Electrochemical sensor for bacterial metabolism based on the detection of NADH by Polythiophene nanoparticles

1 Electrochemical Sensor for Biofilm Based on the Detection of NADH by Polythiophene Nanoparticles Brenda G. Molina,1,2 Luis J. del Valle,1,2 Pau Turon,3 Elaine Armelin,1,2,* and Carlos Alemán1,2,4,* 1 Departament d’Enginyeria Química, EEBE, Universitat Politècnica de Catalunya, C/ Eduard Maristany, 10-14, Ed. I2, 08019, Barcelona, Spain 2 Barcelona Research Center for Multiscale Science and Engineering, Universitat Politècnica de Catalunya, C/ Eduard Maristany, 10-14, Ed. C, 08019, Barcelona, Spain 3 B. Braun Surgical, S.A. Carretera de Terrasa 121, 08191 Rubí (Barcelona), Spain 4 Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028 Barcelona Spain Correspondence to: [email protected] and [email protected] 2 ABSTRACT Composite i-PP/PEDOT films made of isotactic polypropylene (i-PP), which is frequently used for the fabrication of implantable medical devices for internal use, and chemically synthesized poly(3,4-ethylendioxythiophene) (PEDOT) nanoparticles, which are electroactive and biocompatible, have been prepared and used to detect biofilm infection. After chemical and morphological characterization, the properties (interfacial, mechanical, thermal and electrochemical) and biocompatibility of i-PP/PEDOT have been examined. Besides, carbon screen-printed electrodes coated with i-PP/PEDOT have been found to detect the growth of Gram-positive and Gram-negative bacteria through the oxidation of nicotinamide adenine dinucleotide (NADH), which comes from the bacteria metabolism (i.e. the respiration). Thus, as outer bacterial membranes are permeable to cytosolic NADH, this metabolite has been found to be an appropriated target for the detection of growing bacterial infections (biofilms). In addition, the sensor does not respond towards eukaryotic cells. This is because the major NADH pool in eukaryotic cells is located at the mitochondria and, therefore, the concentration of in the medium is not high enough to be detected since the inner mitochondrial membrane is impermeable to NADH or NAD+. 3 INTRODUCTION Bacteria embed themselves in a hydrated extracellular matrix of polysaccharides and proteins, forming a slimy layer known as a biofilm. Biofilms, which are considered as an adaptation of microbes to hostile environments,1 are generated after initial adhesion of bacteria onto any kind of living or inert surface and their subsequent immobilization growth and reproduction. During the growth phase, bacteria produce extracellular biopolymers that extend developing a complex matrix of molecular fibers with unique characteristics. Although some exceptions have been reported,2 the most important one is, in general, its capacity to hinder the access of antimicrobials through it.2-4 As a consequence, adhered microorganisms increase their antimicrobial resistance, becoming up to one thousand times more resistant to antibiotics. In the biomedical field, bacterial biofilm infections, which are typically associated with patients with indwelling devices for the purpose of medical treatments,5 attract significant clinical investigations since one established, biofilm becomes difficult to be eradicated. Thus, with the progress of medical sciences, the application of medical prostheses and implantable devices in the treatment of human diseases is experiencing an exponential growing and, therefore, bacterial biofilm infections become also frequent. Unfortunately, the vast majority of internal (e.g. vascular prosthesis,6 cerebrospinal fluid shunts,7 prosthetic heart valves8 and breast implants9) and external (e.g. dentures10 and contact lenses11) prostheses, as well as hip prosthesis (i.e. when hip joints are replaced by prosthetic implants) may result in biofilm colonization of the devices. New strategies for preventing and detecting in early stage biofilm related infections become therefore challenging and attract significant attention. One of the emerging methods for biofilm detection is the use of electrochemical impedance spectroscopy (EIS) measures by employing polymeric sensors.12-17 In EIS, 4 the electrochemical impedance across the electrode-electrolyte interface is measured over a wide range of frequencies to elicit information about the properties of the interface. In the case of biofilms, successful detection is based on changes linked to charge transfer resistance and capacitance corresponding to the maturing stages of biofilm development. However, analysis of the electrical behaviour of biofilm development is not an easy challenge and development of simplified methodologies is highly desirable to make affordable such objective. In this work cyclic voltammetry (CV) has been used to detect the presence of bacteria while eukaryotic cells remain undetected. As CV is typically employed to identify the redox state of biomolecules, as for example neurotransmitters (e.g. dopamine18,19 and serotonine20), glucose,21,22 and opioids,23,24 we have focused our attention in nicotinamide adenine dinucleotide (NADH). This biomolecule plays an important role not only as cofactor for numerous deshydrogenase enzymes but also in the electron transfer chain in living organisms.25-27 Thus, NADH and its oxidized form (NAD+) mediates many redox reactions, providing the major source of ATP for aerobic organisms.26,27 In addition to energy metabolism of living cells, NADH/NAD+ are closely associated with many pathological states, such as aging, diabetes, cancer and neurological diseases.28-31 Eukaryotic cells present two major NADH pools, the cytosolic and the mitochondrial pools.32 Although aerobic respiration reactions in eukaryotic cells take place in mitochondria, the mitochondrial and cytosolic NADH ratio is cell-type specific.33 However, a distinctive characteristic is that mitochondrial double-membrane is impermeable to NADH or NAD+ (i.e. the outer membrane is quite permeable but the inner membrane is highly folded into cristae), mitochondrial NADH levels being maintained even upon massive depletion of cytosolic NADH.31,34 In opposition, as 5 prokaryotes do not have mitochondria, their respiration occurs in the cytosol or on the inner surfaces of the cell membrane. Therefore, as prokaryote cellular membranes are permeable to NADH and NAD+ and a gradient is expected, the extracellular detection of these species could be an appropriated target for monitoring growing bacterial infections or biofilms. Here we address two questions: (1) Is it possible to distinguish the bacterial growth, and the consequent formation of biofilm, from the growth of eukaryotic cells through the voltammetric detection of NADH (i.e. the oxidation from NADH to NAD+)?; and (2) could such voltammetric sensor be manufactured using biocompatible materials and integrated into smart biomedical prostheses for monitoring bacterial quantification and biofilm colonization?. To address these questions, composites made of isotactic polypropylene (i-PP) and poly(3,4-ethylendioxythiophene) nanoparticles (PEDOT NPs), hereafter denoted i-PP/PEDOT, have been developed. i-PP is used for the fabrication of internal prostheses, as for example surgical meshes for hernia repair while PEDOT NPs, which are biocompatible,35,36 are expected to reduce the overpotential for NADH oxidation, to accelerate the electron transfer between NADH and the electrode, and to minimize the presence of competing reactions. METHODS The materials and the characterization methods, which involved dynamic light scattering (DLS), scanning electron microscopy (SEM), FTIR and micro-Raman spectroscopies, wettability, stress-strain, calorimetric, thermogravimetric and electrochemical measures, are provided in the Electronic Supporting Information (ESI). Synthesis of PEDOT NPs. PEDOT NPs were prepared adapting a previously reported procedure.37 In brief, an aqueous micellar solution was prepared by stirring 6 (750 rpm) a solution of 0.07 g of 4-dodecylbenzenesulfonic acid (DBSA) in 20 mL of milli-Q water for 1 hour. This was followed by the addition of 11.8 mg of 3,4- ethylenedioxythiophene (EDOT) monomer and, again, was stirred (750 rpm) for 1 hour at room temperature. Finally, 0.45 g of ammonium persulfate (APS) dissolved in 5 mL of milli-Q water was added to the solution. Then, the reaction was maintained in agitation at 30 ºC for 24 hours protected from light with aluminium foil. In this process, the colour of the reaction mixture changed from light grey to dark blue. No sedimentation was observed after the reaction, indicating a good colloidal stability. The resultant solution was centrifuged (11000 rpm) for 40 min at 4 °C. The supernatant solution was decanted and the sediment was re-dispersed in milli-Q water using an ultrasonic bath for 15 min at 30 °C. The centrifugation and re-dispersion process was conducted two more times to ensure the elimination of side products and unreacted chemicals, purifying the dispersion medium. Finally, the last pellet was kept under vacuum two days and, subsequently, was weighted and re-dispersed in the corresponding media at the desired concentration. Preparation of i-PP films. In order to dissolve polymer pellets, 3 g of i-PP (Mn = 50000 g/mol, Mw = 190000 g/mol, and polydispersity index= 3.80) and 100 mL of xylene were loaded in a round bottom flask equipped with a magnetic stirrer. The solution was heated on an oil bath set at 130 ºC and continuously stirred (250 rpm) until the pellets were completely dissolved. Then, the temperature was decreased to 120 ºC, maintained at such conditions for 20 min, and cooled to room temperature. After that, the polymer was precipitated by adding 400 mL of methanol. The polymer was separated by filtration, washed with methanol for three times, and put in a ventilation hood during 48 hour for solvent evaporation. The recovered product was dried at 50 ºC for 24 hours. 7 i-PP films were prepared in a hydraulic press of 15 tons equipment with Atlas series heated platens. For this purpose, 0.1 g of polymer powder were placed in the press, heated until 180 ºC and, after 5 min, the pressure was increased from 0 to 5 tons. After 2.5 min, the pressure was increased to 7 tons and maintained for 2.5 min more. Finally, the film was removed from the press and cooled to room temperature. Preparation of porous i-PP films. i-PP powder was composited with 10% w/w NaCl by mechanical stirring at 1000 rpm for 12 hours. Films were obtained by pressing the powder mixture following the procedure previously explained. Porous films were obtained by dissolving the NaCl particles with deionized water. Films were kept immersed for periods of 12 hours until the weight of the dry sample was stable (i.e. water was changed every period). Hereafter, the porous films resulting from such process have been denoted i-PP(p) to differentiate them from compact i-PP films. Preparation of i-PP/PEDOT films. i-PP/PEDOT films were prepared by incorporating PEDOT NPs to the initial mixture, as described for i-PP(p). PEDOT NPs were added considering 40 or 60 % w/w with respect to the i-PP weight. The films resulting from such concentrations were denoted i-PP/PEDOT(40%) and i- PP/PEDOT(60%), respectively. Adhesion and proliferation of bacteria. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), which are Gram-negative and Gram-positive bacteria, respectively, were selected to evaluate the adhesion and initial growth (24 hours) and proliferation (7 days) of prokaryotic cells in contact with the i-PP and i- PP/PEDOT samples. The bacteria were previously grown aerobically to exponential phase in broth culture (5 g/L beef extract, 5 g/L NaCl, 10 g/L tryptone, pH 7.2). 2×108 colony forming units (CFU) per mL were seeded in 10 mL of culture DMEM high glucose supplemented with 2% FBS (pH 8.1) and 0.2% NaHCO3. Bacterial growth 8 curves in that supplemented medium are displayed in Figure S1. After 24 hours, 150 µL were added to 5 mL of NaHCO3 supplemented DMEM in sterile vials containing i-PP, i-PP(p), i-PP/PEDOT(40%) and i-PP/PEDOT(60%) films. Controls were simultaneously performed by culturing cells in sterile vials without material. All samples, including the control, were vortexed for 1 min and incubated at 37 ºC with agitation at 80 rpm for 24 hours and 7 days for cell growth. UV absorbance was measured at λ= 450 nm in a flat-bottomed 96 well-plates with aliquots of 200 µL. The results, derived from the average of three replicates (n=3) for each independent experiment, were normalized with the control for relative percentages. Adhesion and proliferation of eukaryotic cells. Cellular assays were performed using Cos-1 and Vero cells. These cells were selected due to their fast growth. Cells were cultured in DMEM high glucose buffered with 2.5 mM of HEPES, 10% FBS, penicillin (100 units/mL), and streptomycin (100 µg/mL). The cultures were maintained in a humidified incubator with an atmosphere of 5% CO2 and 95% O2 at 37 ºC. Culture media were changed every two days. When the cells reached 80-90% confluence, they were detached using 2 mL of trypsin (0.25% trypsin/EDTA) for 2-5 min at 37 °C. Finally, cells were re-suspended in 5 mL of fresh medium and their concentration was determined by counting with a Neubauer camera using 0.4% trypan blue as a vital dye. i-PP and i-PP/PEDOT films were placed in plates of 24 wells and sterilized using UV irradiation for 15 min in a laminar flux cabinet. Controls were simultaneously performed by culturing cells on the surface of TCPS plates. For adhesion and proliferation assays 2×104 and 1×104 Cos-1 and Vero cells, respectively, were deposited on the film of each well. Then, attachment of cells to the film surface was promoted by incubating under culture conditions for 30 min. Finally, 2 mL of the culture medium were added to each well. After 24 h, cellular adhesion was determined by quantifying 9 the cells attached to the films or the control. Cellular proliferation was evaluated by quantifying the viable cells onto the evaluated materials after 7 days of culture. Cellular viability was evaluated by the colorimetric MTT [3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide] assay, which is described in the ESI. Electrochemical detection of prokaryotic vs. eukaryotic cells. The electrochemical detection of prokaryotic vs. eukaryotic cells was performed by CV using the Autolab PGSTAT302N. Experiments were conducted in the cell culture media at room temperature. The initial and final potentials were −0.20 V while the reversal potential was +0.60 V. A scan rate of 100 mV s−1 was applied in all cases. An Ag|AgCl 3 M KCl and a Pt-wire were used as reference electrode and counter electrode, respectively. The working electrode consisted on a carbon screen-printed electrode (SPE) from Dropsens (DRP-150) coated with a gelatin layer, which was used to adhere the i-PP and i- PP/PEDOT films. Such adhesive layer was prepared by dissolving 50 mg of gelatin from porcine skin in 1 mL of milli-Q water, which was placed in an ultrasonic bath at 40 °C for 15 minutes until a clear solution was acquired. A solution drop (10 μL) was deposited onto the SPE surface and, subsequently, a round film of i-PP or i-PP/PEDOT (4 mm in diameter) was placed onto the resulting gelatin-modified SPE. All experiments were replicated three times. RESULTS AND DISCUSSION Preparation and characterization Figure 1a displays a micrograph of the prepared PEDOT NPs, which present a corallike morphology and exhibit an effective diameter of 48±9 and 91±1 nm as determined by SEM and DLS, respectively. The FTIR spectra of EDOT monomer and PEDOT NPs doped with DBSA are compared in Figure S2. The monomer shows bands at 3109 and 16 of the electroactive CP, which favours the exchange of electrolytes with cell at the interface defined by the surface of the film and the cell membrane. On the detection of bacterial cells while fingerprints of eukaryotic cells remain undetected In this section, the performance of the i-PP/PEDOT composite for the in situ electrochemical detection of biofilm contamination is examined through the oxidation of NADH to NAD+. With the aim of orienting this sensor towards clinical applications, the fingerprints of eukaryotic and prokaryotic cells colonizing a medical device should be differentiated. Therefore, we focused on monitoring the metabolism of prokaryotic and eukaryotic cells using carbon SPEs coated with i-PP/PEDOT(40%) since the load of PEDOT NPs is smaller and display properties similar to i-PP/PEDOT(60%). The growth of bacteria and eukaryotic cells was following by examining the electrochemical response of the medium at different times, which range from 0 (just when the cells are introduced in the culture medium) to 24 hours. Figure 5a displays the response of the sensor to the culture medium without cells at different incubation times. As it can be seen, cyclic voltammograms are similar to those displayed in Figure 3, independently of the incubation time, indicating that the response coming from the oxidization of species contained in the NaHCO3 supplemented DMEM is practically null. Accordingly, the anodic current at the reversal potential (j0.6), 0.60 V, only decreases from 13 to 11 µA/cm2 after 24 hours. In opposition, i-PP/PEDOT(40%) is remarkably affected by the presence of cultured bacteria, even though the response of the sensor against E. coli and S. aureus are different and change with the incubation time (Figures 5b-c). Thus, an oxidation peak at 0.60 V is clearly observed in both cases. This has been attributed to the oxidation of NADH to NAD+. Previous studies on 17 PEDOT-based electrodes reported that the oxidation peak of NADH in a solution without the presence of bacteria can be found between 0.5 V (i.e. processable PEDOT colloidal microparticles)50 and 0.70 V (i.e.electrochemically synthesized graphene- PEDOT:PSS).51 Thus, the detection of the of NADH to NAD+ is indicative of bacterial activity.52,53 Moreover, the current density at 0.60 V varies with the incubation time (Figure 5d), reflecting that i-PP/PEDOT(40%) detects that the bacteria growth is very fast at the first stages of incubation. For E. coli, j0.6 increases from 8 to 171 µA/cm2 after 2 hours, decreasing to 161 µA/cm2 after 24 hours. For S. aureus, the j0.6 value is 298 µA/cm2 after 24 hours of culture, which is consistent with the cell viability measurements displayed in Figure 4a (i.e. adhesion was higher for the Gram-positive bacterium than for the Gram-negative one). However, analysis of the temporal evolution of j0.6 displayed in Figure 5d allows monitoring that in the first stages the growing of E. coli is faster than of S. aureus. It should be remarked that no change of color associated to the pH indicator contained in the DMEM supplement was detected in the culture medium during the bacteria growth. Accordingly, the pH was preserved during the whole detection process and, therefore, the surface of the sensor remained unaffected by acidic metabolism of the bacteria. Analysis of Figure 5d shows that j0.6 reaches a plateau before biofilm formation, which is expected to occur after ∼24 h. This feature is especially striking for E. coli since the plateau is reached very fast. In a first assumption this observation could be attributed to the saturation of PEDOT NPs in the electrode and/or to limitations in the linear dynamic range. More specifically, the adsorption of oxidized analytes onto the electroactive materials used as electrodes and their subsequent saturation is a wellknown limitation of some electrochemical biosensors. Besides, the huge amount of bacteria involved in biofilms requires that a very high interval of CFU/mL over which 18 the sensor response is linear. The plateaus reached in Figure 5d could be interpreted as the loss of the linear regime, which eventually could limit the quantitative utilization of the sensor. However, comparison of the profiles displayed in Figure 5d with Figure 5e, which shows to the growth of E. coli and S. aureus in the culture medium against the incubation time as determined UV-vis spectroscopy, suggest that the limitations discussed above are less decisive than expected. Thus, the variation of the absorbance at 450 nm with the incubation time agrees with the profiles electrochemically obtained for the two bacteria (Figure 5d), evidencing the capacity of the sensor for detecting the presence of growing bacteria. The calibration curve (Figure 5f), which was approximated using the McFarland standard, indicates that the amount of E. coli and S. aureus bacteria grow from 1.6×108 CFU/mL (first measure for both types) to 1.7×108 and 2.4×108 CFU/mL, respectively, after 24 h. These results are fully consistent with the electrochemical sensing measures displayed in Figures 5b-c. The selectivity with respect eukaryotic cells is crucial for the performance and practical application of the sensor in medical devices. Figure 6 proves that the electrochemical response of i-PP/PEDOT(40%) against the growth of eukaryotic cells is completely different to that displayed for bacteria. Cyclic voltammograms recorded just after the addition of Cos-1 and Vero cells are practically identical to those obtained after 24 hours of cell culture (not shown) and very similar to those achieved after 7 days of cell proliferation (Figure 6). Moreover, after such long time j0.6 only increases from 7 to 10 µA/cm2 and from 9 to 11 µA/cm2 for Cos-1 and Vero cells, respectively, evidencing that the concentration of NADH induced by such eukaryotic cells is not high enough to be detected by the i-PP/PEDOT sensor. Results displayed in Figures 5 and 6 clearly indicate that the i-PP/PEDOT(40%) sensor distinguishes bacteria while eukaryotic cells remain undetected. This behavior 19 originates from the sensitivity of the i-PP/PEDOT composite towards the oxidation of NADH, which is high enough to detect the metabolism of bacteria but too low for the respiration of eukaryotic cells. In addition, the oxidation of NADH towards NAD+ probably causes an electrochemically-induced concentration gradient, favouring the exit of cytosolic NADH to the medium through the outer bacterial membrane and, consequently, facilitating the detection of the bacterial metabolism. CONCLUSIONS In this study, simple and highly sensitive biosensors have successfully developed for the detection of bacteria growth through the oxidation of NADH. In a first stage the sensors, which are based on PEDOT NPs supported into an i-PP matrix, have been characterized by FTIR and Raman spectroscopies, SEM, contact angle measurements, mechanical tests, differential scanning calorimetry and thermogravimetry analyses. Although the mechanical and thermal properties of i-PP/PEDOT composites are affected by the interfacial adhesion between their components, the combination of i-PP and PEDOT NPs is very attractive in terms of electrochemical properties. Thus, the electrochemical processes observed for i-PP/PEDOT are similar to those described for neat PEDOT films and, in addition, the composite exhibits self-electrostabilizing behaviour. This unusual characteristic, which consists in the increase of electrochemical activity as the number of consecutive redox cycles grow, is due to an increment in the surface porosity of the films. The i-PP/PEDOT sensor detects the growth of Gram-negative and Gram-positive bacteria through the oxidation of the NADH, which comes from bacterial metabolism and permeates through the outer membrane to the extracellular culture medium. In contrast, the NADH produced by the respiration of eukaryotic cells remains in the 20 mitochondria pool and, therefore, the presence of those cells e not interfering with the readings of the bacterial sensor. The prepared sensor, which can be produced at low cost, is deemed to be of high clinical interest as it might be the first line of defence against biofilm formation benefit in a number of scenarios where bacteria colonize the implanted medical devices competing with healthy eukaryotic cells. ACKNOWLEDGEMENTS Authors acknowledge MINECO/FEDER (MAT2015-69367-R and RTI2018-098951- B-I00), the Agència de Gestió d'Ajuts Universitaris i de Recerca (2017SGR359) and B. Braun Surgical, S.A. company for financial support. Support for the research of C.A. was received through the prize “ICREA Academia” for excellence in research funded by the Generalitat de Catalunya. B. G. M. is thankful to CONACYT for the financial support through a postgraduate scholarship (328467 CVU 621314). SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI:XXXXX. 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