Yun Jung, and YoungRok Kim*

Institute of Life Sciences and Resources & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, 1732 Deogyeongdaero, Giheung-gu, Yongin, Republic of Korea

*Corresponding author. Tel: +82-31-201-3830 Fax: +82-31-204-8116 E-mail address: [email protected]

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Abstract: Polymeric magnetic particles (PMPs) have become a powerful tool for the separation and concentration of microorganism from a heterogeneous liquid matrix. The functionalization of PMPs with polycationic polymers, such as chitosan, provides an effective means of capturing a broad spectrum of pathogenic bacteria through the intrinsic nature of chitosan interacting with the surface components of bacteria. Here we report a fairly simple approach for the preparation of starch magnetic microparticles (SMMPs) through molecular rearrangement of short chain glucans (SCGs) produced by enzymatic debranching of waxy maize starch. The surface of SMMPs were readily functionalized with chitosan through electrostatic interaction and hydrogen-bonding. The chitosan-functionalized SMMPs (CS@SMMPs) showed high capture efficiency (>90%) for both Gram-positive and Gram-negative bacteria. To further investigate the mechanisms of chitosan-bacteria interaction, we employed model bacteria with different surface composition. The outer core lipopolysaccharides as well as the surface charge of bacteria was found to be important for the specific interactions of chitosan to bacteria. The biocompatible paramagnetic materials developed in this study would be of promising means of removing or separating bacteria from a contaminated water for hygienic purpose or subsequent biochemical analysis of certain pathogenic bacteria present in the sample.

Keywords:

magnetic

microparticles,

starch,

chitosan,

lipopolysaccharides, waxy maize.

2


bacteria,

interaction,

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INTRODUCTION Polymeric magnetic particles (PMPs) hold tremendous promise for rapid separation and concentration of bacteria from complex sample matrices.1-3 Effective capture and separation allow the bacterial analysis independent of the sample sizes and media volumes. PMPs are typically functionalized with certain recognition elements, such as antibodies, aptamers, and phages, which can specifically recognize and bind to epitopes on the cell surface.4 The PMPs with specific ligands are advantageous for the separation of specific bacteria with high selectivity but there is also a need for the capture and separation of a broad range of bacteria in food, environment, or clinical samples. For example, the rapid and effective separation of diverse microorganisms from fecal samples is conducive to the study of gut microbial communities that is an important factor affecting human immune systems.5 Capturing whole bacterial species could also render robust tool for the analysis of total number of bacteria in evaluating the microbial quality and safety of drinking water.6 Besides, to reach the full potential of magnetic separation techniques in bacterial analysis, several technical challenges remain to be addressed. For instance, there is a need for methods that can reliably fabricate PMPs, where polymeric materials contain magnetic particles in dispersed or core-shell form, with better control of its shape, size, and surface functionality. Polystyrene-based magnetic microbeads are best known for their excellent morphology and size distribution. However, the hydrophobic nature of their surface could bring about undesirable aggregation and nonspecific binding of non-target components to the surface.7 Therefore, surface passivation of the PMPs is typically required in order to reduce nonspecific adsorption of non-target proteins or cells and thus enhance the selectivity of the PMPs. In addition, their synthesis is 3



typically carried out by emulsion polymerization or sol-gel process,8, 9 which could lead to negative environmental impacts as well as limiting a large-scale production. Starch, the second most abundant carbohydrate in nature,10 would be one of the most promising material for producing the sustainable magnetic particles.11, 12 Debranching enzymes, like pullulanase and isoamylase, have been widely utilized for debranching amylopectin-rich starch, such as waxy maize, waxy potato, and waxy rice starch, in which recrystallizable short-chain glucans (SCGs) can be produced through the debranching reaction of the enzymes that hydrolyze α(1,6)-linkages in amylopectin.13 Owing to the crystallizable nature of SCGs, they can be assembled directly into a spherical microstructure in aqueous environment without any energy consumption,14 which provides a simple, effective and environmentally friendly means of large-scale production of magnetic particles. In addition, we integrated chitosan to the starch magnetic microparticles as a functional component to capture and separate a broad range of bacteria from an aqueous sample. Chitosan, a polycationic polymer composed of β-(1–4)-linked N-acetyl-Dglucosamine, has been reported for its intrinsic antimicrobial activities which is mainly associated with the electrostatic interaction between chitosan and bacteria.15-17 In general, chitosan shows higher affinity towards Gram-positive bacteria in comparison to Gram-negative bacteria, which might be due to the relatively higher charge density of Gram-positive bacteria.18, 19 However, the binding mechanism of chitosan to bacteria in terms of Gram-positive and Gram-negative bacteria still remains to be elucidated. The intracellular leakage hypothesis, in which cationic amine groups of chitosan can bind to negatively charged bacterial surfaces through electrostatic interaction, is widely accepted mechanism to explain the mode of action 4


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leading to the antimicrobial activity of chitosan.20 On the other hand, the interaction associated with hydrogen bonding between chitosan and outer membrane protein A was proposed to be responsible for the antimicrobial activity of chitosan to Escherichia coli O157:H7.21 Chitosan can also invades cell membrane via the hydrogen bonding interaction of its D-glucosamine with the OmpA that is an integral bacterial outer membrane protein embedded as a β-barrel.22, 23 Herein, we prepared monodisperse starch magnetic microparticles (SMMPs) through enzymatic debranching of waxy maize starch using pullulanase in combination with the epitaxial seeding approach using dextran-coated iron oxide nanoparticles (Dex@IONPs). Surface modification of SMMPs with chitosan were carried out through electrostatic interaction between chitosan and the surface of SMMPs in order to prepare chitosan-coated SMMPs (CS@SMMP) for capturing a broad range of bacteria. We also employed Klebsiella pneumoniae 2242 wild type and its isogenic mutants devoid of outer-core LPS and fimbriae as model bacteria to further investigate the binding mechanisms of CS@SMMPs to bacteria in terms of the surface components. EXPERIMENTAL SECTION Materials. Pullulanase, ferrous chloride tetrahydrate (FeCl2·4H2O), chitosan (mol. wt. 50,000-190,000 Da), poly-L-lysine (mol. wt 150,000-300,000 Da) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ferric chloride hexahydrate (FeCl3·6H2O), ammonium hydroxide, and acetone were purchased from Daejung (Siheung, Korea). Sodium acetate trihydrate and waxy maize starch were obtained from Yakuri Pure Chemicals (Kyoto, Japan) and Samyang Co. (Seoul, Korea), respectively. The Luria5



Bertani (LB) broth and agar powder were purchased from Becton, Dickinson and Company (FranklinLakes, NJ, USA). Amine-functionalized silica-based magnetic microbeads (NH2-MMBs) were purchased from BIONEER Co. (Daejeon, South Korea). Preparation of dextran-coated iron oxide nanoparticles (Dex@IONPs). Dex@IONPs were synthesized by coprecipitation of ferrous and ferric salts in the presence of dextran as reported by Luo et al.12 Briefly, 80 mM of FeCl3·6H2O (~433 mg), 40 mM of FeCl2·4H2O (~160 mg), and 150 mg of dextran were dissolved in 20 ml of deionized water (DW). The mixture was purged with nitrogen gas to remove dissolved oxygen in solution, followed by ultrasonication by using a Q500 Sonicator (VC 750, Sonics & Materials Inc., Newtown, CT, USA) with on/off cycle of 3s/3s in an ice bath for 3 min at 30% amplitude through a 6-mm ultrasound probe. During the sonication, 60 % ammonium hydroxide solution was added dropwise into the mixture using pipette until the mixture turned to dark. The synthesized Dex@IONPs were washed by magnetic separation three times with 70 % ethanol and DW to remove residual ammonium hydroxide and dextran, followed by sonication for 10 s. The final product was stored at 4 °C until use. Preparation of starch magnetic microparticles (SMMPs). Waxy maize starch (10% w/v) was suspended in 50 ml deionized water (DW) and subjected to microwave heating for 90 s at 700 W for gelatinization. After cooling to 60 °C, pullulanase was added to the gelatinized starch solution to a final concentration of 8 ASPU/ml and incubated in a shaking incubator at 37 °C and 250 rpm for 24 h. After the incubation, the reaction solution was subjected to a centrifugation at 15000 × g for 5 min. 0.9 ml of the supernatant was transferred to a fresh tube and 0.1 ml of 30 6


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mg/ml Dex@IONPs was added to a final concentration of 3 mg/ml. The mixture was then incubated at 4 °C for 4 h to induce the self-assembly of SMMPs. The synthesized SMMPs were washed by magnetic separation three times with 70 % ethanol and DW to remove residual Dex@IONPs and debranched starch. The final product was stored at 4 °C until use. Preparation of chitosan-coated SMMPs (CS@SMMPs) and poly-L-lysinecoated SMMPs (PL@SMMPs). To prepare CS@SMMPs and PL@SMMPs, the synthesized SMMPs was treated with varying concentration of chitosan (0.02% to 0.5%, w/v) and poly-L-lysine (0.01% to 0.1%, w/v). The surface functionalization of SMMPs with chitosan and poly-L-lysine was carried out in 1% (v/v) acetic acid solution and DW, respectively. After the treatment with chitosan and poly-L-lysine for 30 min, the sample was washed by magnetic separation with DW 3 times to remove residual chitosan and poly-L-lysine. The surface charge of the prepared CS@SMMPs and PL@SMMPs were measured by using dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments). Characterization of Dex@IONPs, SMMPs, and CS@SMMPs. The mean particle size and surface charge of Dex@IONPs were measured by DLS (Zetasizer Nano ZS90). The morphology of the synthesized SMMPs and CS@SMMPs were analyzed by field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM), respectively. The mean particle size of SMMPs was estimated by counting at least 100 particles from the FE-SEM images. Magnetic properties of Dex@IONPs, SMMPs, and CS@SMMPs were measured using physical property measurement system (16 T PPMS Dynacool, Quantum Design, USA) at room temperature from −12000 to 12000 Oe. 7



Fourier transform infrared (FT-IR) analysis. FT-IR spectra for chitosan, SMMPs and CS@SMMPs were recorded using a Perkin-Elmer Spectrum One System spectrometer (Foster City, CA, USA) with KBr pellets in the range of 1500 to 4000 cm−1. Bacterial strains, plasmids and growth conditions. K. pneumoniae KCTC 2242 was obtained from the Korean Collection for Type Cultures (Daejeon, South Korea). K. pneumoniae KCTC 2242 ∆wabG mutant strain and K. pneumoniae KCTC 2242 ∆fimA mutant strain were constructed as reported by Huynh et al.24 Atomic force microscopy. Freshly cultured K. pneumoniae KCTC 2242 cells were washed with DW through centrifugation (7600 × g for 3 min) and resuspended in DW to a final concentration of 108 CFU/ml. An aliquot of resuspended cells (5 µl) was applied onto a clean slide glass, followed by drying at room temperature for imaging. The morphology of the K. pneumoniae KCTC 2242 and its isogenic mutant strains, K. pneumoniae KCTC 2242 ∆fimA and K. pneumoniae KCTC 2242 ∆wabG, were examined by atomic force microscopy (AFM, Park System XE-70) in noncontact mode. Magnetic separation. Overnight cultures of Escherichia coli O157:H7, Vibrio cholerae, Staphylococcus aureus, Bacillus cereus, and K. pneumoniae KCTC 2242 were washed with DW and adjusted to a final concentration of 106 CFU/ml in DW. 0.5% (w/v) CS@SMMPs were introduced to the cell suspensions (1 ml). After incubation at room temperature for 10 min with gentle rotation, the cells were separated along with CS@SMMPs to the side of tube by using a magnet, and the supernatant containing the unbound bacteria was transferred to a fresh EP tube. The 8


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absorbance of the supernatant at 600 nm was measured to determine the capture efficiency of CS@SMMPs by following equation:

%CE=

Ninitial − Nunbound ×100 Ninitial

where %CE is relative capture efficiency to the target bacteria, Ninitial is the initial concentration of bacteria, Nunbound is the concentration of unbound bacteria. Recyclability of CS@SMMPs. After the bacterial capture, CS@SMMPs were treated with 1 ml of 0.1% (v/v) NH4OH solution for 10 min with rotating at 10 rpm, followed by three times of washing with 20% ethanol and DW. The recycled CS@SMMPs were treated again with 0.5% (w/v) chitosan as aforementioned. The capture efficiency of the CS@SMMPs were tested through three successive recycling of the same material. Statistical analysis. Capture efficiency of CS@SMMPs for E. coli O157:H7, V. cholerae, S. aureus, and B. cereus were compared under different pH conditions through two-way analysis of variance (ANOVA) using the GraphPad Prism 7 software package (Graphpad Software, Inc., San Diego, CA). Statistical significance was accepted for P-value of

Surface-Engineered Starch Magnetic ... - ACS Publications

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