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96X Screen-Printed Gold Electrode platform to evaluate electroactive polymers as marine antifouling coatings Hugues Brisset, Jean-François Briand, Raphaëlle Barry-Martinet, The Hy Duong, Pierre Frere, Frédéric Gohier, Philippe Leriche, and Christine Bressy Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00357 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018
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Analytical Chemistry
96X Screen-Printed Gold Electrode platform to evaluate electroactive polymers as marine antifouling coatings §
Hugues Brisset†*, Jean-François Briand†, Raphaëlle Barry-Martinet†, The Hy Duong†,‡, Pierre Frère , Frédéric Gohier§, Philippe Leriche§, Christine Bressy†* †
Laboratoire Matériaux Polymères-Interfaces-Environnement Marin (MAPIEM EA 4323), Université de Toulon, CS 60584, 83041 Toulon cedex 9, France. ‡ University of DANANG, University of Science and Technology, 54 Nguyen Luong Bang, Danang, Vietnam. §
Université d’Angers, MOLTECH-Anjou, UMR CNRS 6200, groupe SCL, UFR Sciences, 2 bd Lavoisier, 49045 Angers, France. E-mail:
[email protected]. Phone: +33494146724 E-mail:
[email protected]. Phone: +33494142580
ABSTRACT: Several alternatives are currently investigated to prevent and control the natural process of colonization of any seawater submerged surfaces by marine organisms. Since few years we develop an approach based on addressable electroactive coatings containing conducting polymers or polymers with lateral redox groups. In this article we describe the use of a screen-printed plate formed by 96 three-electrode electrochemical cells to assess the potential of these electroactive coatings to prevent the adhesion of marine bacteria. This novel platform is intended to control and record the redox properties of the electroactive coating in each well during the bioassay (15h), and to allow screening its anti-adhesion activity with enough replicates to support significant conclusions. Validation of this platform was carried out with poly(ethylenedioxythiophene) (PEDOT) as electroactive coating obtained by electropolymerization of EDOT monomer in artificial seawater electrolyte on the working electrode of each electrochemical cell of the 96-well microplate.
Marine biofouling on materials and equipments leads to deleterious effects in many areas such as civil or military infrastructures, coastal, military and merchant vessels, sailing boats, aquaculture installations, oceanographic equipments, 1 and optical sensors. The consequences of the colonization of ship hulls by marine biofouling are estimated at several billion euros a year, including the costs of maintenance. In this context antifouling (AF) coatings are currently used to inhibit the settlement of marine organisms on surfaces. These coatings are mainly composed of polymer matrixes called binders, which embed inorganic fillers and/or biocides. For many years the most successful AF coatings have been triorganotin-based coatings which are now banned by the International Maritime 2 Organization because of environmental concerns. Chemically-active coatings containing copper-based biocides combined to booster molecules have been developed afterwards. In addition, polydimethylsiloxane elastomer-based fouling release coatings (FRC) have proved to be effective in inhibiting the settlement of marine organisms and enhancing the easy removal of settled organisms without involving chemical reac2,3 tions and biocides. In conjunction with the design of environmentally friendly AF coating, our group aims at developing non-toxic strategies based on electroactive coatings. It is well known that the application of an electrical potential difference between two electrodes can lead to the electrolysis of seawater. This reaction increases locally the pH and/or produces gas bubbles that
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remain on the surface leading to antifouling activity. , , Some polymers mixed with graphite or carbon black particles have been reported to disinfect surfaces from attached marine bacte789 ria, V. alginolyticus. , , Matsunaga’s group has shown that applying directly a potential of 0.8 V vs saturated calomel 10 electrode kills E. coli cells in drinking water. More recently, a conjugated polymer-zwitterionic side chain combination has 11 been shown to have interesting anti-fouling properties. Based on these works, we decided to test organic conducting polymers (CPs) as antifouling coatings. Indeed, their intrinsic electrochemical properties received significant interest for many years in organic electronics, sensors or biomedi12 13 14 cal. , , Among the most successful CPs, poly(ethylenedioxythiophene) (PEDOT) has been selected for its high conductivity and its high chemical, thermal and elec15 16 17 trochemical stabilities. , , By applying an oxidation potential, the ethylenedioxythiophene (EDOT) monomer in organic solutions rapidly electropolymerizes at the surface of the working electrode, resulting in a highly electroactive PEDOT film with good adhesion to the electrode surface and with suitable electrochemical properties for an application compatible with an application in an aqueous media. In this study, we have investigated for the first time the electropolymerization of EDOT in artificial seawater without adding any other electrolyte salts. By this way, we expect that the electroactivity of the PEDOT-based coatings will inhibit
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the settlement of marine organisms without any release of toxic compounds in the environment i.e. organic solvents and electrolyte salts currently used for electropolymerization processes. One of the initial stages in the biofouling colonization process is usually the attachment of marine bacteria which are numerically dominant in the sea. Few years ago, we developed a robust adhesion bioassay to evaluate the efficacy of biocides18 and emerging antifouling coatings toward marine bac19 terial strains. This screening test consists in counting the numbers of bacteria which adhere at the bottom of a well previously covered with the investigated coating. A 96-well microplate method was used to screen the anti-adhesion activity of coatings with enough replicates to support significant conclusions. The challenge of this work was to adapt this multiwell bacterial adhesion bioassay to evaluate electroactive coatings. Electrochemical studies dealing with cell adhesion are generally performed in electrochemical cells. Using such equipment does not allow us to test the repeatability of the biological response, as simultaneous tests done with stimulated and nonstimulated coatings, including controls in the same assay, i.e. with the same inoculum, is not easy handling and timeconsuming. To reach this goal, we used a new 96X screenprinted gold electrodes developed by DROPSENS Company (Figure 1). In each well of this microplate contains a threeelectrode electrochemical cell i.e. gold working and auxiliary electrodes, and silver as reference electrode.
Figure 1. 96X Screen-printed Gold Electrode from DROPSENS Company.
EXPERIMENTAL SECTION Chemicals. Ethylenedioxythiophene (EDOT) was purchased from Sigma–Aldrich (St. Quentin, France) and used as received. Artificial seawater (ASW) was prepared by dissolving Sea Salt (Aldrich, France) in deionized water. Screen-printed Electrodes and potentiostat. 96X Screen-printed Gold Electrode (ref. DRP-96X220), specific connector (ref. CONNECTOR96X and multi Potentiostat (ref. DRP-STAT8000P) were purchased from DROPSENS Company (Oviedo, Spain). 96X Screen-printed Gold Electrode is a screen-printed electrochemical array formed by 96 three-electrode electrochemical cells with gold working (3 mm diameter) and auxiliary electrodes and a silver reference electrode. The screen-printed circuit board is glued on a standard microtiter (300-400µL) ELISA plate with 96 wells. Gold plated contact paths are printed on the backside of the printed circuit board with 96x3 contacts corresponding to the independent working, auxiliary and reference electrodes in each well. This device is fixed on a
specific connector which is the interface between the screenprinted electrodes and the potentiostat. The multipotentiostat allows simultaneous electrochemical measurements in up to 8 wells of one column that act independently. PEDOT is electrogenerated in the 8 wells simultaneously of one column and two columns are prepared for the anti-adhesion test. Electrodeposition of PEDOT in wells. A solution of EDOT at 10-2M in ASW at 20 g.L-1 was previously prepared without adding any other electrolyte salts. 200 µL of EDOT/ASW solution were introduced in the 8 wells of a column of the 96X Screen-Printed Gold Electrode. After connection to the potentiostat 50 recurrent potential scans between 0.5 V and 1.0 V at 100 mV.s-1 were applied (Figures S1 and S2). After removing the polymerization solution, cyclic voltammograms of each well were recorded directly in ASW between -0.3 and 0.4V at 35 mV.s-1 (Figures S3 and S4) Anti-adhesion assay in 96X screen printed electrode. The setup of the bioassay was previously described.18 Bacterial strains were grown on Vaatanen nine-salt solution 20 (VNSS) at 20°C under shaking conditions (120 rpm) and collected at the stationary phase. After centrifugation, cells were suspended in sterile ASW. Bacterial density for the inocula was optimized to reach the highest fluorescence response but remaining in the range where fluorescence was proportional to the bacterial density inoculated (0.2 OD600 nm in the wells). Sterile 96X screen printed electrode and a polystyrene plate (PS) (Nunc, Fisher Scientific, Illkirch, France) used as reference were filled as follows: for the two columns of 96X screen printed electrode with PEDOT and for one column of PS 100 µL of ASW without bacteria are added in border row wells and 100 µL of the bacterial suspension are inoculated on all six wells (Figure S5). The anti-adhesion test was carried out during 15h at 20°C under shaking conditions (120 rpm) (Figure S6). During this time, potential recurrent scans were applied to one of the two columns with PEDOT. After 15h the non-adhered bacteria were eliminated by three successive washes (36 g.L-1 sterile NaCl solution). Staining was performed by adding 200 µL of a SYTO® 61 (Sigma–Aldrich) solution (1µM in a 36 g.L-1 NaCl sterile solution). After 20 min, the excess stain was removed by three washes (36 g.L-1 NaCl solution). Fluorescence intensity (FI) was measured (λexc = 620 nm, λem = 650 nm) using an Infinit 200pro microplate fluorescence reader (Tecan, Lyon, France). Toxicity assay. The live-dead® assay was used to assess the toxicity of chemicals such as EDOT and acetonitrile (ACN). Briefly SYTO® 9 and Propidium Iodide (PI) were used as staining agents. SYTO® 9 stains all the cells whereas the PI only enters into the dead cells. FI was measured for SYTO® 9 (λexc = 470 nm, λem = 515 nm) and PI (λexc = 538 nm, λem = 617 nm) using the Infinit 200pro microplate fluorescence reader (Tecan, Lyon, France). The ratio between SYTO® 9 and PI allows assessing the toxicity in comparison with ethanol. RESULTS AND DISCUSSION PEDOT is currently generated from an EDOT solution on gold, platinum or carbon working electrode by applying a potential or recurrent potential scans in a three-electrodes cell. Organic solvent as acetonitrile is generally used with tetrabutyl ammonium hexafluorophosphate or tetrafluoroborate. In
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our case, organic solvent and ammonium salts cannot be used due to their potential toxicities towards marine bacteria (Figure S7). In this context, the electropolymerization of EDOT has been performed directly in ASW (10-2 M in ASW at 20 g.L-1) without adding any other electrolyte salts. In other words, the ASW solutions used for electropolymerization and bacterial bioassay are the same. Electropolymerization of EDOT was directly carried out by cyclic voltammetry in 8 wells (one column) simultaneously using a solution of EDOT/ASW by applying 50 recurrent potential scans between -0.5 V and 1.0 V at 100 mV.s-1. As expected a new redox system corresponding to the oxidation/reduction of the PEDOT electrogenerated at the surface electrode was recorded simultaneously in 8 wells (Figure 2: example of well A1, Figures S1 and S2: all wells). 500
Electropolymerization of PEDOT in A1
Figure 3. Optical pictures of: gold electrode (left), PEDOT on well 1B before (center) and after (right) 15h of anti-adhesion test.
PEDOT has been generated in ASW preventing the lack of bacterial adhesion due to the toxicity of organic solvent and salts. However, a decrease of bacterial growth can be due to the EDOT monomer released in ASW during the duration of the anti-adhesion test. In these conditions, a toxicity test of EDOT was done preventively and reveals no toxicity of this monomer (data not shown). Results of the anti-adhesion assay are shown on Figure 4.
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Cyclic voltammogram of PEDOT in A1 After 15h 10
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Analytical Chemistry
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Figure 4. Bacterial (TC8) anti-adhesion effect of PEDOT compared to PS without (WP) and with (P) application of recurrent potential scans (p
