Sterilized nutrient broth was inoculated using a single colony of P. mendocina CH50 and was incubated for 16 h at 30°C at 200 rpm. This was then used to inoculate the second stage media which was incubated at 30°C at 150 rpm until the optical density reached 1.6 without dilution. The second stage media comprised of minimal salt media (MSM) which included ammonium sulfate; 0.45 g/L, sodium hydrogen phosphate; 3.42 g/L and potassium dihydrogen phosphate; 2.38 g/L. Glucose at 20 g/L concentration was used as the carbon source. Magnesium sulfate heptahydrate and trace element were also added to the media at a concentration of 0.8 g/L and 1 mL/L, respectively. The inoculated second stage media was used to further inoculate the production media (10% of culture volume) for 48 h at 30°C at 150 rpm which comprised of MSM, glucose, magnesium sulfate heptahydrate and trace elements. The MSM comprised of ammonium sulfate; 0.50 g/L, sodium hydrogen phosphate; 3.80 g/L, potassium dihydrogen phosphate; 2.65 g/L.

After the incubation, the cells were harvested at 48 h by centrifugation at 4600 rpm for 30 min. The centrifuged cells were washed with distilled water followed by 10% ethanol and finally with distilled water. The obtained cells were homogenized for 15 min using a homogenizer. They were then kept at −20°C overnight and were finally placed in the freeze dryer for lyophilization.

The dried cells were used to extract polymer using the soxhlet extraction method. The cells were washed with methanol under reflux conditions for 24 h to remove impurities. Then the methanol was replaced with chloroform and the cells were incubated for 24 h under reflux conditions. This chloroform solution was used to extract polymer by concentrating it in a rotary evaporator. The polymer was then precipitated using ice-cold methanol solution. Although chloroform and methanol were used in this study, it should be noted that several non-chlorinated solvents such as cyclohexane, gamma-butyrolactone, butyl acetate and ethyl acetate have been used to extract PHAs (Aramvash et al., 2015; Jiang et al., 2018). Another environmentally sustainable method used in the extraction of PHAs include the use of supercritical fluid (Kunasundari and Sudesh, 2011).

The composite film was prepared using solvent casting by weighing 0.5 g of MCL-PHA and adding to 10 mL of chloroform. This solution was left stirring overnight until completely dissolved resulting in a 5 wt% solution of the polymer. Once the MCL-PHA was dissolved, different concentrations (15, 20, and 30%) of GnP were added. This was then vortexed for a minute and left to sonicate in a water bath sonicator for 7 h (Figure 1A). After sonication, the contents were immediately poured into a petri dish covered to allow slow and even evaporation and left to air dry in a fume cupboard. Upon drying, water was added in the petri dish and subsequently the composite films were peeled and air dried. The water is required because MCL-PHA is elastomeric in nature and tends to stick to the glass petri dish. Adding water to the petri dish helps in the removal of the film without causing a tear in the film.

A cell proliferation study was carried out to test the cytocompatibility of the composite films with the mammalian cells. It was carried out using HaCaT cells (keratinocyte). Cells at a seeding density of 10,000 cells/mL were added directly on to the sterile graphene composite film samples. They were cultured for 24 h. Cell viability was measured using Alamar Blue assay. TCP was used as a positive control. Neat MCL-PHA was tested for comparative study.

Scanning electron microscopy analysis was performed to evaluate the surface topography of the MCL-PHA/graphene composite films. The SEM images were taken using a beam of 5 keV at 10 cm working distance (JEOL 5610LV-SEM). For the analysis, all the samples were coated with gold for 2 min using a EMITECH-K550 gold spluttering device. This analysis was carried out at the Eastman Dental Hospital, University College London.

Commercially available screen-printed carbon electrodes were purchased from DropSens. Standard carbon electrodes (DRP110), standard carbon modified with carbon nanotubes (DRP110-CNT), and standard carbon modified with graphene (DRP110-GPH) were used. Where the MCL-PHA was used as the sensing substrate, the counter electrode and reference electrode on a standard carbon electrode (DRP110) completed the electrochemical cell. In order to provide a stable reference, the silver reference electrode was modified by incubating the electrode surface for 10 min in a 200 mM solution of Iron(III) chloride in dH₂O. It was found that 3 μl of Iron(III) chloride was sufficient to cover the reference electrode without touching the working or counter electrodes. Following the addition of the chloride, the electrodes were thoroughly rinsed in distilled water.

Electrode chambers were produced through the use of a custom-made PTFE plate (Figure 2A). The 15-mm-thick plate consisted of eight 16-mm-diameter chambers, a base layer of PTFE and a leveling layer to accommodate the thickness of the electrodes. The top plate was mounted on top of the electrodes and the bottom plate using silicon adhesive sealant and allowed to cure for 24 h prior to use. The MCL-PHA/graphene composite was cut into 5 mm strips with a sharp scalpel and held in the prepared chamber with a pair of metal forceps, so that approximately 5 mm of the MCL-PHA/graphene composite was submerged in the media (taking care that the forceps were not in contact with the media). Alternatively, the MCL-PHA/composite was integrated into the bottom of the electrode chamber by cutting a 10-mm-diameter hole through the center. This allowed the biopolymer patch to be integrated over the top of the commercial electrode (Figure 2B). 1 mL of LB was used in the electrode chambers during the experiments.

The complex impedance of the polymers was measured using a PalmSens3 potentiostat (PalmSens) in a two-electrode configuration (Working – Counter/Reference). Two gold electrodes with 1 mm separation were brought into contact with the MCL-PHA/graphene composite patch, where a 0.5 V_DC with a 0.25 V_AC signal amplitude was applied across the frequency range from 0.1 to 10 kHz. In total, 51 frequency points were measured on a logarithmic scale. The reference measurement was performed by measuring the impedance between the two gold electrodes in free space. Both the smooth and rough surfaces (bottom- and top-side) of the MCL-PHA/graphene composite patches were measured, with three randomly positioned measurements, from which an average value was recorded.

Square wave voltammetry (SWV) was used to perform all electrochemical measurements, based upon previously published parameters for the detection of pyocyanin (Sharp et al., 2010). A potential window from −600 to 400 mV (vs. Ag/AgCl) was used, with a step potential of 2 mV and an amplitude of 50 mV. The measurement frequency used was 25 Hz. Electrochemical sweep data was either recorded in PSTrace, or directly in Matlab, by controlling the potentiostat through the PalmSens software development kit¹.

Pyocyanin was diluted in sterile LB media to produce a working stock with a starting concentration of 2.5 mM. 1 mL of sterile LB was added to each electrode chamber and an initial SWV measurement was performed. Following this, 2 μl of pyocyanin working stock was added to the chamber and mixed by aspiration using the pipette, to achieve a final pyocyanin concentration of 5 μM. An SWV measurement was then performed and a further 2 μl of pyocyanin working stock was added in order to increase the concentration to 10 μM pyocyanin, followed by a further SWV measurement. This was repeated until a final calculated concentration of 97 μM pyocyanin existed in the measurement chamber.

Pseudomonas aeruginosa PA14 was used to produce cultures by inoculating 2 mL of sterile LB in a 30 mL universal bottle with a single colony of PA14 from a LB solid agar plate. This was then incubated at 37°C in a shaking incubator for 24 h. The overnight culture was then added to the electrode chamber and SWV measurements were performed with the biopolymer or commercial electrodes as described above.

Medium chain length Polyhydroxyalkanoate was produced by P. mendocina CH50 using glucose as the carbon source. ATR-FTIR was used to identify the purified polymer as an MCL-PHA. Characteristic peaks (1727.4 cm^–1, corresponded to the ester carbonyl bond and 1161 cm^–1 corresponded to the C-O stretching) of MCL-PHA were present in the FTIR spectrum (see Supplementary Data Sheet S1 – supplementary information). Molecular weight, M_w, of the MCL-PHA was measured to be 542 kDa with a polydispersity index (PDI) of 3. The percentage cell viability on the neat biopolymer film at 24 h was 85%. On the composite films with 15, 20, and 30% graphene content, %cell viability were 55, 53, and 49% respectively at 24 h. There was no significant difference between the films. There was a decrease in the cell viability in comparison to the neat biopolymer film.

SEM analysis of the two sides of the polymer film indicated different morphologies, which were influenced by the concentration of graphene added to the MCL-PHA and the side of the polymer (exposed to air or glass while casting in a glass petri dish) was being considered. As the concentration of graphene increased, both the upper and lower surfaces of the biopolymer increased in roughness (Figure 1B). Furthermore, initial electrochemical measurements with the MCL-PHA/graphene composites indicated that the response was dependent upon which side of the composite was used as a sensing surface. To understand the difference between the upper and lower surfaces, conductivity measurements were performed from 0.1 to 10 kHz. The measurements show that the rough, upper (air) side of the composite had a high resistance, regardless of the concentration of graphene present in the sample (Figure 3A). In contrast, it was found that the resistance of the lower (glass) side of the composite was several orders of magnitude less (Figure 3B). Furthermore, the resistance of the smooth lower side of the composite was found to be concentration dependent. As the concentration of graphene increased, the impedance of the layer decreased from a mean 111.8 kOhms (SD 79.4 kOhms) across the frequency range measured with the MCL-PHA/15 wt% graphene composite to 2.18 kOhms (SD 276 Ohms) for the MCL-PHA/30 wt% graphene composite. This suggests that during the curing phase of the MCL-PHA/graphene composite, the graphene settles toward the bottom of the polymer in a concentration dependent manner. Interestingly, the upper, rough surface of the 30 wt% MCL-PHA/graphene composite had a higher resistance than the rough surface with other concentrations of graphene. This is probably caused by the high graphene concentration in the 30 wt% composite, which settled down better toward the lower glass face. As this study is focused on the electrochemical properties and not the mechanical properties, the lower (glass) biopolymer side was used for subsequent electrochemical experiments as this side had the lowest resistance.

The use of graphene as a basis for the detection of pyocyanin relies on a redox reaction, which is linear across the range of interest. A series of standard curve experiments were performed to explore this, using screen printed carbon, graphene and carbon nanotube (CNT) electrodes, across a range of concentrations from 0 to 100 μM pyocyanin. A large background current was observed upon DPV measurements will all electrode types, therefore, a computer script was written in order to remove the background current, so that the peak could easily be resolved (Supplementary Data Sheet S2). In our study, across all types of carbon electrodes, a peak was observed between −265 and −292 mV vs. Ag-AgCl (Figure 4A), which is similar to SWV peaks previously reported for pyocyanin (Sharp et al., 2010; Bellin et al., 2014; Sismaet et al., 2016). For the graphene modified electrodes, the pyocyanin-current curve was linear up to 45 μM pyocyanin, at which point the rate of current increase changed (Figure 4B). In order to assess the implications of this for our sensor, we measured an overnight culture of P. aeruginosa PA14 after growth in conditions that promoted high concentrations of pyocyanin (Figure 4C). These measurements demonstrate that a redox peak relating to pyocyanin can be observed with a peak height of 49.36 μA, corresponding to approximately 42 μM pyocyanin, on the basis of the standard curve when a spline interpolation was used. In other studies (Wilson et al., 1988; Muller et al., 2009), clinically relevant concentrations of pyocyanin have been found to be less than 50 μM, highlighting that further development would be required to use these commercial screen printed electrodes as a sensor substrate for the linear detection of pyocyanin.

The MCL-PHA/graphene composites with different concentrations of graphene were tested in conjunction with pyocyanin in order to determine the electrochemical performance, in contrast to the commercially available electrodes measured above. This was achieved by placing a strip of the MCL-PHA/graphene composite film, connected using forceps, into a well with a commercial screen-printed carbon electrode in the bottom. The screen-printed carbon electrode was used as the counter electrode and Ag-AgCl reference electrode, whereas the MCL-PHA/graphene composite formed the working electrode. The results indicate that a peak can be resolved in the SWV plot at −287 mV (Figure 5A), which is close to the location of the peak observed on graphene and CNT modified electrodes. This suggested that a similar electron transfer mechanism to that observed with the DRP110-GPH commercial electrodes was occurring. The 30% MCL-PHA/graphene composite showed the largest peak response to pyocyanin and was therefore explored further through the production of a standard curve (Figure 5B). This showed that the MCL-PHA/graphene composite has a lower current response to pyocyanin than the commercially modified electrodes. However, the response to increasing concentrations of pyocyanin is linear and peaks can be resolved from a low concentration of approximately 5 μM pyocyanin.

The stationary phase culture of P. aeruginosa PA14 was strongly pigmented with a blue-green color, indicating the production of pyocyanin. Measurement of the overnight culture with a strip of the MCL-PHA/graphene composite indicated that a clear peak can be observed when PA14 is present (Figure 6). Much like the measurements performed using the commercial electrodes, these results confirmed that it is possible to identify the presence of P. aeruginosa PA14, once high concentrations of pyocyanin have been produced. Although the pyocyanin peak for the MCL-PHA/graphene composite was much lower than that observed with the commercial screen-printed electrodes, the results demonstrate the potential of the composite for the indirect identification of P. aeruginosa PA14.

In contrast to an overnight culture of P. aeruginosa, wound exudate is a complex matrix consisting of potential interferents including, glucose, pH, proteins, human cells, and other microorganisms. A previous study has demonstrated the ability to detect pyocyanin electrochemically in wound exudate with a carbon based electrode (Sismaet et al., 2016). In terms of polymicrobial infections, we have shown previously that it is possible to detect pyocyanin in a polymicrobial competition model in conjunction with Staphylococcus aureus (Ward et al., 2014). The production rate of pyocyanin in the presence of other common clinical isolates has also been investigated. In this study it was found that there was no statistical difference in pyocyanin concentration when P. aeruginosa was co-cultured alongside Enterococcus faecalis, S. aureus, or Staphylococcus epidermidis (Santiveri et al., 2018). Hence, literature suggests that it will be possible to electrochemically identify the presence of pyocyanin in wound exudate. In future studies, we will explore if this is also possible with our MCL/PHA graphene composite.

Electrochemical detection of pyocyanin has been explored in other studies with a view to producing low cost diagnostic tests. Sharp et al. (2010) demonstrate the ability to detect the presence of pyocyanin in concentration ranges between 1 and 100 μM using a carbon fiber tow in buffer. Alatraktchi et al. (2016) used commercially available screen printed gold electrodes to detect pyocyanin in a concentration range between 2 and 100 μM with cyclic voltammetry in buffer. This shows that the results reported in our study with the biocompatible polymer are comparable to other work. Interestingly, Ciui et al. (2018) produced a “swipeable” sensor, capable of detecting pyocyanin between 10 and 100 nM with a carbon electrode screen printed onto a disposable glove. This demonstrates that much higher sensitivities can be achieved with low cost, carbon-based substrates.

The MCL-PHA described in this paper has previously been demonstrated to be non-cytotoxic toward HaCaT cells (keratinocytes) in vitro. This study potentially adds additional benefits to the MCL-PHA by allowing it to be used as a sensing substrate to detect the presence of one potentially pathogenic microorganism. In order to fully exploit the benefits of MCL-PHA based graphene composites as a novel sensing circuit, we explored a feedback and control system that could be used to release antimicrobial agents into the wound bed when an infection is identified. Heavy metal ions, such as silver (Jung et al., 2008; Fromm, 2013; Sharma et al., 2015) have antimicrobial properties and could be electrochemically dissolved by applying a potential between two silver electrodes. This would cause silver ions to be dissolved into the wound bed and exert a toxic effect on the invading pathogen. Integration of a printed sliver substrate within the wound dressing could therefore provide a mechanism through which an antimicrobial metal ion could be electrochemically released on demand, as soon as pyocyanin is detected. The approach is conceptually attractive as the same electrochemical instrumentation used for the measurement could also be used to release silver ions from the silver substrate.

To achieve this, we designed an overall structure of the feedback control system for regulating the concentration of silver to a toxic level while maintaining the concentration at a minimum used as shown in Figure 7. The neural network is trained off-line using simulated data, generated using the dynamic model represented in a set of ordinary differential equations (Dockery and Keener, 2001), to estimate the uncertainties. Training the network using simulated data is common practice when there is limited real measurement data available to train the network. The trained network determines the model uncertainty using the measurement from the sensing circuit. The dynamic model predicts the future toxic level after correcting the model error to identify whether the toxic level was achieved or not. The graph in Figure 7 demonstrates the prediction capability of the algorithm where the blue lines are the true toxic level and the red is the predicted one. This could be used to determine when and how much of the antimicrobial agents should be released to exert a toxic effect. Note that the true toxic level is generated using a stochastic molecular interaction model in Barbuti et al. (2008). It is our aim to build and verify this model further in the future through the production of in-depth experimental data.