Electrochemical impedance response of the nanostructured Ti–6Al–4V surface in the presence of S. aureus and E. coli

Implant infections associated with biofilm are a significant problem in current healthcare. Early detection of the development of bacterial infection would allow the deployment of antibiotic treatment to suppress complications. Biofilm detection can be based on the changes in the electrochemical response of a metal oxide sensor. The nanostructured surface of titanium alloys provides a large active/geometric surface area ratio and may respond to the presence of bacteria by changing its behaviour. In this work, the changes in impedance response of nanostructured Ti–6Al–4V alloy surface in the presence of E. coli and S. aureus were studied. The nanostructures were prepared by anodic oxidation in a fluoride ions-containing environment. The change in impedance spectra and open circuit potential of the prepared surfaces was monitored for 48 h. Furthermore, a series of measurements in model systems were carried out to help identify the processes leading to the change in the electrochemical behaviour of the surface. The measurements showed significant changes in the surface impedance response over a wide range of frequencies and for both bacterial strains. Based on the results, the implantable sensor based on the nanotubular titanium oxide seems to be a possible and simple way how to detect bacterial infection.


Introduction
Biofilm-related infections (BRI) are an overwhelming issue for health care systems in Europe and worldwide.As publicly announced by the US National Institutes of Health "Biofilms are medically important, accounting for over 80% of microbial infections in the body" [1].Common examples of biofilm-related infections include implant-related infections, like those that may follow joint replacement, osteosynthesis, mechanical heart valves.[2].BRIs are characterised by a chronic course or frequent recurrences, antibiotic resistance, complex and prolonged treatment, poor prognosis, high social and economical costs and difficult diagnosis.
Biofilm is a structured microbial community and a quite natural form of growth of most microorganisms that preferably colonises the implant as a foreign interface in the human body.Adhesion of microbial cells, subsequent biofilm formation, and maturation of its structure are connected with changes in cell phenotype, which are generally manifested by new metabolic pathways, increased resistance to toxic compounds and, in the case of pathogens, increased virulence [3].Up to a 1000-fold increase in resistance to antibiotics in biofilm populations is also associated with the ability of the biofilm matrix to prevent antibiotics from reaching the cells and influence the function of individual genes that are preferentially expressed in biofilms [4].In vitro experiments showed that young biofilm could be easily eliminated by antibiotic treatment compared to matured biofilm [5].Therefore, early and aggressive antibiotic treatments by cefazolin, nafcillin, oxacillin, vancomycin, daptomycin and linezolid or its combination are recommended for biofilm infections [6].Infections of orthopaedic implants are usually caused by microbial contamination during surgery [7].Early postoperative infections develop within 3 months after surgery.Delayed and late infections can develop over months or years, quite often as a result of haematogenous spread.This is the transport of bacteria through the bloodstream from another site in the body affected by the infection.The most common microorganisms that cause orthopaedic implant infection are Gram-positive, facultatively anaerobic Staphylococcus aureus and Staphylococcus epidermidis [8].Less common are Gram-negative aerobic Escherichia coli, Proteus mirabilis or Pseudomonas aeruginosa.A major problem today is the increasing resistance of staphylococci to antibiotics [9].One reason for the predominance of staphylococci at the site of inflammation is their ability to switch to fermentative metabolism in the absence of oxygen.Various inflammatory diseases have to be often characterised by tissue hypoxia [10].
Most bacteria require nourishing iron to successfully infect human tissues [11].Obtaining it is quite difficult because most of the iron present in the body is tightly bound to proteins [12].For that reason, bacteria have evolved a system that they use during periods of starvation.Under these conditions, S. aureus redirects its metabolism using regulatory proteins so that large amounts of acidic products, especially lactate or formate, are produced.The accumulation of these acidic products facilitates the release of iron from host proteins and also leads to a significant decrease in pH at the site of inflammation.In proportion to the local decrease in pH, the reduction potential is decreased, causing the reduction of the insoluble host Fe 3+ to the more biologically advantageous Fe 2+ [13].The presence of acidic products also results in the so-called nitrate respiration, where nitrate is reduced to nitrite, nitrogen gas or ammonia [14].Here, lactate, formate and other acidic substances act as electron donors.The initial products of NO 3 − reduction differ depending on the type of bacteria, E. coli and S. aureus reduce to NO 2 − , P. aeruginosa mainly to N 2 or NH 4+ [10,14,15].
The ability to acquire real-time measurements of environmental parameters of interest, including within the implant surface in situ, would significantly enhance the information available to medical professionals to detect and affect early inflammation.The biofilm formation process can be detected by many electrochemical methods [16,17].Monitoring by these techniques may include impedance spectroscopy, voltammetry or amperometry, among others [18][19][20][21].
The recording of open circuit potential may represent a useful and simple technique for continuous monitoring of biofilm formation [22].Souza et al. [23] tested an environment that simulates oral biofilms and their effect on the wear and the corrosion resistance of titanium.The work published by Ben-Yoav [18] successfully studied biofilm growth using impedance spectroscopy.Liu et al. [24] detected the bacterial adhesion of E. coli and Salmonella to gold chips in the Luria-Bert broth environment.Muñoz et al. in their work [25] tested a working electrode made of platinum, in an arrangement with a platinum counter electrode and an Ag/AgCl reference electrode in a minimal essential medium inoculated with E. coli.Bressel et al. in their work [16] describe the possibility of documenting the influence of bacteria by electrochemical methods.A decrease in potential was observed during exposure of a platinum electrode in a 1 mol L −1 Na 2 SO 4 solution with the addition of a biofilm from drinking water.The evolution of the measured quantity was due to the change and catalysis of charge transfer reactions at the phase interface.The cause may be due to active metabolites of the bacteria and changing nutritional conditions in the vicinity of the emerging biofilm.A large electrochemically active surface could increase the sensitivity of detecting changes at the sensor-bacteria/biofilm interface.One possibility to increase the exposed area is the formation of nanotubular oxide surfaces on titanium [25] and its alloys [26,27].
This work aims to describe the electrochemical behaviour of the nanotubular Ti-6Al-4V surface in the presence of different bacteria strains and its potential usefulness as an implantable sensor for detecting inflammation in the human body (typically on the foreign surface of a bone implant).The main advantages of this approach include the simple design of the sensor and the material used, which complies with medical device legislation.In addition, most of the concepts currently studied are based on polymer layers that are sensitive to specific proteins produced by bacteria.The specific response of the sensor can then be inhibited by other physiological processes [28,29].Polymeric materials can be unstable in the body environment with long-term use, which is required for implantable sensors [29,30].Another problem is the specific reaction of the organism to a foreign object; this risk would be eliminated by placing the sensor directly on the implant or in its close vicinity [28,31].

Materials
The Ti-6Al-4V (Grade 5, ASTM B391-13, hereinafter TiAlV) pins with 5.5 mm diameter and 50 mm length were used as working electrodes.The surface was ground to P2500 SiC paper and anodic nanostructuring followed.The nanostructuring was carried out using Jaissle IMP 88 PC-200V potentiostat/galvanostat with a PGU-AUTO Extern control unit.The nanostructure was formed by polarisation to 20 V/SSCE with a polarisation sweep rate of 100 mV s −1 , and a dwell time of 2200 s at this potential followed.The electrolyte was prepared with deionized water contained 1 mol L −1 (NH 4 ) 2 SO 4 and 0.2 wt% NH 4 F.After exposure, the samples were cleaned for 10 min in an ultrasonic bath with distilled water and then in 70% ethanol.
The TiAlV pins with the surface ground with P320 SiC paper were used as reference specimens.
The surface of samples was documented by scanning electron microscopy (SEM) using a VEGA 3 LMU (TES-CAN).The inner diameter of the nanotubes was evaluated from SEM images using ImageJ software [32].The histogram represents data from three different areas with a minimum of 50 measurements in each.

Bacteria cultivation and pin contamination
Indicator strains of the following bacteria were used for pin contamination: Escherichia coli ATCC (American Type Culture Collection) 700728 (hereinafter EC) and Staphylococcus aureus ATCC 6538 (hereinafter SA).All strains were cultured in BHI (Brain Heart Infusion) broth (HiMedia, India) with pH adjusted to 7.2.The culture medium was inoculated with 1% (v/v) inoculum and cultured aerobically at 37 °C for 18 h.The indicator strains were re-inoculated once a week and stored in a refrigerator at 5 °C.The initial concentration in the nutrient solution was 10 9 colony-forming unit (CFU) mL −1 .A fresh bacterial culture was diluted with saline solution (8.5 g L −1 NaCl and 1 g L −1 peptone) to 10 8 CFU mL −1 .In the case of S. aureus, a lower concentration of 10 5 CFU mL −1 was also tested.Surface contamination was achieved by immersion of specimen in the bacterial suspense for 10 s followed by 10 min drying in the laminar flow box.
After exposure, samples were removed and the biofilm was fixed by immersion in 3% glutaraldehyde for 2 h at 4-6 °C.The pins were then rinsed 3 times with physiologic solution (PS, 9 g L −1 NaCl) and dried in a series of ethanol solutions (70% and 96% vol.) for 10 min.Before observing the surfaces with a scanning electron microscope, 3 nm of gold was sputtered onto the samples in a Quorum 150R vacuum sputter.

Electrochemical measurement
Electrochemical measurements were performed in the standard three-electrode setup with a silver-silver chloride reference electrode filled with 3 mol L −1 KCl (hereinafter SSCE) and platinum wire as the counter electrode.The test electrolyte was agar (5 g L −1 ) with the addition of 13 g L −1 Nutrient (Nutrient Broth, Oxoid).The mixture was boiled for 5 min and then transferred to a 50 mL electrochemical cell.The filled and sealed cell was sterilised by hot steam for 20 min at 121 °C.A contaminated pin was inserted into the sterile electrochemical cell with agar medium and measurement starts immediately.Electrochemical measurements consisted of repetitive sequences of the one-hour open circuit potential (OCP) stabilisation and potentiostatic electrochemical impedance spectra (EIS) over a frequency range from 20 kHz to 0.0075 Hz with DC potential equal to OCP and AC potential 0.01 V rms −1 (root mean square).The selection of the frequency range was chosen with respect to the length of the measurements to allow sufficiently intensive OCP recording in the interim.The spectra were evaluated using Zview 3.5f software and then the effective capacitances of the constant phase elements present were calculated according to Brug et al. [33].To determine the charge exchange resistance on the titanium alloy, the polarization resistance was also measured after the EIS measurements in the range of + − 0.020 V/OCP with a scan rate of 0.0001 V/s.
The impedance spectra will be also presented as a normalised impedance (IPN) when the selected impedance parameter (IP) is divided by its value at the beginning of the exposure (IP 0 ) [34,35].
The potentiodynamic curves were measured in agar medium with non-contaminated specimens.The potential range was from − 0.5 V/OCP to 1 V/SSCE with potential sweep rate 1 mV s −1 .The cyclic polarisation on the platinum was realized in the potential range − 0.45 V/OCP to 1 V/ SSCE and back with potential sweep rate 1 mV s −1 .
The measurements simulating the presence of the bacteria metabolites were performed in the same set-up, but

Results and discussion
The nanostructured surface of the TiAlV alloy is shown in Fig. 1.The surface is formed by nanotubular mixed oxide and cavities belonging to the etched beta phase [36].Nanotube diameters ranged from 20 to 90 nm, with 35 to 60 nm being the most frequent (Fig. 1).
The basic electrochemical characterisation of the studied system was realised by potentiodynamic curves measurement.The potentiodynamic curves of the nanostructured surface, ground surface and platinum as an inert material are presented in Fig. 2. The both samples show passive behaviour.The higher current density of the nanostructured surface is caused by higher real than geometrical surface [27].The increase of the current densities on the titanium samples was caused by reactions of the environment, as evidenced by cyclic polarisation on inert platinum.
Figure 3 shows the recordings of the open circuit potential during 48 h of exposure in an agar environment with and without bacterial contamination of the specimen surface.The green curve is a record of the electrochemical response of the nanostructured surface in the bacteria-free environment (BFE) and its slight increasing trend is evident.The growth corresponds to small positive changes in the system and the measured values are consistent with the predicted stability of the modified surface.
Controlled contamination leads to an overall decrease in open circuit potential in all cases.In the case of surface contamination with E. coli with an initial concentration of 10 8 CFU, the OCP begins to decrease slightly after 6 h with a pattern corresponding to a parabolic dependence and an overall change of 0.200 V throughout the exposure.In the case of contamination of the reference ground surface with the S. aureus (black curve, SA 10 8 ref), the situation is similar, but the sharp decrease is more pronounced.Subsequently, there is a very slight increase.For the nanostructured surface, very similar trends were observed, but the changes are more pronounced and the end of the sharp decline phase occurs 12 h later.Reducing the initial concentration to 10 5 CFU leads to a prolongation of the initial lag phase from 6 to 13 h resulting in a time shift of the whole process.Moreover, these values are about 0.100 V higher throughout the measurement.All changes are probably a reflection of the change in the redox potential of the environment.Given the decreasing trend, this is probably a decrease in oxygen concentration caused by the metabolic activity of the bacteria.The same course of the open circuit potential was also observed by Souza et al. [37].
Fig. 5 The impedance spectra analysis of the ground surface in the selected media Despite the significant changes in OCP measurements, the impedance spectra on the reference ground surface (Fig. 4) did not show notable changes during exposure to both bacterial species, although the microorganisms were clearly present on the surface.Two time constants are evident in the spectra with a notable capacitive character at low frequencies, which is a typical response of passive titanium alloys [38,39].In the case of exposure with S. aureus, a small phase shift at the highest frequencies is still evident, with the form of the microorganisms identified as a fully mature biofilm with a 3D structure (Fig. 4a).Furthermore, the communication channels are clearly visible in the figure, allowing the biofilm to communicate with the environment even for deeper layers [40,41].In the case of E. coli, there is no formation of a continuous biofilm (Fig. 4b).
A detailed analysis of the impedance spectra, together with the equivalent circuits used for fitting spectra of the ground surface, are summarized in Fig. 5. Spectra in an environment free of bacterial contamination were fit using an equivalent circuit describing the alloy surface by constant phase element (CPEeld) and a warburg diffusion element parallel with CPE ((CPEenv/Wenv)) characterizing the environment close to the electrode and bulk agar resistance (Ragar).Analysis of the individual components of the circuit shows that the material behaves stable throughout the exposure, with only a slight increase in the diffusion element, which is likely related to oxygen transport to the agar/ passive layer interface.This corresponds to an increase in the effective capacity of the medium.In the presence of E. coli, the ground surface shows only minimal changes and the response corresponds to the passive layer.The spectra were evaluated with the same equivalent circuit as in the BFE case.Compared to the previous situation, the trend of the diffusion is reversed, i.e. the environment is affected by the presence of bacteria.The equivalent circuit used to fit the impedance spectra of the ground alloy in the environment with S. aureus describes a two-layer structure of the passive titanium layer, it is an outer porous layer (Reldout/CPEeldout) and an inner compact layer (CPEeldin) [42].During the exposure, the capacitance of the inner layer decreased, this process is associated with an increase in its thickness.In the case of the diffusion and the resistance of the outer layer, a change in the trend between the eighth and twelfth hour can be seen.Since the capacity of the outer layer remains de facto constant, this is probably a manifestation of a faradaic Fig. 6 The impedance spectra of the NT-TiAlV without bacterial contamination in agar media Fig. 7 The impedance spectra of the nanostructured TiAlV in the presence of bacteria and surface coverage by bacteria E. coli after 48 h exposure reaction other than a decrease in the corrosion resistance of the material.As the time interval correlates with the change in electrode potential, a change in the cathodic reaction from oxygen depolarisation to a different mechanism can be assumed.
The impedance spectra of the nanostructured surface in an agar medium without the presence of bacteria are summarised in Fig. 6.The measurement was performed to obtain the information about electrochemical behaviour of the nanostructured surface in a relatively high viscous electrolyte.The spectra show the presence of at least two time constants, and the effect of nanostructuring is evident here, where the local minimum of the phase shift affecting the response at higher frequencies (which describes the surface porosity effect) is further shifted towards even higher frequencies.This corresponds to the effect of the significantly larger pores of the modified surface material compared to the ground material.The slight decrease in impedance modulus compared to the ground sample is due to the increase in real surface area.Changes in the spectra within 48 h of exposure are evident in both time constants, but the changes were only minor.The change in the region around 50 Hz probably includes the effect of not perfect wetting of the nanostructured surface by the relatively viscous electrolyte.The slight increase in capacitance in the low-frequency region is indicative of the stabilisation of the system.
Contamination with E. coli leads already in the initial part of the exposure to a significant influence on the response at higher frequencies, where the local minimum of the phase shift shifts an order of magnitude higher compared to the uncontaminated surface.(Fig. 7).Thus, there has been an increase in surface sensitivity to the extent that comparable contamination effects are exhibited by DLC [43] coatings or the growth of massive layers of defective hydroxyapatite [44], where the typical electrolyte response was also suppressed.Compared to a ground surface, where changes in the impedance response at high frequencies are not apparent, a thicker layer of the culture medium with bacteria was probably formed.The nature of the layer may be affected by the significantly higher wettability of the nanostructured surface [45].
The evolution of the bacteria film on the nanostructure leads to a complex progress in impedance spectra shape.In the lag phase of bacterial development, the phase shift decreases in the mid frequencies and the spectra in this region approach the spectra of the uncontaminated surface in character.After the lag phase, there is a significant increase at low frequencies and mid frequencies, and the trends in the region below 1 Hz correspond reciprocally to the trends of the OCP course.Changes in time constants also reflect the phase shift, however, with significantly lower sensitivity.It reaches the most significant change in the region between 100 Hz and 1 kHz and probably reflects changes in transport characteristics both in the bacteria-occupied environment and, in part, as the reducible component moves deeper into the pore.The change in the thickness of the bacterial film Fig. 8 The impedance spectra of the nanostructured TiAlV in the presence of bacteria and surface coverage by bacteria S. aureus (10 5 CFU) after 48 h exposure was also reflected at these frequencies, affecting the surface capacitance [46].After exposure, bacteria and the sealing of the nanotubes by the culture medium were visible on the surface (Fig. 7).However, as in the case of the ground surface, the bacteria do not form a continuous biofilm.
S. aureus contamination also affects the region above 1 kHz, but the effect is less pronounced in the case of exposure with E. coli which is reflected both by the more similar Zmod slopes and the absolute values of the local minimum of the phase shift and their frequency location between exposure without bacteria and with S. aureus.During cultivation, there are very important changes in the impedance response (Fig. 8).In the initial hours of exposure, a phase shift is evident at the highest frequencies, with virtually no further evolution, and this phenomenon is also evident in the normalised modulus.In the case of S. aureus surface contamination with an initial concentration of 10 8 CFU, bacterial multiplication and biofilm growth occur very rapidly.The most important changes occur in the low to medium frequency region, where the modulus decreases by more than 80%.This change may be due to the presence of redox couple, as a product of bacterial metabolism and whose reaction masks the material response.The similar behaviour was observed in our previous work with nanotubular surface [27].Furthermore, the colonisation of the surface itself is also evident.However, the manifestations of surface colonisation are reported in the literature over the entire range of frequencies from mHz to hundreds of kHz [47][48][49].The trends of these changes are consistent with the OCP changes, but are shifted by about 6 h.The SA bacteria switch from an aerobic to an anaerobic mechanism, the decrease in OCP reflects the consumption of oxygen by the bacteria, then switch to an anaerobic metabolic pathway that causes the formation of a redox couple in the close vicinity of the surface, which subsequently stabilises the OCP.Nitrate and nitrite can be used as terminal electron acceptors under anaerobic conditions [50].All bacterial nitrate reductases catalyse the reduction of nitrate to nitrite [51] and require microaerobic (or low oxygen) conditions to properly function [52].Burke et al. [53] and Heinemann et al. [54] describe the metabolic processes associated with the formation of nitrate, nitrite and lactate.As a result of these processes, a hydrogen donor is formed, which can then lead to hydrogen depolarization.This is in agreement with the OCP values (Fig. 3).
In the case of lower SA concentration, the nanostructured surface's behaviour is in principle the same as in the previous case but delayed.Stabilisation of the system occurs after 40 h compared to 30 h for 10 8 CFU (Fig. 9).The changes are also slightly less pronounced.The large electroactive surface is able to detect even minor changes in the system.The shape of the spectrum after 48 h is close to the previous one after 24 h, it is associated with slower colonisation of the surface in the case of lower initial bacterial concentration.The shift in impedance response changes compared to Fig. 9 The impedance spectra of the nanostructured TiAlV in the presence of bacteria and surface coverage by bacteria S. aureus (10 8 CFU) after 48 h exposure OCP is approximately 4 h even in the case of lower bacterial concentration.The impedance changes are detectable even if the surface is not fully covered by the 3D biofilm.
A detailed analysis of the impedance spectra, together with the equivalent circuits used for fitting spectra of the nanostructured surface, are summarized in Fig. 10.The equivalent circuit used to evaluate the spectra is more complex compared to the ground surface.The circuit reflects the nanotubular nature of the oxide and takes into account diffusion in the nanotubes (Cwall/Rpor-Wtube/(Cdl/Rct)).The circuit also describes the characteristics of the electrolyte in the close vicinity of the electrode (CPEenv/Renv-Wenv) and Fig. 10 The impedance spectra analysis of the nanostructured surface in the selected media the agar bulk (Ragar).The results of the analysis show that in an environment free of bacterial contamination, there are no significant changes in the electrochemical behaviour of the system during exposure.In the presence of E. coli, both the electrolyte near electrode and the parameters describing the surface show changes.The slight decrease and subsequent increase in the resistance of the Renv environment is probably related to the gradual colonization of the surface by bacteria.A similar trend can be observed for the electrode surface.However, after 24 h no significant changes occur and the system is in steady state.The situation is more complicated in the case of surfaces contaminated with S. aureus.The trend of the monitored parameters for both bacteria concentrations is similar, however, in the case of lower initial bacterial concentrations, changes occur later.In both cases there is an increase in diffusion parameters, this trend can be associated with an increase in biofilm thickness.In both cases there is also a sharp change in the capacitance at the interface of the nanostructure.Due to the relatively rapid change, it can be assumed that the electrode reaction mechanism changes during exposure.Due to the passive nature of the electrode surface, it can be assumed that this is a change in the depolarization reaction, in which bacterial metabolites start to participate.This would be consistent with a steady state trend after 36 h of exposure.
Since changes in surface impedance characteristics can be caused not only by the physical presence of bacteria but also by their metabolic processes [46], measurements have been made in easily modelled systems created by redox couples Fe 2+ /Fe 3+ and NO 3 − /NO 2 − .Commercial culture media should act as a source of iron and nitrogen compounds in the case of in vitro experiments [13,55].Simulating the effect of bacteria on the environment by externally influencing the NOx ratio leads to manifestations dominantly at higher frequencies (Fig. 11).In contrast, the effect of changing the iron ion concentration is evident over the entire range of the impedance spectrum.Thus, it is clear that the measured changes in the impedance response in the presence of bacteria are related to their physical presence as well as to their metabolic processes.Another possible influence may be due to the polarisation of the bacteria due to the external electric field during the measurement of the EIS spectra [49,56].
Normalised impedance parameters of the nanostructured surfaces in the presence of E. coli and S. aureus are presented in Fig. 12.The main advantage of this presentation is that it is possible to clearly display changes in the full range of frequencies and time.The back projection represents the maximum of the relative change in impedance parameter and side projection is relative change of impedance parameter in time.The 3D graph allows direct comparison of changes for each frequency of the impedance spectrum.Another advantage is the practical use for the impedance response of the  impedance could also be used to determine the type of surface contamination.
Based on the evaluation of the normalized impedance, taking into account the magnitude of changes during the measurement and the sufficiently short measurement time for each frequency, two frequencies were selected: 1 and 100 Hz.The time course is shown in Fig. 13.A sensor that would evaluate the impedance change at the given frequencies would be applicable.A relative change in the impedance parameter of 0.1 would then indicate the development of a biofilm on the implant surface.From the application point of view, there are several issues, that must be solved, respectively tested before the clinical use.In particular, the development of a stable bioinert reference electrode, the response of the surface to mixed bacterial strains must be tested and interference phenomena that could distort the sensor response must be identified.Finally yet importantly, the problem of a stable high-capacity power supply for the sensor must be solved.

Conclusion
It was found that the nanostructured surface of the Ti-6Al-4V alloy exhibits changes in impedance behaviour in the presence of E. coli and S. aureus.These changes reflect both the physical presence of the bacteria, i.e. surface colonisation and biofilm growth and their metabolic processes.Based on the evaluation of the relative changes in impedance characteristics, frequencies were selected that could be used for in situ detection of biofilm formation.Frequencies of 1 Hz and 100 Hz meet the sensor requirements for measurement speed, energy consumption and detectability of changes.The course of the changes could be used to determine the bacteria type contamination.
IPN t=n = IP t=n ∕IP 0 .with different electrolytes.Both tested solutions were PS based, the first one with the addition of the K 4 [Fe(CN) 6 ] and K 3 [Fe(CN) 6 ] (concentration of the Fe 3+ /Fe 2+ couple 0.01 and 0.1 mol L −1 ) and the second one containing KNO 3 and KNO 2 (concentration of the NO 3 − /NO 2 − couple 0.01, 0.1 and 1 mol L −1 ).The EIS was measured after 12 h of OCP stabilisation in the frequency range from 60 kHz to 0.002 Hz with E DC = 0/OCP and E AC = 10 mV rms −1 .

Fig. 1 Fig. 2 Fig. 3
Fig.1The SEM image of the nanostructured TiAlV surface and nanotubes pore diameter distribution

Fig. 4
Fig. 4 The impedance spectra of the ground TiAlV in the presence of bacteria and surface coverage by bacteria after 48 h exposure (SEM): a SA 10 8 CFU, b EC 10 8 CFU

Fig. 12
Fig. 12 Normalised impedance of the nanostructured surface in the presence of a E. coli and b S. aureus 10 8 CFU