Electrochemiluminescence for Electric-Driven Antibacterial Thera

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Electrochemiluminescence for Electric-Driven Antibacterial Therapeutics Shanshan Liu, Huanxiang Yuan, Haotian Bai, Pengbo Zhang, Fengting Lv, Libing Liu, Zhihui Dai, Jianchun Bao, and Shu Wang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b12140 • Publication Date (Web): 21 Jan 2018 Downloaded from http://pubs.acs.org on January 21, 2018

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Journal of the American Chemical Society

Electrochemiluminescence for Electric-Driven Antibacterial Therapeutics Shanshan Liu,a,b Huanxiang Yuan,a Haotian Bai,a Pengbo Zhang,a Fengting Lv,a Libing Liu,a Zhihui Dai,b* Jianchun Bao,b Shu Wanga* a

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100910, P. R. China b School of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China ABSTRACT: The employment of physical light source in clinical photodynamic therapy (PDT) system endow it with crucial defect in the treatment of deeper tissue lesions due to the limited penetration depth of light in biological tissues. In this work, we constructed for the first time an electric driven luminous system based on electrochemiluminescence (ECL) for killing pathogenic bacteria, where ECL is used for the excitation of photosensitizer instead of physical light source to produce reactive oxygen species (ROS). We named this new strategy as ECL-therapeutics. The mechanism for the ECL-therapeutics is dependent on the perfect spectral overlap and energy transfer from the ECL generated by luminol to photosensitizer, cationic oligo (p-phenylene vinylene) (OPV), to sensitize the surrounding oxygen molecule into ROS. Furthermore, taking into account the practical application of our ECL-therapeutics, we used flexible hydrogel to replace the liquid system to develop hydrogel antibacterial device. Because the chemical reaction is a slow process in the hydrogel, the luminescence could last for more than ten minutes after only electrifying for five seconds. This unique persistent luminescence characteristic with long afterglow life makes them suitable for persistent antibacterial applications. Thus, stretchable and persistent hydrogel devices are designed by integrating stretchable hydrogel, persistent ECL and antibacterial function into hydrogel matrices. This novel strategy avoids the employment of external light source, making it simple, convenient and controllable, which exploits new field for ECL beyond sensors and also opens up a new model for PDT.

INTRODUCTION Photodynamic therapy (PDT), which has emerged as a clinical modality for the treatment of various solid tumors and dermatology-related diseases, employs a photosensitizer and a suitable excitation light to sensitize the surrounding molecular oxygen to generate reactive oxygen species (ROS), and then kill cancer cells or pathogenic bacteria.1-4 Compared to traditional therapies, PDT holds various advantages, such as noninvasive nature, high temporal and spatial resolution and absence of drug-resistance.5 Nevertheless, the external light source with fixed wavelength needed in the PDT limits its application in the treatment of deeper tissue lesions, because of the limited penetration depth of light in biological tissues.6 Although near-infrared photosensitizers possess better penetration ability,7 it is still a challenge to obtain super penetrating capacity and robust ROS production efficiency.8 Due to such limitations, our group developed an original chemical molecule-activated PDT system, which employed the in-situ chemiluminescence instead of the external light source to activate the photosensitizer and efficiently kill cancer cells and pathogenic microbes.9 However, it is difficult to control the chemiluminescence process to enhance temporal and spatial resolution. Therefore, development of other novel driving mode for traditional PDT is extremely required. Electrochemiluminescence (ECL), involving a light emission process in the redox reaction of electrogenerated reactants, combines the advantages of chemiluminescence with the ability to control the temporal, space, and intensity of the light emitting reaction, which is afforded by electrochemistry.10-12

During the past decades, this technique has been widely applied in various sensor and imaging fields, such as immunoassays,13 DNA detection,14 cancer screening,15 food safety and environmental analysis,16 which were obtained by collecting the signal via photomultiplier tubes or charge-coupled devices.17 The mainly merits of ECL, such as simple operation, persistent luminescence and convenient control, are expected to make it ideal replacement of the external light source in PDT system. To the best of our knowledge, there is no report about the exploration of ECL in therapeutics. Because the diseases induced by bacteria infections have posed a major threat to human health,18, 19 it is highly desirable to develop novel and highly efficient antibacterial systems.20-22 Herein, we develop an electric-driven ECL system for the excitation of photosensitizer to generate ROS, and then kill pathogenic bacteria (named as ECL- therapeutics). This novel strategy avoids the employment of external light source, making it simple, convenient and controllable, which exploits new field for ECL beyond sensors and also opens up a new model for PDT.Furthermore, taking into account the practical application of our ECL-therapeutics, we used flexible hydrogel to replace the liquid system to develop hydrogel antibacterial device. The chemical reaction becomes slow in hydrogel in comparison to in liquid,23,24 thus the resulting ECL in hydrogel will last for longer time after electrifying. This unique persistent luminescence characteristic with long afterglow life makes them suitable for persistent antibacterial applications.

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Scheme 1. Schematic illustration of the electric-driven mechanism for ECL-therapeutics to produce ROS and kill pathogenic bacteria. Ag/AgCl electrode: reference electrode; Pt electrode: counter electrode; glassy carbon electrode (GCE): working electrode. As shown in Figure S1A, the oxidation peak at 0.5 V in the cyclic voltammogram (CV) for solution only containing luminol-H2O2 (L-H2O2) demonstrated that the electrochemical oxidation reaction of luminol in the assistant of H2O2 occurred on the surface of glassy carbon electrode (GCE). On continuous cyclic potential scan from 0.2-0.6 V, the intensity of the ECL emission decreases gradually (Figure 1A). This is attributed to the reactive intermediates produced by the betatopic luminol that can effectively react with O2·- to form excited state 3-aminophthalate dianion followed by light emitting in alkalescence environment. 23 Once OPV was added into L-H2O2 solution, the ECL intensity decreases more rapidly as the redox reaction went on (Figure S1B and Figure 1B), due to the occurrence of energy transfer from ECL to OPV.30 Moreover, similar result was obtained for the L-H2O2porphyrin solution (Figure S1C and Figure 1C), except for the lower transfer efficiency in comparison to that of L-H2O2-OPV solution. Control experiments with photosensitizer only (OPV or porphyrin) showed no ECL emission signal (Figure S1D and S1E), which indicates that OPV and porphyrin themselves do not have such electrochemical property. Therefore, effective ECL reaction could occur in L-H2O2 system, and the generated energy was absorbed by added OPV or porphyrin. In order to testify the energy transfer between ECL and OPV or porphyrin, we recorded their ECL emission intensity at fixed wavelengths after electrifying for 50 seconds and studied the emission spectra of luminol. As shown in Figure 1D, the ECL emission spectrum of L-H2O2 exhibited two main peaks at 420 nm and 510 nm, which overlap well with the absorption of cationic OPV (Figure S2).9 The addition of OPV caused significant quenching of ECL and emission of OPV was observed at 540 nm probably due to efficient energy transfer. After the addition of porphyrin which possesses a broad absorption spectrum from 300 nm to 700 nm (Figure S2), the intensity of ECL emission at 510 nm decreases slightly, which indicates that porphyrin seemed to absorb a

RESULTS AND DISCUSSION The electric-driven mechanism for ECL-therapeutics system to produce ROS and kill pathogenic bacteria is shown in Scheme 1. Luminol (5-amino-2,3-dihydro-1,4phthalazinedione) is one of the most popular ECL reagents, which emits luminescence (400 nm-550 nm) in the presence of hydrogen peroxide (H2O2) in alkalescent solution.25, 26 A cationic oligo (p-phenylene vinylene) (OPV) possessing a relatively broad absorption (350−550 nm) and a maximal emission at 550 nm9 was employed as photosensitizer in this work.27 It is known that ROS can’t be generated very well directly by ECL process because, the electrogenerated free radical preferentially produces excited states and emits ECL. As an efficient photosensitizer, OPV can transfer the absorbed energy to the surrounding oxygen molecules to produce more cytotoxic ROS. Figure S1A shows the anodic oxidation of luminol in the potential region from 0.2 V to 0.6 V at the scan rate of 100 mV/s on the surface of glassy carbon electrode (GCE). H2O2 as co-reactants in this system is oxidized to superoxide anions with scan voltage from 0.2 to 0.6 V.28 The reactive intermediate produced by the anodic oxidation of luminol in the alkalescence environment can react with O2·- to form excited state 3-aminophthalate dianion which falls to the ground state with light emission (reactions shown in Scheme S1). The spectral overlap between the absorption spectrum of photosensitizer and the ECL spectrum of luminol guarantees the occurrence of energy transfer from ECL to OPV to generate ROS (Scheme S2).29 The photosensitizer could absorb the ECL emission to sensitize oxygen and further produce ROS. The cationic OPV can bind to the negative membrane of pathogenic bacteria via electrostatic and/or hydrophobic interactions, and the generated ROS kills the pathogenic bacteria simultaneously. 9 We firstly studied the electrochemistry and ECL responses of ECL-therapeutics system containing luminolH2O2-OPV (L-H2O2-OPV). In comparison to commercial photosensitizer, porphyrin, the responses of luminol-H2O2porphyrin (L-H2O2-porphyrin) system were also investigated.

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Figure 1. ECL intensity curve of L-H2O2 (A), L-H2O2-OPV (B), L-H2O2-porphyrin (C) solutions under a continuous cyclic potential scan from 0.2 to 0.6 V for 400 s at 100 mV s-1. The emission window was placed in front of the photomultiplier tube, which was biased at 600 V. (D) The ECL spectra of L-H2O2, L-H2O2+OPV, L-H2O2+porphyrin, OPV and porphyrin solutions. (E) Fluorescence spectra and intensity of DCFH for L-H2O2 before and after adding OPV or porphyrin as functions of photosensitizer concentration (0-1 µM). (F) Fluorescence spectra and intensity of DCFH for L-H2O2 before and after adding OPV or porphyrin with different electrifying time (0-5 min). part of energy of luminol emission spectrum leading to fluorescence quenching. Note that porphyrin did not showed any emission of its own probably due to weak energy transfer compare to OPV. By calculating the energy transfer ratio of emission intensity at 540 nm and 420 nm (I540 nm/I420 nm), the LH2O2-OPV exhibits 2.9-fold higher ratio than that of L-H2O2porphyrin. This indicates that the energy transfer from ECL to

OPV or porphyrin occurred, and the energy transfer efficiency to OPV is much better than that to porphyrin. According to previous reports,9, 31 photosensitizers can absorb the luminescence of luminol to form excited state followed by sensitizing surrounding oxygen molecules to generate ROS. Herein, we used 2,7-dichlorofluorescein (DCFH), which could be transformed into highly fluorescent 2,7-



dichlorofluorescein (DCF) in the presence of ROS,32 to evaluate the ROS production ability of L-H2O2 system before and after addition of OPV or porphyrin (Figure 1E,1F, and Figure S3). For L-H2O2-OPV, upon charging with direct current for three minutes, the fluorescence intensity of DCF increased apparently as a function of OPV concentration, which indicates that OPV could absorb the energy of ECL and sensitize surrounding oxygen molecules to produce ROS (Figure S3AC). At the same experimental condition, the fluorescence intensity of control group (OPV only) was much lower than those of tested groups. The similar results were obtained for LH2O2-porphyrin and porphyrin, respectively. Furthermore, the ROS production efficiency could be enhanced by expanding the charging time from 0 to 5 min, as shown in Figure S3D-E and Figure 1F. It is expected that the generation efficiency of ROS for L-H2O2-porphyrin is much weaker than that of LH2O2-OPV, suggesting the good performance of OPV as photosensitizer in our ECL therapeutics system. The antibacterial mechanism of ROS as strong oxidant is dependent in its close contact with pathogenic microorganism.33 Therefore, the zeta potentials (ζ) were obtained to investigate the interactions between photosensitizers and pathogenic microorganisms (Table S1 in supporting information). Upon the addition of OPV, the ζ potentials of E. coli showed more positive shift compared with blank group, while the ζ potentials of S. aureus and C. albicans did not exhibit obvious shift. E. coli, as Gramnegative bacteria, owns the thin negatively charged cell membrane to adsorb positively charged OPV via electrostatic interactions, resulting in the positive shift of membrane potential. However, S. aureus which belongs to Gram-positive bacteria have a relatively thick and poriferous cell wall in the range of 20∼80 nm. When the positively charged OPV bind to the negatively charged teichoic acids on the membrane surface of S. aureus, OPV could insert into the poriferous thick cell wall and major positively charged groups could be hidden. Therefore, no distinct change of the ζ potential was observed for the Gram-positive S. aureus.34 For C. albicans, the thick cell wall components comprises of glucans, chitin and mannoproteins could also hide the positive group, hence, the minor change of the ζ potential was observed.9, 31 As a result, there was no obvious change in the ζ potentials for S. aureus and C. albicans before and after binding cationic OPV. To further confirm the binding event through an additional experiment, we incubated the bacteria (E.coli, S.aureus) and fungus (C.albicans) with OPV and visualize it under confocal laser scanning microscope (CLSM) (Figure S4). The bright green fluorescence images of OPV-stained bacteria and fungi indicate that OPV binded well with these microbes and also consistent with the previous reports.35 For the photosensitizer porphyrin, the general variation tendency of ζ potentials was similar to OPV. These results suggest that the close contacting interactions between microorganism and photosensitizers are in favor of antibacterial activity induced by ROS. To prove that ECL-therapeutics system works with different kinds of pathogens, the antibacterial experiments were investigated towards E. coli (Gram-negative bacteria), S. aureus (Gram-positive bacteria) and C. albicans (fungi). As the primary contributors for the pathogen infection; fungi and

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Figure 2. (A) Plate photographs for E. coli on LB agar plate treated with various ECL-therapeutics systems. (B) Antibacterial activities of various ECL-therapeutics systems to E. coli. (C) Antibacterial activities of various ECL-therapeutics systems to different kinds of pathogenic microorganisms. bacteria cause numerous chronic and ultimately fatal infections, seriously affect the human life and health. 9,36 As shown in Figure S5, the antibacterial activity towards E. coli was enhanced by increasing the concentration of luminol or OPV, where we maintained 0.2 mM of luminol and 1.0 µM of OPV as the optimal con centrations, respectively. Utilizing L-H2O2OPV ECL-therapeutics system, after electrifying for 15 min, the bactericidal efficiency reached to about 80% (Figure 2A-B and Table S2) in comparison to those of control groups with L-H2O2 (16%) and OPV itself (9%). Although the reactive intermediates O2·- generates for L-H2O2 system upon electrifying, it is not effective in killing bacteria, since it only occurs on the electrode surface within 3 nm range. It is also expected that the L-H2O2-porphyrin system shows bactericidal efficiency less than 17 %. Moreover, we also investigated the antibacterial activities of ECL-therapeutics system towards grampositive bacterium (S. aureus) (Figure S6 and S8) and fungi (C. albicans) (Figure S7 and S8). The results showed that our novel L-H2O2-OPV ECL-therapeutics system also exhibited remarkable ability in killing S. aureus and C. albicans with efficiency large than 66% (Figure 2C), which demonstrated its broad spectrum antibacterial ability. It is noted that the bactericidal efficiencies (66% ∼ 80%) in this work are comparable to those of other reported PDT strategies.37-39 If some effective measures such as prolong the charging time and increasing electrode area are taken, the bactericidal efficiency will be further improved. Considering the practical application of our ECLtherapeutics, we used flexible hydrogel to replace the liquid system to meet the requirement. Furthermore, since the redox reaction becomes slow in hydrogel in comparison to in


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Figure 3. (A) ECL-therapeutics device based on hydrogel. (B) FSEM image of hydrogel. Scale bar: 5 µm (left), 1 µm (right). (C) Charged hydrogel film with bending and stretching deformation modes for persistent luminescence up to 10 minutes. liquid,23,24 the ECL in hydrogel will last for longer time. This unique persistent luminescence characteristic with long afterglow life will be advantageous to our ECL-therapeutics system. Due to the ability of absorbing plenty of chemical molecules, the porous polyacrylamide hydrogel was selected as our flexible substrate that was prepared by theclassical radical polymerization of acrylamide.40 As shown in Figure 3A, the transparent elastic and porous hydrogel was sandwiched between the two thin copper sheet electrodes connected with direct current (DC) to form the antibacterial therapeutic device. Frozen scanning electron microscopy (FSEM) images revealed the porous morphology characteristic of hydrogel as shown in Figure 3B. After absorbing chemical molecules including luminol and H2O2, we charged the hydrogel with the direct current and collected the luminescence spectra of the luminous system as shown in Figure 3C. After electrifying for 5 seconds, the luminescence was observed immediately for charged hydrogel. Because the transformation of luminol molecules from excited triplet state back to excited singlet state is a slow process, the luminescence could last for more than ten minutes (also see Figure 4A). This unique persistent luminescence characteristic with long afterglow life provides is advantageous to the energy transfer from ECL to photosensitizers in our ECL- therapeutics system. It is noted that the luminescent hydrogel could undergo anthropogenic bending and stretching deformation modes (Figure 3C), which would make it possible for making wearable antibacterial devices.

For the purpose of certifying the energy transfer from ECL to OPV for L-H2O2-OPV in the flexible hydrogel, the luminescent spectrum of luminol was measured in absence and presence of OPV. As shown in Figure 4B, the luminescent intensity of luminol at 420 nm markedly decreased with the addition of OPV, which was accompanied by the generation of luminescent spectrum in the range from 500 nm to 650 nm. The intensity ratio at 540 nm and 420 nm (I540 nm /I420 nm) for L-H2O2-OPV in the charged flexible hydrogel was higher than that of charged flexible hydrogel without OPV, which suggested the satisfactory transfer efficiency between ECL and OPV in charged hydrogel (Figure 4C). We introduce DCFH to evaluate the ROS production ability of L-H2O2-OPV system in the charged hydrogel. As shown in Figure S9, compared with the control groups, DCF treated with the charged hydrogel contained L-H2O2-OPV exhibited the highest fluorescence intensity at 540 nm, which demonstrated that L-H2O2-OPV system also had excellent ability to sensitize oxygen to generate ROS in the hydrogel substrate. The bactericidal efficiency of the therapeutic hydrogel towards C. albicans was nearly about 80% (Figure 4D-E and Table S3). While the control group using the charged hydrogel only with L-H2O2 exhibited low efficiency (25%), and that of charged hydrogel itself was about 5%. Thus, our ECL-therapeutics system could also work in flexible hydrogel substrate, especially offering a persistent antibacterial mode with temporary electricity processing. CONCLUSION In summary, we developed an ECL-therapeutics system for the excitation of photosensitizer to generate ROS, and then



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Figure 4. (A) The intensity of ECL spectra of the charged hydrogel absorbing luminol and H2O2 as a function of time. [luminol] = 2 mM, [H2O2] = 0.5 mM. (B) ECL spectra of the charged hydrogel absorbing luminol and H2O2 before and after adding OPV. [luminol] = 0.2 mM, [H2O2] = 0.5 mM, [OPV] = 50 µM. (C) Energy transfer ratio of ECL to OPV for the charged hydrogel absorbing luminol and H2O2 before and after adding OPV. (D) Plate photographs for C. albicans on YPD agar plate treated with different charged hydrogel systems. (E) Antibacterial activity to C. albicans of the charged hydrogel absorbing luminol and H2O2 before and after adding OPV. kill pathogenic bacteria. The perfect spectral overlap guarantee the energy transfer from the ECL generated from luminol to photosensitizer, cationic oligo (p-phenylene vinylene) (OPV), which can effectively sensitize the surrounding oxygen molecule into ROS and then kill the pathogenic bacteria. Since the redox reaction becomes slow in hydrogel in comparison to in liquid, the ECL in hydrogel could last for more than ten minutes after only electrifying for five seconds. The charged hydrogel could undergo bending and stretching deformation modes. The therapeutic device was constructed by making the hydrogel sandwiched between the two thin copper sheet electrodes. The unique persistent luminescence characteristic with long afterglow life will be advantageous to our ECLtherapeutics system. In addition, this novel strategy avoids the employment of external light source, making it simple, convenient and controllable, which exploits new fields for ECL beyond sensors. To the best of our knowledge, the PDT system driven by electricity is first reported in this work, which opens up a new model for PDT. The developed hydrogel antibacterial ECL device could be applied in the skin infection

treatment. Moreover, combining the wireless charging technology with instrument miniaturization (such as smart microelectrode), the ECL-therapeutics system may be implanted in vivo and driven by remote control manner in the future.

EXPERIMENTAL SECTION Materials and instruments: All the chemicals used in the experiments were purchased from Acros, Aldrich Chemical Company or Alfa-Aesar. The cationic OPV was synthesized according to previous report.9 The ECL emission was conducted with an MPI-EII electrochemiluminescence (ECL) workstation (Xi′An Remax Electronic Science &Technology Co. Ltd., Xi′An, China). All experiments were carried out at room temperature using a conventional three-electrode system with a bare glassy carbon electrode (GCE) (2 mm diameter) as working electrode, a platinum electrode as the auxiliary electrode and an Ag/AgCl electrode as reference electrode. UVVis absorption spectra were taken on evolution 201 spectro-


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photometers. The fluorescence measurements were recorded on a Hitachi F-4500 fluorimeter equipped with a Xenon lamp excitation source. The images of the plate counting were taken with a Bio-Rad Molecular Imager ChemiDocXRS system. The zeta potentials were measured on the Nano ZS (ZEN3600) system.

dried hydrogel absorbed this mixed solution, the amount of luminol, H2O2, OPV uptake into the hydrogel could be measured. The weight (0.06 g) of 15×15 mm2 dried hydrogel increases to 0.79 g, after soaking in the ECL solution for 4 hours. Therefore, amount of luminol, H2O2, OPV absorbed in hydrogel was about 0.15 µmol, 0.37 µmol and 7.3×10-4 µmol, respectively.

Electrochemiluminescence measurement procedure for liquid system: The GCE was polished carefully with 0.05 µm α-Al2O3 powders on chamois leathers and washed ultrasonically with water and ethanol, respectively. Then the electrode was transferred into water containing 0.2 mM luminol, 0.5 mM H2O2 and 0.2 mM 4-iodophenol in the detection cell (Figure S10) made up polymethyl methacrylate with a chamber volume of about 500 µL. The detection cell was placed in front of the photomultiplier tube that was biased at 600 V, and then the ECL was recorded between 0.2 and 0.6 V at 100 mV s-1 with emission window in the range from 360 ∼ 680 nm.

Reactive oxygen species (ROS) measurements: (a) 2,7dichlorofluorescin (DCFH) could turned into highly fluorescent 2,7-dichloro fluorescein (DCF) in the presence of ROS.32 The L-H2O2 luminescence system, single photosensitizer (OPV or porphyrin) and luminescence system with photosensitizer (final concentration is 1.0 µM) were added separately into the solution of activated DCFH solution (40 µM), respectively. The fluorescence spectra were measured for each sample after electrifying by ECL workstation at different time intervals. Then, the activated DCFH solution (40 µM) with luminol, H2O2 and photosensitizer in detection cell was immediately transferred to cuvette to collect the fluorescence. The fluorescence spectra of DCF solution was recorded in 500-700 nm emission range with the excitation wavelength of 488 nm. (b) The blank DCFH, the blank hydrogel without being charged, the charged blank hydrogel, the charged hydrogel with soaking treatment (0.2 mM luminol and 0.1 mM hydrogen peroxide) and the charged processed hydrogel with solutions (0.2 mM luminol, 0.1 mM hydrogen peroxide and 1 µM OPV) immersed in 1 mL activated DCFH solution (40 µM) for 3 min. After interacting with processed hydrogel, the DCFH solution was immediately transferred to disposable cuvette to collect the fluorescence in 500–700 nm emission range with the excitation wave-length of 488 nm.

Synthesis of hydrogel: Hydrogel was synthesized according to a previous report.40 In particular, 0.9384 g acrylamide (monomers), 0.564 mg N,N-methylenebisacrylamide (crosslinkers), 1.598 mg ammonium persulfate (photo initiator) and 3 µL N,N,N’,N’-tetramethylethylenediamine (crosslinking accelerator) were dissolved in 6 mL deionized water first. After degassing with sonication, poured the solutions into a 85×100×1 mm3 glass mold. Then the solutions in the mold were cured with UV light for 30 min with power 8 W and 254 nm wavelength. The obtained hydrogel been cut into different size and dried in the vacuum freeze drier for further use. Spectral measurement of electrochemiluminescence resonance energy transfer (ERET) from luminol to photosensitizer: 15 µL of 20 mM luminol, 75 µL of 10 mM H2O2 and 15 µL of 50 mM 4-iodophenol were added into 1395 µL of water to obtain the ECL intensity of L-H2O2 luminescence system between 0.2 and 0.6 V at 100 mV s-1 at different wavelength with fixed wavelength filter (340 nm, 405 nm, 475 nm, 505 nm, 545 nm, 575 nm, 620 nm and 670 nm). Then, we added 150 µL of 300 µM OPV or porphyrin to 1350 µL water to measure the ECL response of photosensitizer with the above method. Finally, photosensitizer (OPV or porphyrin) was added to L-H2O2 luminescence system to collect the ECL intensity with fixed wavelength filter.

Preparation of bacterial solutions: A single colony of Ampr E. coli on a solid Luria-Bertani (LB) agar plate was transferred to 5 mL of liquid LB culture medium with 50 µg/mL ampicillin and was grown at 37 ℃ for 6 hours. A single colony of S. aureus was transferred from a solid Nutient-Broth agar plate to 5 mL of liquid NB culture medium and was grown at 37 ℃ overnight. A single colony of C. albicans on a solid Yeastextract Tryptone Dextrose (YTD) agar plate was transferred to 5 mL of liquid YTD culture medium and was grown at 30 ℃ overnight. Then, Bacteria were harvested by centrifuging (7100 rpm for 2 min) and washing with phosphate buffer saline (PBS, 10 mM, pH=7.4) for three times. The supernatant was discarded and the remaining bacteria were resuspended in PBS and diluted to an optical density of 1.0 (E. coli for 1.0; S. aureus for 0.8 and C. albicans for 2.0) at 600 nm.

Electrochemiluminescence measurement procedure for hydrogel system: 15×15 mm2 dried hydrogel was soaked in 2 mL solution contains 2 mM luminol and 0.5 mM hydrogen peroxide for 4 hours. After soaking in the ECL solution, the hydrogel was placed between two copper electrodes to form sandwich construction for charging with the battery. The electrochemiluminescence spectra were measured after the processed hydrogel was charged with alternating current (3 V) for 5 second. The luminescence spectra and intensity in different time (0 to 10 min, once a minute) were recorded in the range of 300-700 nm. The luminescence spectra and intensity of hydrogel containing 0.2 mM luminol, 0.1 mM hydrogen peroxide and OPV (0 and 1 µM) were also measured using the same method. The mixture of luminol, H2O2 and OPV was added into water to prepare a clear solution (their concentration is 0.2 mM, 0.1 mM and 1.0 µM, respectively). After the

Zeta potential measurements: Bacteria (E. coli; S. aureus or C. albicans) in PBS (10 mM, pH=7.4) was incubated separately with OPV or porphyrin (final concentration is 1.0 µM) for 30 min at 37 ℃. The bacteria were harvested by centrifuging (7100 rpm for 2 min) and the supernatant were then suspended in ultrapure water for zeta potential measurements. The untreated bacteria (without incubate with OPV or Porphyrin) were also disposed with above process as negative controls. CLSM characterization: 100 µL of bacteria (E. coli for 1.5 OD; S. aureus for 1.2 OD or C. albicans for 2.0 OD) was in-



cubated with OPV (1.0 µM) for 30 min in dark at 37 ℃. Then, the bacteria were harvested by centrifuging (7100 rpm for 2 min) and the sediments were then resuspended in 20 µL of PBS (10 mM). The suspensions were then examined by confocal laser scanning microscopy using a 488 nm laser (FV5LAMAR). The fluorescence of OPV was highlighted in green.

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Author Contributions The manuscript was written through contributions of all authors. /All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT

Antibacterial experiments: (a) Firstly, 100 µL bacteria (E. coli for 1.0 OD; S. aureus for 0.8 OD and C. albicans for 2.0 OD) was incubated with photosensitizer (1.0 µM OPV or porphyrin) for 30 min in dark. Then, the substrate (0.2 mM luminol and 0.5 mM hydrogen peroxide) was added into the bacteria suspensions with 500 µL final volume and transferred to detection cell. After electrifying for 15 min in the electrochemical luminescence detection cell from 0.2 V to 0.6 V, the bacteria suspensions were serially diluted 1 × 104 folds with PBS (C. albicans for 5000 folds). Then, the bacteria suspensions were serially diluted 1 × 104 fold with PBS (C. albicans for 5000 fold). A 100 µL portion of the diluted bacteria suspensions was spread on the solid agar plate (E. coli for LB agar plate; S. aureus for NB agar plate and C. albicans for YTD agar plate), and the colonies formed after 12 h incubation at 37 ℃ were counted (E. coli and S. aureus for 37 ℃, C. albicans for 30 ℃). (b) Since the fungi was not easy to kill, we chose C. albicans as a representative for the hydrogel experiment. The hydrogel was placed between two copper electrodes connecting with power supply. After turning on the power, the sandwiched hydrogel charged and emit light. After electrifying for 5 seconds, the hydrogel would be taken out and placed in the bacteria solution. C. albicans was respectively incubated with blank hydrogel, the charged blank hydrogel, the charged hydrogel contains luminol and H2O2 in the presence and absence of OPV at 37 ℃ for 15 min in dark. The size of the charged hydrogel was about 24×24 mm2 that was placed in 300 µL of solution containing bacteria. Then, the bacteria suspensions were serially diluted 5000 fold with PBS. A 100 µL portion of the diluted bacteria suspensions was spread on the YPD solid agar plate, and the colonies formed after 12 h incubation at 30 ℃ were counted. (c) The inhibition ratio was determined by dividing the number of colony-forming unite (CFU). The Inhibition ratio (IR) was calculated according to the following equation: ஼ ି஼ IR = బ ×100%

The authors are grateful to the National Natural Science Foundation of China (Nos. 21533012, 91527306, 21625502 and 21661132006), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA09030306).

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where C is the cfu of the experimental group treated with OPV or porphyrin in the presence and absence of luminol-H2O2 ECL system, and C0 is the cfu of the control group without any treatment.

ASSOCIATED CONTENT Supporting Information Additional experimental procedures and supplementary Figures S1-10. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author


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