Antibacterial and Antibiofouling Properties of Light Triggered

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Antibacterial and Antibiofouling Properties of Light Triggered Fluorescent Hydrophobic Carbon Quantum Dots Langmuir-Blodgett Thin Films Nenad Stankovic, Michal Bodik, Peter Siffalovic, Mario Kotlar, Matej Micusik, Zdenko Spitalsky, Martin Danko, Dusan Milivojevic, Angela Kleinova, Pavel Kubat, Zdenka Capakova, Petr Humpolí#ek, Marián Lehocký, Biljana M Todorovi# Markovi#, and Zoran Mitar Markovic ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04566 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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ACS Sustainable Chemistry & Engineering

Antibacterial and Antibiofouling Properties of Light Triggered Fluorescent Hydrophobic Carbon Quantum Dots Langmuir-Blodgett Thin Films Nenad K. Stanković1, Michal Bodik2, Peter Šiffalovič2, Mario Kotlar3, Matej Mičušik4, Zdenko Špitalsky4, Martin Danko4, Dušan D. Milivojević5, Angela Kleinova4, Pavel Kubat6, Zdenka Capakova7, Petr Humpoliček7, Marian Lehocky7, Biljana M. Todorović Marković5 and Zoran M. Marković4,5* 1

The School of Electrical Engineering, University of Belgrade, Bulevar kralja Aleksandra 73, 11000 Belgrade, Serbia 2

Institute of Physics, Slovak Academy of Sciences, Dubravska cesta 9, 84541 Bratislava, Slovakia 3

4

Center for Nano-diagnostics STU, Vazovova 5, 81243 Bratislava, Slovakia

Polymer Institute, Slovak Academy of Sciences, Dubravska cesta 9, 84541 Bratislava, Slovakia

5

Vinča Institute of Nuclear Sciences, University of Belgrade, POB 522, 11001 Belgrade, Serbia 6

J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 3, 182 23 Praha 8, Czech Republic

7

Centre of Polymer Systems, Tomas Bata University in Zlín, Trida Tomase Bati 5678, Zlín, Czech Republic

*corresponding author: e-mail: [email protected]; [email protected]; phone: +421-2-3229 4326 SYNOPSIS: Carbon quantum dots thin films exhibits strong blue light triggered antibiofouling activity with high application potential. ABSTRACT: Inimitable properties of carbon quantum dots as well as a cheap production contribute to their possible application in biomedicine especially as antibacterial and antibiofouling 1 ACS Paragon Plus Environment

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coatings. Fluorescent hydrophobic carbon quantum dots are synthesized by bottom-up condensation method and used for deposition of uniform and homogeneous Langmuir-Blodgett thin films on different substrates. It is found that this kind of quantum dots generates singlet oxygen under blue light irradiation. Antibacterial and antibiofouling testing on four different bacteria strains (Escherichia Coli, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa) reveal enhanced antibacterial and antibiofouling activity of hydrophobic carbon dots thin films under blue light irradiation. Moreover, hydrophobic quantum dots show non-cytotoxic effect on mouse fibroblast cell line. These properties enable potential usage of hydrophobic carbon quantum dots thin films as excellent antibacterial and antibiofouling coatings for different biomedical applications. KEYWORDS: Hydrophobic carbon quantum dots, Langmuir-Blodgett thin films, photodynamic therapy, singlet oxygen INTRODUCTION Deposition of uniform and homogeneous hydrophobic carbon quantum dots (hCQDs) thin films on different substrates is of great importance for studying various properties of this material including their antibacterial potentials. Carbon quantum dots (CQDs) belong to a family of carbon nanomaterials with small lateral dimension (less than 10 nm), facile synthesis, very good aqueous solubility, chemical stability, high photoluminescence.1 Hydrophilic and hydrophobic CQDs can be fabricated by a few different methods: laser ablation of carbon target2, single layer graphene treatment in oxygen plasma3 electrochemically oxidizing graphite rods4, cutting graphene sheets in the hydrothermal reactor5, carbon fibers treated with acid6, pyrolysis of polyethylene glycol 200 and saccharide in the microwave reactor7, facile synthesis of hCQDs from a poloxamer, polyoxyethylenepolyoxypropylene-polyoxyethylene

(PEO-PPO-PEO)

block

co-polymer

pluronic

F-68

in

microwave plasma.8 Application of CQDs is limited in organic electronics due to their 2 ACS Paragon Plus Environment

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hydrophilicity and poor solubility in organic solvents.9 It is very difficult to deposit uniform and homogeneous hydrophilic CQDs thin films due to the inability to deposit them by typical methods: vacuum filtration, electrophoretic deposition or spin-coating. Langmuir-Blodgett (LB) films are deposited by transfer from the water surface onto a solid substrate and are typically soluble in chloroform. Thus, it could be obtained homogeneous, ordered and uniform films.10 Films thickness is controlled easily by this deposition method, too. Amphipathic or amphiphilic compounds form stable monolayers on the water surfaces due to very large non-polar moiety of compound molecules. This moiety prevents water dissolving of the formed layer. The long chain alkanoic acids (stearic acid) belong to the simplest amphiphilic materials.11 One of the problems to deposit continuous and uniform hydrophilic CQDs thin films is their water solubility, which prevents the formation of Langmuir layer at water sub-phase. To solve this problem we prepared hCQDs colloids and deposit monolayers via modified LB method.12 Nile red is often used to mark hydrophobic domains in bacteria due to its strong fluorescence. But, its usage as hydrophobic probe is limited due to photo-bleaching.13 Instead of it, the hCQDs can be used because they show inertness to photo-bleaching and have very strong photoluminescence.14 In our previous reports we found that hydrophilic graphene quantum dots (GQDs) produce singlet oxygen under blue light irradiation15 and act bactericidal towards Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli).16 Therefore, we assumed that hCQDs can act in a similar way on different types of bacteria strains. Microbes can be repelled or killed in contact with antibacterial surfaces.17,18 These repelling antibacterial surfaces are made either from neutral polymers such as (poly(ethyleneglycol) or from charged anionic polymers. Thus, antimicrobial surfaces prevent bacteria strains to attach. Neutral polymers do not allow bacteria adherence on surfaces by steric hindrance whereas anionic polymers repel negatively charged bacteria membrane. The other type of antimicrobial surfaces (contact killing surfaces) can be designed either by modification with cationic polymers or by embedding photoactive substances into polymer matrix. 3 ACS Paragon Plus Environment


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Photoactive substance kill bacteria either by increase of surface temperature above 42°C (gold nanorods decorated by platinum nanodots19, Fe3O4@SiO2 decorated with gold, iron magnetic-gold shells (Fe@Au), SiO2@Au nanoparticles functionalized by carboxylate-terminated organosulfur ligands)20 or surface emission of reactive oxygen species by photoactive materials.21‒23 Singlet oxygen, molecular type of oxygen induces damage of various cellular membranes including cancer cells and microbes.24 Photosensitizers are materials which are used in photodynamic therapy (PDT) for treatment cancers and bacterial or fungal infections.25 They are activated by visible light and excited to singlet state. From singlet state, they transit to the long-lived excited triplet state. In photochemical reaction with oxygen, reactive oxygen species (singlet oxygen, super oxide or hydroxyl radicals) are formed.20 In this paper we succeeded for the first time to deposit uniform and homogeneous LB hCQDs thin films on different substrates (SiO2/Si, glass and mica substrates). We have studied their structural and optical properties and considered their potentials for possible antibacterial application. The hCQDs were synthesized from polyoxyethylene – polyoxypropylene - polyoxyethylene block copolymer pluronic F-68 (PF-68) by bottom-up condensation method. Their properties were investigated by high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), UV-Vis spectroscopy, photoluminescence (PL), confocal PL mapping, X-ray photo electron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), electron paramagnetic resonance (EPR) and contact angle measurements. We have also tested possible antibiofouling activities of these films toward two types of bacteria strains. EXPERIMENTAL PROCEDURE Preparation of the hCQDs. In a typical synthesis of hydrophobic carbon dots (hCQDs), 1 g of polyoxyethylene-polyoxypropylene-polyoxyethylene Pluronic 68 (Interchim, France) was allowed to dissolve in 100 mL of water for about 15 minutes. Then 200 mL of phosphoric acid (Sigma Aldrich, Germany) was added to solution and stirred to form a homogeneous reaction mixture. The 4 ACS Paragon Plus Environment

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reaction mixture was then heated on magnetic stirrer at 250 degrees for about 120 minutes. During this time period a brown colored product was obtained. After that the reaction mixture was cooled down at ambient temperature and then 250 mL of water was added to it to obtain a brownish black colored precipitate. On top of it, 300 mL of toluene or chloroform (HPLC purity) (Sigma Aldrich, Germany) is poured. Water and organic phases are mixed on stirrer until toluene or chloroform obtained yellow color. Organic phase was decanted and filtered. Deposition of the hCQDs thin films. The hCQDs thin films were deposited by a modified Langmuir-Blodgett (LB) technique12 using a computer-controlled LB trough (Nima Technology). Before the particles were spread onto the water-sub-phase, substrate must be dipped inside the trough. A micro syringe was used to distribute the hCQDs onto air/water interface of LB trough. The hCQDs were dissolved in chloroform previously. After 30 minutes chloroform evaporated and the nanoparticle layer was compressed to a surface pressure Π=22 mN/m, which corresponds to a surface pressure of a closed hCQDs monolayer. A Wilhelmy plate was used to monitor the surface pressure of nanoparticle layer. After that, the hCQDs monolayer was transferred onto SiO2/Si or mica substrates. The formation of the hCQD Langmuir layer directly at water sub-phase was monitored by Brewster angle microscopy (Model EP3, Accurion). Characterization of the hCQDs and LB hCQDs thin films. HRTEM (ARM200CF, Jeol) and AFM (MultiMode 8, Bruker) were used to determine the size, shape and local ordering of the hCQDs. For TEM imaging, the hCQDs sample was placed dropwise onto an amorphous carbon support grid. Shape and lateral dimension of the hCQDs were determined by atomic force microscopy (AFM- Bruker, Germany). This microscope is equipped with ScanAsyst probes. A spin-coating method was used to deposit the hCQDs on freshly cleaved mica. All measurements were conducted at ambient temperature in air. The imaging mode used was PeakForce QNM. The determination of lateral dimension and height size of hCQDs was performed by Gwyddion software.26

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Surface morphology of LB hCQDs thin films deposited on SiO2/Si was monitored by AFM-Bruker, Germany at ambient temperature in air. Root-mean-square roughness (rms) of LB hCQDs thin films were calculated by Gwyddion software.26 A Shimadzu UV-Vis-NIR SolidSpec-3700 spectrophotometer was used to measure the UV-Vis spectrum of the hCQDs colloid in the range of 200‒500 nm at ambient temperature. For UV-Vis measurements, the concentration of hCQDs was 0.1 mg/mL. A RF-5301PC (Shimadzu, Japan) spectrofluorophotometer was used to measure the PL spectra of the hCQDs colloid. The excitation wavelength was set in range from 320 to 480 nm. The 1×1 cm quartz cuvettes set in the right angle arrangement or a triangular cuvette in the case of a high probe concentration were used to measure the PL of the hCQDs colloids. For PL measurements, the concentration of hCQDs was 0.3 mg/mL. PL mapping using confocal Raman microscope (Alpha 300 R, Witec) working in scanning mode was used to verify the homogeneity of the deposited LB hCQDs layers over the large area. The UV excitation laser at wavelength of 355 nm was focused onto sample by achromatic optical objective with NA=0.8 (50x). The spatially-resolved PL of hCQDs was analyzed by fiber-coupled spectrometer employing optical grating with 150 g/mm and Peltier-cooled CCD chip. The scanned area was set to 50x50 µm2 with 100x100 point density. The XPS of hCQDs thin films was performed on Thermo Scientific K-Alpha XPS system (Thermo Fisher Scientific). Monochromatic Al Kα X-ray source (hν=1486.6 eV) was used. The diameter of X-ray beam was set to 400 µm at a sample. The high-resolution XPS spectra were acquired using 50 eV pass energy and energy step size of 100 meV. The XPS data were evaluated in the Thermo Scientific Avantage software. A Nicolet 8700 spectrometer with spectral resolution of 4 cm-1 was used to measure the micro ATR FTIR spectra of the hCQDs thin films deposited at ambient temperature. Films were deposited on aluminum foil. ATR FTIR measurements were conducted in the spectral range from 400 to 4000 cm-1.


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The generation of singlet oxygen in the organic solutions and on surfaces was monitored by EPR measurements of the hCQDs colloid. All measurements were performed at ambient temperature using a Magnetech spectrometer operating at a nominal frequency of 9.5 GHz. A mixture containing 0.18 mL TMP (Sigma) and 2 mL of Milli-Q water was used. Aliquot of 100µL was dropped on surface of hCQD film on SiO2/Si surface. A blue light at 470 nm with power of 15 W was used to irradiate films for 2 h at ambient temperature. Aliquots (30 µL) of the TMP-water mixtures was taken from the surface and transferred into 3 mm i.d. quartz tubes. After that Tempol signal was analyzed by EPR. To quantify the EPR signals, mean value of their amplitudes was calculated and the data are expressed in arbitrary units. Similar procedure was applied to determine singlet oxygen generation by the hCQD colloids in chloroform. Time-resolved near-infrared luminescence spectroscopy was used to measure the formation of singlet oxygen of the hCQDs. The excitation of individual samples was achieved by a Lambda Physik FL 3002 laser (wavelength 425 nm, pulse width ~28 ns). Luminescence of singlet oxygen at 1270 nm was recorded in the right angle to the excitation using a Judson Ge diode and interference filters. A 600 MHz oscilloscope (Agilent Infiniium) was used to collect the signal from the detector. The signal was transferred to a computer for further analysis. The signal-to-noise ratio of the signals was improved by the averaging of at least 2000 individual traces. Due to a large scattering of the laser pulse and luminescence of the hCQDs and other compounds, the initial part (up to 2 µs) was omitted, and it was not used for evaluation. The details of measurement and evaluation of data were described in our previous paper.27 The Surface Energy Evaluation System (SEE System; Advex Instruments, Czech Republic) was used to measure the contact angle data of the hCQDs thin films. As the testing liquids, deionized water, ethylene glycol and diiodomethane were used. To avoid errors connected with the gravity acting to the sessile drop, the droplets volume was set to 5 µL for all experiments. The SEE System software was used for all calculations. Six contact angle readings were taken and averaged to obtain one value. 7 ACS Paragon Plus Environment


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Bacteria strains and culture conditions. The antibacterial activity of the hCQDs thin films was tested toward Escherichia coli CCM 4517 and Staphylococcus aureus CCM 4516. Used bacteria strains originate from CCM (Czech Collection of Microorganisms). Two types of samples were tested: a) LB hCQDs thin films deposited on glass - sterilization prior to treatment by UV light (258 nm) for 30 min.; b) reference - pure glass - sterilization prior to treatment by pouring into the 70 % ethanol. All samples were treated by blue light at 470 nm with power of 15 W for 1 hour. The samples were placed 50 cm from on the lamp. Methodology for determination of antibacterial activity. Antibacterial activity was evaluated according to ISO 22196 “Measurement of antibacterial activity on plastics and other non-porous surfaces” with modification. The modification lays in the smaller size of tested samples than ISO prescribes. Sample dimension was 20x10 mm. The set-up of the test was adjusted to correspond to smaller sample sizes. Used inoculum of S. aureus was 7.8x106 cfu/mL and of E. coli was 2.0x107 cfu/mL. The antibacterial activity of all samples tested was compared mutually. The following equation was used to calculate the number of viable bacteria recovered per cm2 per specimen (N) N= (100 x C x D x V) / A

(1)

were C = the average plate count for the duplicate plates; D = the dilution factor for the plates counted; V – the volume (mL), of SCDLP added to the specimen; A = the surface area (mm2) of the LB hCQDs film; The following equation was used to calculate antibacterial activity (R): R = Ut - At

(2)

were Ut – the average of the common logarithm of the number of viable bacteria (cells/cm2), recovered from the untreated test specimens after 24 h; At ‒ the average of the common logarithm of the number of viable bacteria (cells/cm2) recovered from the treated test specimens after 24 h. Quantification of biofilm formation and effect of the hCQDs on formed biofilms. Two bacteria strains (Bacillus cereus CCM 2010 and Preudomonas aeruginosa CCM3955) were used to investigate antibiofouling effect of the LB hCQDs thin films. Selected species of bacteria originate 8 ACS Paragon Plus Environment

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from CCM (Czech Collection of Microorganisms). Bacteria were grown on the Nutrient agar No.2 with glucose (HiMedia, India). Bacterium Bacillus cereus (B. cereus) was incubated at 37 °C for 24 hours and Pseudomonas aeruginosa (P. aeruginosa) at 30°C for 48 hours. By seeding the bacterial strains to the physiological solution, bacterial suspensions were prepared. The density of suspensions was modified on the densitometer (Biosan) to 2. degree of McFarland standard. The samples were sterilized using UV light for 30 min. 2 mL of Trypton soya Broth (Himedia, India) was pipetted to each Petri dish with samples (TPP, Switzerland). Subsequently, it was seeded by 20 µL of the bacterial suspensions. The Petri dishes with samples were incubated according to requirements of individual bacterial strains at 30 °C (P. aeruginosa) and 37 °C (B. cereus) for 48 hours. The content of Petri dishes was removed after the incubation and the samples were rinsed with the physiological solution and air dried. For releasing of the cells from the surface, 100 µL of biological water + 100 µL of Extractant B/S (BioThema, Sweden) were pippeted on the samples surfaces. 40 µL of this solution was pipetted to the Eppendorf test-tube with 160 µL of ATP Reagent HS + diluent B solution (BioThema, Sweden). The light emission (Ismp1/2) was measured at Luminometer (Turner BioSystems). Then, 10 µL of 100 nmol/L ATP Standard (BioThema, Sweden) was added to the Eppendorf test-tube. The light emission was measured once more (Ismp+std). The amount of ATP (pmol) was calculated in the sample by the equation: ATPsmp = Ismp1 / (Ismp+std – Ismp2)

(3)

For testing of antibiofouling effect of LB hCQDs thin films, the blue light at 470 nm with power of 15 W was used. After 24 hours of incubation, the tested specimens were enlightened for 2 hours from a height of 50 centimeters by the blue light. Then, the specimens were incubated for another 24 hours. The statistical differences were determined by unpaired t-test. Cytotoxicity determination. The influence of the LB hCQDs thin films irradiated with blue light at 470 nm with power of 15 W for 2, 4 and 6 hours on the growth of mouse embryonic fibroblast cell 9 ACS Paragon Plus Environment


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line (ATCC CRL-1658 NIH/3T3, USA) was evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay measured at 570 nm and the reference wavelength was adjusted on 690 nm. The ATCC–formulated Dulbecco's Modified Eagle's Medium (PAA Laboratories GmbH, Austria) was used as the culture medium. It contains 10 % of calf serum (BioSera, France) and 100 U mL−1 Penicillin/Streptomycin (GE Healthcare HyClone, United Kingdom). Cells were incubated at 37°C in 5% CO2 in humidified air. To determine cell morphology DNA staining with Hoechst 33258 (Invitrogen, USA) was used. Cells were fixed and permeabilized before staining. Cells were fixed using 4% formaldehyde (Penta, Czech Republic) for 15 minutes, washed by PBS and subsequently poured with 0.5% Triton X-100 (Sigma-Aldrich, USA) for 5 minutes to permeabilization. Then cells were washed 3 times by PBS (Invitrogen, USA). Required amount of PBS and 5µg mL-1 of Hoechst 33258 were added and left to incubate for 30 minutes in the dark. The cytotoxicity is presented as a reduction of cell viability (%) in comparison to cell cultivated in medium without the extracts of tested materials. An inverted Olympus phase contrast microscope (IX 81) was used to visualize cell morphology from the culture plates. Concentration of cells seeded onto samples was 2.5x104 cells/ cm2. The statistical differences were determined by using unpaired t-test. RESULTS AND DISCUSSION TEM and AFM were used to visualize the shape and height of hCQDs as well as for determination of their mean diameters. Figure 1a presents TEM micrograph of hCQDs nanoparticles on amorphous carbon support film. The hCQDs have oblate spheroid shape with the average diameter of 5 nm along the longer axis, which was determined by statistical analysis of more than 20 micrographs. The HRTEM and SAED pattern analysis revealed polycrystalline structure of individual hCQDs with unperturbed graphite lattice (Figure 1b,c). Figure 1b shows magnified view of single hCQD nanoparticle. The measured separation of (100) lattice planes of 0.211 nm gives the lattice parameter a=2.44 Å (space group: P63mc), which is close to the undistorted lattice parameter a=2.456 Å of graphite.28 An AFM image of randomly displaced hCQDs on mica substrate is given 10 ACS Paragon Plus Environment

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in Figure 2a. The inset of Figure 2a shows the height line scan across an individual hCQDs. The hCQD diameter along the shorter axis of oblate spheroid was determined26 to be 1±0.3 nm (Figure 2b). Average diameter of hCQDs determined from AFM images was 20 nm. This value of average diameter indicates slight agglomeration of hCQDs when compared to the value obtained from TEM analysis.

Figure 1. a) TEM micrograph of hCQDs, b) HRTEM micrograph of selected hCQD nanoparticle with polycrystalline structure and c) SAED pattern of the hCQDs.

Figure 2. a) Top view AFM image of hCQDs, b) The height distribution histogram of hCQDs deposited on mica substrate. The UV-Vis absorption spectrum of hCQDs nanoparticles dispersed in chloroform is presented in Figure 3a. An absorption peak at 271 nm is π-plasmon excitation29 common for graphitic structures,



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which is further supported by observation of graphite lattice in hCQDs using HRTEM (Figure 1b). Optical image of hCQDs colloidal solution in chloroform is presented on the inset of Figure 3a. The hCQDs show strong photoluminescence at different excitation wavelengths-Figure 3b. The highest PL intensity was obtained at excitation wavelength of 380 nm with the emission peak at 467 nm. The Stokes shift of the hCQDs dispersed in chloroform is 87 nm. These hCQDs emit bluegreen light depending on the excitation wavelength. Based on many reports strong photoluminescence of the hCQDs originates from radiative recombination of electron-hole pairs in localized electronic states of the sp2 domains as well as zigzag effect.1,30 Besides these mechanisms, surface defects i.e. oxygen functional groups which are distributed over the edges and on basal planes of the hCQDs can induce red shift of photoluminescence emission.31 ”Surface states” on the hCQDs, with energy levels between π and π* states of C=C bonds are formed by these functional groups. Furthermore, radiation due to recombination of trapped excitons contributes to red shift of photoluminescence emission.

Figure 3. a) UV-Vis spectra of hCQDs dispersed in chloroform, b) PL of hCQDs dispersed in chloroform. Figure 4a shows XPS spectrum of LB hCQDs film deposited on SiO2/Si substrates. The chemical shifts of C1s core level clearly show different chemical bonds over the surface and at the edges of the hCQDs. In Table 1 is presented a detailed list of identified chemical bonds. XPS analysis revealed three types of carbon atoms bonds: sp2 (21.6 atomic %), sp3 (66.6 atomic %) and 12 ACS Paragon Plus Environment

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oxygenated (11.8 atomic %). Obtained XPS and PL results indicate that strong photoluminescence of the hCQDs originates from the confinement effects and recombination of electron-hole as well as surface defects caused by the presence of oxygen functional groups over the surface and edges of the hCQDs. 31,32

Figure 4. a) XPS spectrum of hCQDs, b) FTIR spectrum of hCQDs, c) EPR spectra of singlet oxygen produced by LB hCQDs thin film and reference, d) Comparison of time-resolved singlet oxygen luminescence after laser excitation of hCQDs-curve 1, tetraphenylporphyrin-curve 2, fullerene C60 in air-saturated chloroform-curve 3 and hCQDs in argon-saturated chloroform-curve 4. All samples were adjusted to the same absorbance (A = 0.155±0.002) at the excitation wavelength of 425 nm. Red lines are single exponential fits into experimental data.



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Table 1. Atomic concentrations of various carbon bonds in the hCQDs based on XPS measurements of chemical shifts of C1s core level binding energy.

Name

Atomic %

C1s sp2

21.6

C1s sp3

66.6

C1s C‒O

6.8

C1s C=O

1.9

C1s O‒C=O

3.1

C1s π‒π*

0.0

Figure 4b presents FTIR spectrum of LB hCQDs thin film deposited on SiO2/Si substrate. As can be noticed from this figure, the peaks at 2857, 2871, 2927 and 2958 cm-1 belong to C‒H stretching vibrations.32,33 The peak at 1720 cm-1 stem from to C=O groups whereas the peaks at 1410, 1577, 1600 cm-1 indicate C=C vibrations.34 The peaks at 1017, 1075 1125 and 1456 cm-1 originate C‒O bonds 32,34 whereas the peaks at 873 and 1377 cm-1 indicate C‒H bending vibrations.32,35,36 Photo-generation of singlet oxygen was confirmed by two independent methods based on EPR spectroscopy and singlet oxygen luminescence. Figure 4c shows the EPR spectrum of blue light irradiated TEMP/water solution dropped on LB hCQDs thin films deposited on SiO2/Si. As a reference we used TEMP/water solution. A selective trap agent TEMP was used to detect the singlet oxygen production of the hCQDs.15 Due to reaction between TEMP and singlet oxygen a stable, EPR active compound, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) is formed. The singlet oxygen production in sample was monitored before and after photo-excitation at wavelength of 470 nm. As can be seen from Figure 4c, hCQDs generate singlet oxygen under blue light irradiation (irradiation time was 120 minutes). Without irradiation there was no singlet oxygen generation. The EPR peaks intensity of singlet oxygen generated by hCQD thin film is almost ten times higher than that of the reference. 14 ACS Paragon Plus Environment

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Figure 4d shows time-resolved luminescence of singlet oxygen formed by irradiation of hCQDs by a blue light in chloroform-curve 1. The hCQDs in anaerobic condition did not produce any singlet oxygen (curve 4). The large value of quantum yield of singlet oxygen formation for hCQDs, Φ∆ = 0.31, was estimated by comparison of luminescence amplitudes with tetraphenylporphyrin standard (curve 2, Φ∆ = 0.50 in chloroform).37 Note that fullerene C60 (curve 3, calculated Φ∆ = 0.86) is more efficient producer of singlet oxygen. The same lifetimes of long-lived luminescence of singlet oxygen in chloroform (τL ~ 160 µs) for all hCQDs, tetraphenylporphyrin and C60 corresponds with literature data38 and indicates that these compounds did not significantly quench singlet oxygen. Singlet oxygen production enables promising application of hCQDs as an antibacterial agent. To monitor the formation of hCQDs Langmuir layer at water sub-phase, the Brewster angle microscopy (BAM) was used. We observe homogenous nucleation of the hCQDs into well separated nanoparticle clusters at surface pressure of 1 mN/m - Figure 5a. At higher surface pressure of 3 mN/m (Figure 5b), spatially homogeneous distributed hCQDs islands are observed as a result of coalescence of smaller hCQDs islands. At even higher surface pressure of 13 mN/m percolated network of hCQDs islands is formed (Figure 5c). The hCQDs monolayer closes at the surface pressure of approximately 22±2 mN/m as indicated by the maximum of elastic modulus.39 Figure 5d presents surface morphology and corresponding surface profile of bare SiO2/Si substrate. Bare SiO2/Si substrate is rather smooth with rms of 0.186 nm. Figure 5e presents surface morphology and corresponding surface profile of deposited hCQDs monolayer on SiO2/Si substrates measured by AFM. The LB hCQDs thin films are uniform and homogeneous with the rms of 0.3 nm. Optical micrograph of LB hCQDs thin films with overlay of PL mapping is presented in Figure 5f. The PL mapping shows homogenous PL across the whole layer with the exception of prolonged streaks with higher intensity. The observed oriented streaks correspond to the locally collapsed hCQDs monolayer due to uni-directional local surface pressure above the monolayer collapse threshold. 15 ACS Paragon Plus Environment


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Figure 5. Brewster angle microscopy images of the LB hCQD layers at the surface pressure: a) 1mN/m, b) 3 mN/m, c) 13 mN/m, d) Top-view AFM image of bare SiO2/Si substrate and corresponding surface profile, e) Top-view AFM image of uniform and homogeneous LB hCQDs thin film and corresponding surface profile, f) Optical micrograph of LB hCQDs thin film with overlay of photoluminescence mapping. The wettability of the surface of LB hCQDs thin films is determined by static contact angle measurements as shown in Figure 6 (a,b). Water droplets were deposited on LB hCQDs films on 16 ACS Paragon Plus Environment

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SiO2/Si and mica substrates. The average values of static contact angles were 99.9±0.5˚, 98.9±0.5˚, for hCQDs/SiO2/Si and hCQDs/mica samples, respectively.

Figure 6. Water contact angle measurements on: a) hCQDs thin films/SiO2/Si, b) hCQDs thin films/mica. Bacteria adhesion and subsequently biofilm formation depend on the substrate properties (surface roughness and hydrophobicity). It is considered that hydrophobic surfaces are more appropriate for bacteria biofilm formation.40,41 Therefore, hydrophobic LB hCQDs thin films with photodynamic features are ideal surfaces for antibacterial applications, such as antibacterial windows, floors and ceilings in oncology, haematology and burn units in hospitals. To verify expected antibacterial activity of LB hCQDs thin films, antibacterial tests have been conducted on LB hCQDs thin films deposited on glass and pure glass, respectively with and without blue light irradiation. Table 2 shows results of antibacterial activity of LB hCQDs thin films.



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Table 2. Antibacterial activity of LB hCQDs thin films compares to pure glass. Sample

S. aureus Blue light

N [cfu/cm2](a)

applied

E.coli (b)

R compared to

(a)

N [cfu/cm2]

(b)

R compared

non-irradiated

to non-irradiated

pure glass

pure glass

LBhCQDs

No

2.8x104

1.1

4.2x103

1.8

LBhCQDs

Yes

1.2x104

1.5

8.0x102

2.5

Pure glass

No

3.4x105

Ut = 5.5

2.5x105

Ut = 5.4

Pure glass

Yes

8.8x104

0.59

2.8x104

0.96

(a) N = the number of viable bacteria recovered per cm2 per test sample; (b) R = the antibacterial activity; Based on the results presented in Table 2 we can conclude that there is several orders of magnitude inhibition of bacterial growth on the surfaces coated with LB hCQDs, but the effect was different in the case of S. aureus and E. coli. E. coli is more sensitive to the surface of LB hCQDs thin films compared to S. aureus. Singlet oxygen which the hCQDs produce under blue light irradiation diffuses from the LB hCQDs thin films and kill tested bacteria strains. Results from Table 2 indicate that a large number of E.coli bacteria strains are dead after 1 hour. Thus we suppose that possible mechanism of antibacterial action of LB hCQDs thin films is the following: generated singlet oxygen attacks membrane wall, damages it by increasing its porosity and allowing radical products to reach the cytoplasmic membrane and causing its lipid peroxidation.42 Meziani et al. demonstrated that carbon quantum dots could be bactericidal after irradiation by visible light (or even under ambient light conditions).43 Walker et al. claim that a medical grade silicone incorporating crystal violet, methylene blue and 2 nm gold nanoparticles act bactericidal toward

Staphylococcus epidermidis, Saccharomyces cerevisiae and MS2 Bacteriophage only under white light.42 Felgentrager et al. report that photosensitizer molecules deposited on surfaces generate singlet oxygen and effectively kill S. aureus (efficiency >99.9 %).21 18 ACS Paragon Plus Environment

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Further investigations referring on antibiofouling behavior of LB hCQDs thin films deposited on SiO2/Si have been conducted on B. cereus and P. aureginosa bacteria strains-Figure 7 (a and b). Hydrophobicity, surface roughness as well as a predisposition to protein adsorption affect generally on bacteria ability to form biofilm.44 There are opposite results concerning the effect of hydrophobicity to colonize bacteria strains, but a general trend is that hydrophobic surfaces attract more bacteria. Surface conditions of LB hCQDs thin films referring to hydrophobicity and rms (99.9o and 0.3 nm) were convenient for used bacteria. Namely, both used bacterial strains formed biofilm on LB hCQDs thin films. Formation of Pseudomonas biofilm is promoted on surfaces with surface roughness below 1 nm.44 After blue light irradiation for 2 h, the metabolic activity, correlating to number of viable bacterial cells, of B. cereus strains was reduced by 50 % (Figure 7a), whereas metabolic activity of P. aureginosa on LB hCQDs thin films was reduced slightly over the same period of time (2 h)-Figure 7b. Reduction of number of cells was statistically significant in both cases: B. Cereus (P-value 0.0042) and P. Aureginosa (P-value 0.0069).

Figure 7. The antibiofouling effect of LB hCQDs thin films on) B. cereus and P. aureginosa bacterial strains without (0) and with (2 h) blue light irradiation. Although numerous publications concerning cytotoxicity of CQDs have been already reported cytotoxicity test has been performed on mouse embryonic fibroblast cell line-Figure 8.16,43 The statistical significant differences were found only between the non-irradiated sample and sample irradiated for 6 h (P-value 0.0313). Results have confirmed previous results that cytotoxicity of LB hCQDs thin films has been changed slightly during 6 h of blue light irradiation. Figure 9 shows cell 19 ACS Paragon Plus Environment


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morphology of untreated cells (Figure 9a) and treated cells by hCQDs (Figure 9b). As can be seen from Figure 9b cell morphology stays intact after treatment with the hCQDs. Here we established by two different methods that the hCQDs produce singlet oxygen which may damage living systems by oxidizing critical cellular macromolecules. In our previous investigation it was established that cancer cells and normal cells might be more resistant to GQDs phototoxicity than bacteria.16,45 Kim et al. found that level of isocitrate dehydrogenase (IDPc) in the cells affect the cell death by singlet oxygen.46 They claim that cells with low level of IDPc are more sensitive to death by singlet oxygen. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. Based on these facts and obtained results we can conclude that used bacteria strains and mouse embryonic fibroblast cells use different mechanisms to cope with oxidative stress.16,47

Figure 8. Cytotoxicity of LB hCQDs thin films determined as a percentage of viable NIH/3T3 cells after irradiation by blue light for 2, 4 and 6 hours. The dashed lines highlight the limits of viability according to EN ISO 10993-5: viability > 80 corresponds to no cytotoxicity, > 60 – 80 mild cytotoxicity, > 40 – 60 moderate toxicity and < 40 severe cytotoxicity. 20 ACS Paragon Plus Environment

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Figure 9. The NIH/3T3 cells untreated (a) and treated (b) with the LB hCQDs thin films. DNA stained blue (Hoechst 33258), actin filaments stained red (ActinRed™ 555). Magnification is 200x. Based on data referring to antibacterial and antibiofouling activities of LB hCQDs thin films, we can conclude that the hCQDs had higher efficacy toward Gram negative bacteria, E. coli, whereas better antibiofouling effect is directed toward Gram positive bacteria, B. cereus. Schematic diagram of possible antibacterial and antibiofouling activities of LB hCQDs thin films is presented in Figure 10.

Figure 10. Schematic diagram of possible antibacterial and antibiofouling activities of LB hCQDs thin films. CONCLUSION In this work, we present for the first time deposition of uniform and homogeneous LB hCQDs thin films on SiO2/Si, glass and mica substrates. The hCQDs are produced from polyoxyethylene – polyoxypropylene - polyoxyethylene block co-polymer pluronic F-68 by bottom-up condensation 21 ACS Paragon Plus Environment


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method. It was found that hCQDs have shape of oblate spheroids with the average diameter of 5 nm and 1 nm along the long and short axis, respectively. The most important feature of these hCQDs is visible blue light triggered generation of singlet oxygen confirmed by two different methods. The conducted antibacterial and antibiofouling tests on ultra-smooth films show moderate antibacterial activity of LB hCQDs thin films toward E. coli and B. cereus especially during blue light irradiation. The obtained results enable hCQDs usage in different fields, especially biomedical applications. AUTHOR INFORMATION Corresponding author: *e-mail: [email protected]; [email protected]; phone: +421-2-3229 4326 Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS This research was supported by the SASPRO Programme project 1237/02/02-b. The research leading to these results has received funding from the People Programme (Marie Curie Actions) European Union’s Seventh Framework Programme under REA grant agreement No. 609427. Research has been further co-funded by the Slovak Academy of Sciences. Research was also supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (project no. 172003), bilateral project Serbia-Slovakia SK-SRB-2016-0038, and multilateral scientific and technological cooperation in the Danube region (DS021). We also acknowledge support of the APVV-15-0641 and VEGA (2/0093/16). Authors also appreciated the project of Czech Science Foundation (17-05095S). Authors thank Nikola Mikušova for technical support. REFERENCES 1. Wang, Y.; Hu, A. Carbon Quantum Dots: Synthesis, Properties and Applications. J. Mater. Chem. C 2014, 2, 6921‒6939, DOI 10.1039/C4TC00988F.


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For Table of Contents Use Only Synopsis Carbon quantum dots thin films exhibits strong blue light triggered antibiofouling activity with high application potential.



Figure 1. a) TEM micrograph of hCQDs, b) HRTEM micrograph of selected hCQD nanoparticle with polycrystalline structure and c) SAED pattern of the hCQDs. 183x57mm (300 x 300 DPI)

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Figure 2. a) Top view AFM image of hCQDs, b) The height distribution histogram of hCQDs deposited on SiO2 substrate. 232x88mm (300 x 300 DPI)



Figure 3. a) UV-Vis spectra of hCQDs dispersed in chloroform, b) PL of hCQDs dispersed in chloroform. 187x69mm (300 x 300 DPI)


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Figure 4. a) XPS spectrum of hCQDs, b) FTIR spectrum of hCQDs, c) EPR spectra of singlet oxygen produced by LB hCQDs thin film and reference, d) Comparison of time-resolved singlet oxygen luminescence after laser excitation of hCQDs-curve 1, tetraphenylporphyrin-curve 2, fullerene C60 in air-saturated chloroformcurve 3 and hCQDs in argon-saturated chloroform-curve 4. All samples were adjusted to the same absorbance (A = 0.155±0.002) at the excitation wavelength of 425 nm. Red lines are single exponential fits into experimental data. 205x145mm (300 x 300 DPI)



Figure 5. Brewster angle microscopy images of the hCQD Langmuir layers at the surface pressure: a) 1mN/m, b) 3 mN/m, c) 13 mN/m, d) Top-view AFM image of bare SiO2/Si substrate and corresponding surface profile, e) Top-view AFM image of uniform and homogeneous LB hCQDs thin film and corresponding surface profile, f) Optical micrograph of LB hCQDs thin film with overlay of photoluminescence mapping. 193x199mm (300 x 300 DPI)


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Figure 6. Water contact angle measurement on: a) hCQDs thin films/SiO2, b) hCQDs thin films/mica. 163x61mm (300 x 300 DPI)



Figure 7. The antibiofouling effect of LB hCQDs thin films on) B. cereus and P. aureginosa bacterial strains without (0) and with (2 h) blue light irradiation. 146x56mm (300 x 300 DPI)


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Figure 8. Cytotoxicity of LB hCQDs thin films determined as a percentage of viable NIH/3T3 cells after irradiation by blue light for 2, 4 and 6 hours. The dashed lines highlight the limits of viability according to EN ISO 10993-5: viability > 80 corresponds to no cytotoxicity, > 60 – 80 mild cytotoxicity, > 40 – 60 moderate toxicity and < 40 severe cytotoxicity. 159x113mm (300 x 300 DPI)



Figure 9. The NIH/3T3 cells untreated (a) and treated (b) with the LB hCQDs thin films. DNA stained blue (Hoechst 33258), actin filaments stained red (ActinRed™ 555). Magnification is 200x. 155x57mm (300 x 300 DPI)


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Figure 10. Schematic diagram of possible antibacterial and antibiofouling activities of LB hCQDs thin films. 459x150mm (300 x 300 DPI)


Antibacterial and Antibiofouling Properties of Light Triggered

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