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1 Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2017 Supporting Information Title Chloride-Accelerated CuO Nanoparticle-Based Fenton Chemistry for Biofilm Removal Author(s), and Corresponding Author(s)* Li Wang*, Yanni Miao, Mingsheng Lu, Zhi Shan, Shan Lu, Jiaojiao Hou, Qiumei Yang, Xinle Liang, Tao Zhou, Dennis Curry, Ken Oakes, Xu Zhang* Experimental Section Materials Chitosan (low molecular weight), DNA (Herring sperm), bovine serum albumin (BSA), sodium chloride (NaCl, 99.5%), Copper-oxide nanoparticles (CuO NPs), and Coomassie blue G250 were purchased from Sigma-Aldrich, China. Cinnamyl alcohol was obtained from Matheson Coleman & Bell (Norwood, Ohio, USA). GelRed was supplied by Biotium, USA. Glutaraldehyde (50%), hydrogen peroxide (H 2, 30% stock), crystal violet (0.1%), fluorescein diacetate (97%), ethanol (CH 3 CH 2 OH), dichloromethane (CH 2 Cl 2 ), sodium hydroxide (NaOH, 96%), copper sulfate (CuSO 4 ), copper acetate (Cu(Ac) 2 ), acetic acid (HAc) ( 99.5%), hydrochloric acid (HCl), sodium phosphate dibasic (Na 2 HPO 4 ), monosodium phosphate (NaH 2 PO 4 ), and sodium acetate (NaAc) were purchased from Aladdin (Shanghai, China). All chemicals and reagents were of analytical or higher grade and used without further purification. Ultrapure water was prepared by a Milli-Q purification system (Millipore, USA). All experiments were carried out in acetate buffer solution (0.1 M HAc-NaAc, ph 4.5). Instrumentation A VICTOR X3 microplate reader (PerkinElmer, USA) was used to obtain the optical density of bacterial cells (flat bottom clear 96-well polystyrene plates). UV-vis spectra of Cu(Ac) 2 and CuO in H 2 O and HAc-NaAc (0.1 M ph 4.5) buffer was recorded by a 3600 UV-vis spectrophotometer (SHIMADZU). Biofilms werevisualized with an inverted fluorescent microscope (Nikon Eclipse Ti, Tokyo, Japan), a FE-SEM (field emission scanning electron microscope, Carl Zeiss vltra55, Germany), and a widefield high-content analysis system

2 (Image Xpress Micro XLS, Molecular Devices, USA) with a FITC filter cube [Ex 482/35, Em 536/40; center wavelength (nm)/bandpass width (nm)]. The color of the crystal violet stained biofilm was recorded with a digital camera. Identification of chlorinated compounds was performed with an Agilent 7890A GC coupled to a 5975C Inert Mass Spectrometer. Cleavage of Nucelic Acids and Proteins Unless indicated otherwise, nucleic acid cleavage assays were performed at 37 for 30 min in 50 µl HAc-NaAc buffer (0.1 M, ph 4.5) containing 5 mm CuO NPs (with a final concentration of 5 mm Cu 2+ released), 1 mm H 2, 0.7 M NaCl and 25 µg DNA (Herring sperm). Nucleic acid cleavage products were identified by agarose gel electrophoresis and GelRed staining. Protein cleavage assays were performed at 37 for 1 h in 50 µl HAc- NaAc (0.1 M, ph 4.5) containing 5 mm CuO NPs, 5 mm H 2, 0.7 M Cl -1 and 80 µg BSA prior to separation by SDS-PAGE and Coomassie blue G250 staining. Cleavage of Polysaccharides Chitosan was chosen as a model polysaccharide, and chitosan hydrogel was prepared with glutaraldehyde cross-linking. Chitosan (1%) was dissolved in 500 µl HAc-NaAc (0.1 M, ph 4.5) and incubated with 0.2% glutaraldehyde at 37. The formed hydrogel appeared yellow in glass vials and its mass was recorded as before cleavage. Next, 300 µl HAc-NaAc (0.1 M, ph 4.5) containing 5 mm CuO NPs, 15 mm H 2 and 0.7 M Cl - was added to cover the gel in the glass vial prior to sample incubation at 37 for 1 h. The supernatant was then discarded and the remaining gel was rinsed 3 times with water. Inversion to filter paper for 1 h was performed to remove water and un-crosslinked chitosan, and the mass of the remaining gel was recorded as after cleavage. The volume of gel remaining in the glass vial was visualized after inversion and the mass difference was recorded. Cell-killing of CuO-H 2 -Cl -1 systems for Escherichia coli and Staphylococcus aureus The antibacterial efficacy of the reagents in the CuO-H 2 -Cl system was tested on DH5a cells of Escherichia coli. DH5a cells were cultured in liquid LB medium and incubated at 37

3 overnight, where the OD 600 measurement was ~ 3.0. Next, 5 ml DH5a cells were transferred to centrifuge tubes for a 2-min centrifugation at 3000 rpm. In each tube, the supernatant was then removed and DH5a cells were re-suspended in 0.8 ml sterile water to obtain concentrated bacterial cells. Thereafter, 5 µl concentrated bacterial cells were pipetted in HAc-NaAc buffer (0.1 M, ph 4.5, final volume 50 µl), containing different combinations of the following reagents: 5 mm CuO NPs, 12 mm H 2, and 0.7 M Cl -1. After incubation for 1 h at 37, the mixture was centrifuged and supernatant discarded. The pellet was then resuspended in 1 ml LB medium before being partially (200 µl) transferred into each of the 96 wells in the polystyrene plates. The OD 600 was recorded after incubation at 37 for 7 h to measure population size. The experiments were performed in triplicate. Killing of Staphylococcus aureus by the CuO-H 2 -Cl -1 system was tested using D48 cells following the aforementioned procedure except that the concentration of H 2 used was 15 mm. Killing of Escherichia coli by the system with different copper speciation such as CuO, Cu(Ac) 2 and CuSO 4 (containing the same amount of Cu 2+ ), and different reagent concentrations was investigated in a similar way. Biofilm Elimination Biofilm formation assay of Pseudomonas aeruginosa (PA01) on plastic disks (made of silicone, 12 mm diameter, 2 mm thickness) was performed both in a batch static mode and in a simulated flow cell model. Briefly, PA01 cells were cultured in liquid LB medium and incubated at 37 C overnight, where the OD 600 measurement was ~3.0. PA01 cells were diluted in LB liquid media at a ratio of 1 to 100 and cultured at 37 C for 1 h. For quantitative analysis of biofilm growth, PA01 cells were transferred to 24-well polystyrene plates (each well contained 1 plastic disk) and then incubated in batch static mode at 37 C to form biofilm. In another batch, PA01 cells were transferred to 50-mL centrifugal tubes (each tube contained 1 plastic disk) and incubated at 37 C on a horizontal shaker (50 rpm/min) to form biofilm. This system effectively stimulated the shearing force and medium convection that the biofilm would experience in a

4 flow-cell system. Old liquid LB medium was discarded and fresh medium was added every 24 h. After 72 h, plastic disks were carefully collected and dried. Thereafter, the plastic disk with attached biofilm was immersed in 1 ml HAc-NaAc buffer (0.1 M, ph 4.5) containing varying combinations of 0.5 mm CuO NPs, 0.3 M H 2, and 0.7 M NaCl for 1 h at 37 C. For visualization of the bioflims on the surface of the plastic disks, the disks were rinsed before being fixed with 2.5% glutaraldehyde overnight; afterwards, the biofilms were dehydrated through a series of graded ethanol baths, then dried and gold coated, and finally imaged using a FE-SEM (field emission scanning electron microscope, Carl Zeiss vltra55, Germany). To image the biofilm with the digital camara, the plastic disk was rinsed, dried, and then stained with 0.1% crystal violet for 0.5 h. Subsequently 10% acetic acid was used to solubilize the dye for quantification, where the OD 600 absorbance values of the acetic acid samples were measured using a microplate reader. Biofilm formation assay of Pseudomonas aeruginosa (PA01) on 24-well plates without the plastic disks in a batch static mode was performed following the same cell cluturing and rinsing steps mentioned above. An inverted microscope (Nikon Eclipse Ti, Tokyo, Japan) and widefield high-content analysis system (Image Xpress Micro XLS, Molecular Devices, USA) with a FITC filter cube [Ex 482/35, Em 536/40; center wavelength (nm)/bandpass width (nm)] at 20 magnification were employed for visual observations. For biofilm imaging with the Image Xpress Micro XLS system observation, fluorescein diacetate (FDA) was used to stain the biofilms. For the biofilm viability assay, PA01 cells were transferred to 24-well polystyrene plates that did not contain plastic disks in the wells for a 72-h incubation at 37 C to form biofilms. Following a similar procedure, liquid LB medium was discarded and fresh medium was added every 24 h. Finally, liquid LB medium was carefully removed and the wells were gently rinsed using sterile water. Afterwards, 2 ml HAc-NaAc (0.1 M, ph 4.5) containing 0.5 mm CuO NPs, 0.3 M H 2, and 0.7 M NaCl was added. After incubation for 1 h at 37 C, the wells were rinsed with sterile water prior to 1 ml of liquid LB medium being added in to each well.

5 Dilutions were made and 100 µl aliquots of each dilution were plated onto LB agar plates and cultured for 20 h at 37 C. Colonies were counted and imaged, with the number of CFU recorded. Chlorination of Cinnamyl alcohol with CuO NP-based CA-Fenton To confirm the generation of reactive chloride species (RCSs) by the CuO NP system, oxidized and chlorinated cinnamyl alcohol species in the presence of NaCl were identified by mass spectrometry. To prevent potential interference, the reaction was performed in 1 ml pure water adjusted to ph 4.0 using hydrochloric acid. The concentrations of the reagents were as follows: cinnamyl alcohol: 1.5 mm; CuO: 400 μm; NaCl: 100, 250 or 500 mm; H 2 : 500 mm. The mixtures were incubated at room temperature for 45 h. The products were extracted with 400 μl of dichloromethane for instrumental analysis. GC-MS analysis was performed on an Agilent 7890A GC equipped with a DB-17ms capillary column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness), connected to a 5975C inert massspectrometer. The injector temperature was 270 C, and the injection volume was 1 μl. Helium was used as a carrier gas (1 ml/min), and the temperature program was initiated for 1 min at a constant temperature of 45 C followed by an increase to 300 C at 10 C/min. Supporting Data:

6 Figure S1. (A) Chitosan before (upper series) and after (lower series) treatment with CuO- H 2 -Cl - combinations. Treatments in (A) correspond to (B) in the same order (left-to-right). (B) Chitosan degradation quantified by mass (mg) loss from vial surface after treatment, with statistic analysis showing significant (, p < 0.05) and highly significant (, p < 0.01) differences. Figure S2. The growth of planktonic (A) Escherichia coli and (B) Staphylococcus aureus upon treatment with various combinations of reagents involved in CuO NP-based chloride accelerated Fenton Chemistry.

7 Figure S3. The concentration effects of (A) CuO, (B) Cl - and (C) H 2 on E. coli viability. Figure S4. Microscope images of Pseudomonas aeruginosa biofilms following CuO NP-CA Fenton treatment (1. Blank control; 2. CuO NP; 3. NaCl; 4. H 2 ; 5. CuO+ H 2 ; 6. H 2 +CI - ; 7. CuO+Cl - ; 8. CuO+ H 2 +CI -

8 Figure S5. Field emission scanning electron microscopic (FE-SEM) images of Pseudomonas aeruginosa biofilms following various treatments: (A) Blank control, (B) H 2, (C) CuO + H 2 and (D) CuO + H 2 + Cl -. Figure S6. The wide-field high-content analysis images of Pseudomonas aeruginosa biofilms after the following treatments: (A) Control, (B) CuO, (C) Cl -, (D) H 2, (E) CuO + H 2, (F) H 2 + Cl -, (G) CuO + Cl - and (H) CuO + H 2 + Cl -.

9 2500 Number of CFU X CK CuO Cl - H 2 CuO H 2 +H 2 +Cl - CuO +Cl - CuO +H 2 +Cl - Figure S7. The viability (colony forming capability) of the EPS- protected resident cells within the Pseudomonas aeruginosa biofilms after various treatment was illustrated in terms of the colony forming units (CFUs) OD E.coli E.coli E.coli E.coli +CuO +CuSO 4 +Cu(CH 3 COO) 2 +H 2 +Cl - +H 2 +Cl - Figure S8. Effect of various ion sources (CuO NPs and Cu 2+ ) in CA-Fenton on inhibiting the growth of E.coli. +H 2 +Cl -

10 Figure S9. UV-Vis spectrum of Cu(Ac) 2 and CuO NPs in H 2 O and HAc-NaAc (ph 4.5) buffer. Figure S10. Mass spectrometry analysis of the products of cinnamyl alcohol after exposure to the CuO NP-based CA-Fenton system.