Antibacterial and Antibiofilm Surfaces through Polydopamine-Assisted Immobilization of Lysostaphin as an Antibacterial Enzyme
Abstract

Antibiotic resistance and the colonization of bacteria on surfaces, often as biofilms, prolong hospitalization periods, increase mortality, and are thus major concerns for health care providers. There is an urgent need for antimicrobial and antibiofilm surface treatments that are permanent, can eradicate both biofilms and planktonic pathogens over long periods of time, and do not select for resistant strains. In this study, we have demonstrated a simple, robust, and biocompatible method that utilizes the adhesive property of polydopamine (PDA) to covalently attach the antimicrobial enzyme lysostaphin (Lst) to a variety of surfaces to generate antibacterial and antibiofilm interfaces. The immobilization of the recombinant Lst onto PDA-coated surfaces was carried out under physiological conditions, most probably through the C-terminal His6-tag fragment of the enzyme, minimizing the losses of bioagent activity. The modified surfaces were extensively characterized by X-ray photoelectron spectroscopy and peak force quantitative nanomechanical mapping (PeakForce QNM) AFM-based method, and the presence of Lst on the surfaces was further confirmed immunochemically using anti-Lst antibody. We also found that, in contrast to the physically adsorbed Lst, the covalently attached Lst does not leach from the surfaces and maintains its endopeptidase activity to degrade the staphylococcal cell wall, avoiding most intracellular bacterial resistance mechanisms. Moreover, the Lst-coated surfaces kill hospital strains of Staphylococcus aureus in less than 15 min and prevent biofilm formation. This immobilization method should be applicable also to other proteins and enzymes that are recombinantly expressed to include the His6-tag fragment.
Cited By
This article is cited by 66 publications.
- Wen Ma, Liuqing Yang, Tiantian Chen, Zhibin Ye, Nathalie Tufenkji, Md. Saifur Rahaman. Engineering Polymer Forest on Membranes: Tuning Density, Thickness, and Architecture for Biofouling Control. ACS Applied Polymer Materials 2020, 2 (11) , 4592-4603. https://doi.org/10.1021/acsapm.0c00676
- Xia Wu, Keith Fraser, Jian Zha, Jonathan S. Dordick. Flexible Peptide Linkers Enhance the Antimicrobial Activity of Surface-Immobilized Bacteriolytic Enzymes. ACS Applied Materials & Interfaces 2018, 10 (43) , 36746-36756. https://doi.org/10.1021/acsami.8b14411
- Qiang Zeng, Yiwen Zhu, Bingran Yu, Yujie Sun, Xiaokang Ding, Chen Xu, Yu-Wei Wu, Zhihui Tang, Fu-Jian Xu. Antimicrobial and Antifouling Polymeric Agents for Surface Functionalization of Medical Implants. Biomacromolecules 2018, 19 (7) , 2805-2811. https://doi.org/10.1021/acs.biomac.8b00399
- Mei Liu, Jiao Li, and Baoxin Li . Mannose-Modificated Polyethylenimine: A Specific and Effective Antibacterial Agent against Escherichia coli. Langmuir 2018, 34 (4) , 1574-1580. https://doi.org/10.1021/acs.langmuir.7b03556
- Chao Zhou, Yang Wu, Kishore Reddy Venkata Thappeta, Jo Thy Lachumy Subramanian, Dicky Pranantyo, En-Tang Kang, Hongwei Duan, Kimberly Kline, Mary B. Chan-Park. In Vivo Anti-Biofilm and Anti-Bacterial Non-Leachable Coating Thermally Polymerized on Cylindrical Catheter. ACS Applied Materials & Interfaces 2017, 9 (41) , 36269-36280. https://doi.org/10.1021/acsami.7b07053
- Lei Jiao, Zijian Xu, Wenwen Du, He Li, and Miao Yin . Fast Preparation of Polydopamine Nanoparticles Catalyzed by Fe2+/H2O2 for Visible Sensitive Smartphone-Enabled Cytosensing. ACS Applied Materials & Interfaces 2017, 9 (34) , 28339-28345. https://doi.org/10.1021/acsami.7b10564
- Jiangna Guo, Qiming Xu, Rongwei Shi, Zhiqiang Zheng, Hailei Mao, and Feng Yan . Polyanionic Antimicrobial Membranes: An Experimental and Theoretical Study. Langmuir 2017, 33 (17) , 4346-4355. https://doi.org/10.1021/acs.langmuir.7b00185
- Pegah Kord Forooshani Hao Meng Bruce P. Lee . Catechol Redox Reaction: Reactive Oxygen Species Generation, Regulation, and Biomedical Applications. 2017,,, 179-196. https://doi.org/10.1021/bk-2017-1252.ch010
- Xueli Sun, Zhiyong Qian, Lingqiong Luo, Qipeng Yuan, Ximin Guo, Lei Tao, Yen Wei, and Xing Wang . Antibacterial Adhesion of Poly(methyl methacrylate) Modified by Borneol Acrylate. ACS Applied Materials & Interfaces 2016, 8 (42) , 28522-28528. https://doi.org/10.1021/acsami.6b10498
- Nagaraj Patil, Daniela Cordella, Abdelhafid Aqil, Antoine Debuigne, Shimelis Admassie, Christine Jérôme, and Christophe Detrembleur . Surface- and Redox-Active Multifunctional Polyphenol-Derived Poly(ionic liquid)s: Controlled Synthesis and Characterization. Macromolecules 2016, 49 (20) , 7676-7691. https://doi.org/10.1021/acs.macromol.6b01857
- Gil Yeroslavsky, Ronit Lavi, Abraham Alishaev, and Shai Rahimipour . Sonochemically-Produced Metal-Containing Polydopamine Nanoparticles and Their Antibacterial and Antibiofilm Activity. Langmuir 2016, 32 (20) , 5201-5212. https://doi.org/10.1021/acs.langmuir.6b00576
- Fang Xie, Xiaoen Bian, Yiwen Lu, Tian Xia, Duoduo Xu, Yanfeng Wang, Jie Cai. Versatile antibacterial surface with amphiphilic quaternized chitin-based derivatives for catheter associated infection prevention. Carbohydrate Polymers 2022, 275 , 118683. https://doi.org/10.1016/j.carbpol.2021.118683
- Li-Sheng Wang, Shirley Xu, Sneha Gopal, Eunsol Kim, Domyoung Kim, Matthew Brier, Kusum Solanki, Jonathan S. Dordick. Facile fabrication of antibacterial and antiviral perhydrolase-polydopamine composite coatings. Scientific Reports 2021, 11 (1) https://doi.org/10.1038/s41598-021-91925-6
- Testaverde S. Kim, Jahyun Nam, Dae Woo Kim, Hee-Tae Jung, Kyung-Min Yeon, Jungbae Kim. Antifouling membranes employing a 2D planar nanobiocatalyst of crosslinked glucose oxidase aggregates wrapping extra-large graphene oxide. Chemical Engineering Journal 2021, 424 , 130343. https://doi.org/10.1016/j.cej.2021.130343
- Mohamed G. Aboelmaati, Sara A. Abdel Gaber, Wafaa E. Soliman, Walid F. Elkhatib, Amr M. Abdelhameed, Heba A. Sahyon, Maged El-Kemary. Biogenic and biocompatible silver nanoparticles for an apoptotic anti-ovarian activity and as polydopamine-functionalized antibiotic carrier for an augmented antibiofilm activity. Colloids and Surfaces B: Biointerfaces 2021, 206 , 111935. https://doi.org/10.1016/j.colsurfb.2021.111935
- Buket Alkan-Tas, Ayse Durmus-Sayar, Z. Efsun Duman, E. Billur Sevinis-Ozbulut, Aise Unlu, Baris Binay, Serkan Unal, Hayriye Unal. Antibacterial hybrid coatings from halloysite-immobilized lysostaphin and waterborne polyurethanes. Progress in Organic Coatings 2021, 156 , 106248. https://doi.org/10.1016/j.porgcoat.2021.106248
- Yu Fu, Lei Yang, Jianhua Zhang, Junfei Hu, Gaigai Duan, Xianhu Liu, Yiwen Li, Zhipeng Gu. Polydopamine antibacterial materials. Materials Horizons 2021, 8 (6) , 1618-1633. https://doi.org/10.1039/D0MH01985B
- Shiquan Lin, Zhong Lin Wang. Scanning triboelectric nanogenerator as a nanoscale probe for measuring local surface charge density on a dielectric film. Applied Physics Letters 2021, 118 (19) , 193901. https://doi.org/10.1063/5.0051522
- Indu Singh, Gagan Dhawan, Seema Gupta, Pradeep Kumar. Recent Advances in a Polydopamine-Mediated Antimicrobial Adhesion System. Frontiers in Microbiology 2021, 11 https://doi.org/10.3389/fmicb.2020.607099
- Marta Martínez-Pérez, Jaime Esteban, Concepcion Pérez-Jorge Peremarch. The role of antibacterial coatings in the development of biomaterials. 2021,,, 1-36. https://doi.org/10.1016/B978-0-444-63237-1.00001-2
- Marie Van der Gucht, Merve Kübra Aktan, Hanne Hendrix, Greetje Vande Velde, Jan Paeshuyse, Annabel Braem, Rob Lavigne. qDNase assay: A quantitative method for real-time assessment of DNase activity on coated surfaces. Biochemical and Biophysical Research Communications 2021, 534 , 1003-1006. https://doi.org/10.1016/j.bbrc.2020.10.050
- Zeynep Efsun Duman-Özdamar, Aişe Ünlü, Hayriye Ünal, John M. Woodley, Barış Bi̇nay. High-yield production of active recombinant S. simulans lysostaphin expressed in E. coli in a laboratory bioreactor. Protein Expression and Purification 2021, 177 , 105753. https://doi.org/10.1016/j.pep.2020.105753
- Audrey Beaussart, Chloé Retourney, Fabienne Quilès, Raphael Dos Santos Morais, Claire Gaiani, Henri-Pierre Fiérobe, Sofiane El-Kirat-Chatel. Supported lysozyme for improved antimicrobial surface protection. Journal of Colloid and Interface Science 2021, 582 , 764-772. https://doi.org/10.1016/j.jcis.2020.08.107
- Aqsa Shahid, Bilal Aslam, Saima Muzammil, Nosheen Aslam, Muhammad Shahid, Ahmad Almatroudi, Khaled S Allemailem, Muhammad Saqalein, Muhammad Atif Nisar, Muhammad Hidayat Rasool, Mohsin Khurshid. The prospects of antimicrobial coated medical implants. Journal of Applied Biomaterials & Functional Materials 2021, 19 , 228080002110403. https://doi.org/10.1177/22808000211040304
- Shilin Mei, Xiaohui Xu, Rodney D. Priestley, Yan Lu. Polydopamine-based nanoreactors: synthesis and applications in bioscience and energy materials. Chemical Science 2020, 11 (45) , 12269-12281. https://doi.org/10.1039/D0SC04486E
- Amala Bhagwat, Monica Mixon, Cynthia H. Collins, Jonathan S. Dordick. Opportunities for broadening the application of cell wall lytic enzymes. Applied Microbiology and Biotechnology 2020, 104 (21) , 9019-9040. https://doi.org/10.1007/s00253-020-10862-y
- Vincent De Maesschalck, Diana Gutiérrez, Jan Paeshuyse, Rob Lavigne, Yves Briers. Advanced engineering of third-generation lysins and formulation strategies for clinical applications. Critical Reviews in Microbiology 2020, 46 (5) , 548-564. https://doi.org/10.1080/1040841X.2020.1809346
- Inseon Lee, Hong Jae Cheon, Manab Deb Adhikari, Tai Duc Tran, Kyung-Min Yeon, Moon Il Kim, Jungbae Kim. Glucose oxidase-copper hybrid nanoflowers embedded with magnetic nanoparticles as an effective antibacterial agent. International Journal of Biological Macromolecules 2020, 155 , 1520-1531. https://doi.org/10.1016/j.ijbiomac.2019.11.129
- Qian Bao, Ayaka Hosoe, Masaaki Hosomi, Akihiko Terada. Quorum quenching acylase impacts the viability and morphological change of Agrobacterium tumefaciens cells. Journal of Bioscience and Bioengineering 2020, 130 (1) , 82-88. https://doi.org/10.1016/j.jbiosc.2020.02.005
- Gil Yeroslavsky, Kyohei Okubo, Masakazu Umezawa, Karina Nigoghossian, Doan Thi Kim Dung, Keiji Miyata, Koki Nomura, Masao Kamimura, Kohei Soga. Energy Transfer Between Rare Earth-doped Ceramic Nanoparticles for Gauging Strain and Temperature in Elastic Polymers. Journal of Photopolymer Science and Technology 2020, 33 (1) , 129-137. https://doi.org/10.2494/photopolymer.33.129
- Yuxue Cao, Mahdi Naseri, Yan He, Chun Xu, Laurence J. Walsh, Zyta M. Ziora. Non-antibiotic antimicrobial agents to combat biofilm-forming bacteria. Journal of Global Antimicrobial Resistance 2020, 21 , 445-451. https://doi.org/10.1016/j.jgar.2019.11.012
- Hong-Jyuan Jian, Jiantao Yu, Yu-Jia Li, Binesh Unnikrishnan, Yu-Fen Huang, Li-Jyuan Luo, David Hui-Kang Ma, Scott G. Harroun, Huan-Tsung Chang, Han-Jia Lin, Jui-Yang Lai, Chih-Ching Huang. Highly adhesive carbon quantum dots from biogenic amines for prevention of biofilm formation. Chemical Engineering Journal 2020, 386 , 123913. https://doi.org/10.1016/j.cej.2019.123913
- Zahra Mehrabi, Asghar Taheri-Kafrani, Mohsen Asadnia, Amir Razmjou. Bienzymatic modification of polymeric membranes to mitigate biofouling. Separation and Purification Technology 2020, 237 , 116464. https://doi.org/10.1016/j.seppur.2019.116464
- Kai Zeng, En-Jie Sun, Ze-Wen Liu, Junhui Guo, Chengqing Yuan, Ying Yang, Hao Xie. Synthesis of magnetic nanoparticles with an IDA or TED modified surface for purification and immobilization of poly-histidine tagged proteins. RSC Advances 2020, 10 (19) , 11524-11534. https://doi.org/10.1039/C9RA10473A
- Zoe Harrison, Leslie Pace, Rukhsana Awais, J. Amber Jennings. Local Delivery of Anti-biofilm Therapeutics. 2020,,, 477-510. https://doi.org/10.1007/978-3-030-34475-7_21
- Basma A. Omran, Mohamed Omar Abdel-Salam. The Catastrophic Battle of Biofouling in Oil and Gas Facilities: Impacts, History, Involved Microorganisms, Biocides and Polymer Coatings to Combat Biofouling. 2020,,, 47-99. https://doi.org/10.1007/978-3-030-49532-9_2
- Rui Zhai, Xiangxue Chen, Mingjie Jin, Jinguang Hu. Synthesis of a polydopamaine nanoparticle/bacterial cellulose composite for use as a biocompatible matrix for laccase immobilization. Cellulose 2019, 26 (15) , 8337-8349. https://doi.org/10.1007/s10570-019-02588-6
- Javad Yasbolaghi Sharahi, Taher Azimi, Aref Shariati, Hossein Safari, Melika Khanzadeh Tehrani, Ali Hashemi. Advanced strategies for combating bacterial biofilms. Journal of Cellular Physiology 2019, 234 (9) , 14689-14708. https://doi.org/10.1002/jcp.28225
- Amala Bhagwat, Cynthia H. Collins, Jonathan S. Dordick. Selective antimicrobial activity of cell lytic enzymes in a bacterial consortium. Applied Microbiology and Biotechnology 2019, 103 (17) , 7041-7054. https://doi.org/10.1007/s00253-019-09955-0
- Chaoming Xie. Bio‐inspired nanofunctionalisation of biomaterial surfaces: a review. Biosurface and Biotribology 2019, 5 (3) , 83-92. https://doi.org/10.1049/bsbt.2019.0009
- Xue-Na Zou, Xiao Han, Qian Zhang, Jun-Jiao Yin, Li-Qiang Chu. Preparation and antibacterial activity of silver-loaded poly(oligo(ethylene glycol) methacrylate) brush. Journal of Biomaterials Science, Polymer Edition 2019, 30 (9) , 756-768. https://doi.org/10.1080/09205063.2019.1603066
- Zeynep Efsun Duman, Aişe Ünlü, Mehmet Mervan Çakar, Hayriye Ünal, Barış Binay. Enhanced production of recombinant Staphylococcus simulans lysostaphin using medium engineering. Preparative Biochemistry & Biotechnology 2019, 49 (5) , 521-528. https://doi.org/10.1080/10826068.2019.1599393
- Malay K. Das, Anupam Sarma, Trinayan Deka. Polydopamine-Based Simple and Versatile Surface Modification of Polymeric Nano Drug Carriers. 2019,,, 369-389. https://doi.org/10.1007/978-3-030-06115-9_19
- Xiangping Hao, WenHui Wang, Zhaoqing Yang, Longfei Yue, Haiyun Sun, Hongfen Wang, Zhanhu Guo, Frank Cheng, Shougang Chen. pH responsive antifouling and antibacterial multilayer films with Self-healing performance. Chemical Engineering Journal 2019, 356 , 130-141. https://doi.org/10.1016/j.cej.2018.08.181
- Shamsun Nahar, Md. Furkanur Rahaman Mizan, Angela Jie-won Ha, Sang-Do Ha. Advances and Future Prospects of Enzyme-Based Biofilm Prevention Approaches in the Food Industry. Comprehensive Reviews in Food Science and Food Safety 2018, 17 (6) , 1484-1502. https://doi.org/10.1111/1541-4337.12382
- Gavin Hazell, Leanne E. Fisher, W. Andrew Murray, Angela H. Nobbs, Bo Su. Bioinspired bactericidal surfaces with polymer nanocone arrays. Journal of Colloid and Interface Science 2018, 528 , 389-399. https://doi.org/10.1016/j.jcis.2018.05.096
- Wen Ma, Magdalena Panecka, Nathalie Tufenkji, Md. Saifur Rahaman. Bacteriophage-based strategies for biofouling control in ultrafiltration: In situ biofouling mitigation, biocidal additives and biofilm cleanser. Journal of Colloid and Interface Science 2018, 523 , 254-265. https://doi.org/10.1016/j.jcis.2018.03.105
- Christopher T. Johnson, James A. Wroe, Rachit Agarwal, Karen E. Martin, Robert E. Guldberg, Rodney M. Donlan, Lars F. Westblade, Andrés J. García. Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing. Proceedings of the National Academy of Sciences 2018, 115 (22) , E4960-E4969. https://doi.org/10.1073/pnas.1801013115
- Arnau Bassegoda, Kristina Ivanova, Eva Ramon, Tzanko Tzanov. Strategies to prevent the occurrence of resistance against antibiotics by using advanced materials. Applied Microbiology and Biotechnology 2018, 102 (5) , 2075-2089. https://doi.org/10.1007/s00253-018-8776-0
- Pan Cao, Ying Yang, Fidelia Uche, Sarah Hart, Wen-Wu Li, Chengqing Yuan. Coupling Plant-Derived Cyclotides to Metal Surfaces: An Antibacterial and Antibiofilm Study. International Journal of Molecular Sciences 2018, 19 (3) , 793. https://doi.org/10.3390/ijms19030793
- Huisoo Jang, Palan Lee, Seokjae Kim, Sun Min Kim, Tae-Joon Jeon. An antibacterial microfluidic system with fish gill structure for the detection of Staphylococcus via enzymatic reaction on a chromatic polydiacetylene material caused by lysostaphin. Microchimica Acta 2017, 184 (11) , 4563-4569. https://doi.org/10.1007/s00604-017-2517-4
- Thomas G. Barclay, Hanaa M. Hegab, Stephen R. Clarke, Milena Ginic‐Markovic. Versatile Surface Modification Using Polydopamine and Related Polycatecholamines: Chemistry, Structure, and Applications. Advanced Materials Interfaces 2017, 4 (19) , 1601192. https://doi.org/10.1002/admi.201601192
- Karol Kyzioł, Łukasz Kaczmarek, Agnieszka Kyzioł. Surface Functionalization of Biomaterials. 2017,,, 457-490. https://doi.org/10.1002/9781119441632.ch80
- Zihnil Adha Islamy Mazrad, Insik In, Kang-Dae Lee, Sung Young Park. Rapid fluorometric bacteria detection assay and photothermal effect by fluorescent polymer of coated surfaces and aqueous state. Biosensors and Bioelectronics 2017, 89 , 1026-1033. https://doi.org/10.1016/j.bios.2016.10.027
- Xiangping Hao, Shougang Chen, Hongzheng Zhu, Longqiang Wang, Yue Zhang, Yansheng Yin. The Synergy of Graphene Oxide and Polydopamine Assisted Immobilization of Lysozyme to Improve Antibacterial Properties. ChemistrySelect 2017, 2 (6) , 2174-2182. https://doi.org/10.1002/slct.201601794
- Mihyun Lee, Si-Hwa Lee, Il-Kwon Oh, Haeshin Lee. Microwave-Accelerated Rapid, Chemical Oxidant-Free, Material-Independent Surface Chemistry of Poly(dopamine). Small 2017, 13 (4) , 1600443. https://doi.org/10.1002/smll.201600443
- Diana Alves, Andreia Magalhães, Daria Grzywacz, Damian Neubauer, Wojciech Kamysz, Maria Olívia Pereira. Co-immobilization of Palm and DNase I for the development of an effective anti-infective coating for catheter surfaces. Acta Biomaterialia 2016, 44 , 313-322. https://doi.org/10.1016/j.actbio.2016.08.010
- Jianming Pan, Yue Ma, Jun Zeng, Xianghen Niu, Tao Zhang, Fengxian Qiu, Yinxian Peng, Yongsheng Yan. Facile assembly of hollow polydopamine capsules onto macroporous poly(glycidyl methacrylate) foams for simultaneous removal of λ-cyhalothrin and copper ions. Chemical Engineering Journal 2016, 302 , 670-681. https://doi.org/10.1016/j.cej.2016.05.102
- Lei Su, Yang Yu, Yanshuang Zhao, Feng Liang, Xueji Zhang. Strong Antibacterial Polydopamine Coatings Prepared by a Shaking-assisted Method. Scientific Reports 2016, 6 (1) https://doi.org/10.1038/srep24420
- Yuanfeng Pan, Huining Xiao, Pingxiong Cai, Meaghan Colpitts. Cellulose fibers modified with nano-sized antimicrobial polymer latex for pathogen deactivation. Carbohydrate Polymers 2016, 135 , 94-100. https://doi.org/10.1016/j.carbpol.2015.08.046
- Meiying Liu, Guangjian Zeng, Ke Wang, Qing Wan, Lei Tao, Xiaoyong Zhang, Yen Wei. Recent developments in polydopamine: an emerging soft matter for surface modification and biomedical applications. Nanoscale 2016, 8 (38) , 16819-16840. https://doi.org/10.1039/C5NR09078D
- Huimin Yuan, Bingran Yu, Li-Hai Fan, Meng Wang, Yiwen Zhu, Xiaokang Ding, Fu-Jian Xu. Multiple types of hydroxyl-rich cationic derivatives of PGMA for broad-spectrum antibacterial and antifouling coatings. Polymer Chemistry 2016, 7 (36) , 5709-5718. https://doi.org/10.1039/C6PY01242F
- Yixiao Cai, Hu Li, Mikael Karlsson, Klaus Leifer, Håkan Engqvist, Wei Xia. Biomineralization on single crystalline rutile: the modulated growth of hydroxyapatite by fibronectin in a simulated body fluid. RSC Advances 2016, 6 (42) , 35507-35516. https://doi.org/10.1039/C6RA04303H
- Runnan Zhang, Yanlei Su, Linjie Zhou, Tiantian Zhou, Xueting Zhao, Yafei Li, Yanan Liu, Zhongyi Jiang. Manipulating the multifunctionalities of polydopamine to prepare high-flux anti-biofouling composite nanofiltration membranes. RSC Advances 2016, 6 (39) , 32863-32873. https://doi.org/10.1039/C6RA04458A
- A. I. Mazrad Zihnil, Insik In, Sung Young Park. Reusable Fe 3 O 4 and WO 3 immobilized onto montmorillonite as a photo-reactive antimicrobial agent. RSC Advances 2016, 6 (59) , 54486-54494. https://doi.org/10.1039/C6RA08180K
- Zhen Li, Xiaoyong Zhang, Shiqi Wang, Yang Yang, Benye Qin, Ke Wang, Tao Xie, Yen Wei, Yan Ji. Polydopamine coated shape memory polymer: enabling light triggered shape recovery, light controlled shape reprogramming and surface functionalization. Chemical Science 2016, 7 (7) , 4741-4747. https://doi.org/10.1039/C6SC00584E



