Direct Laser-Functionalized Au-LIG Sensors for Real-time Electrochemical Monitoring of Response of Pseudomonas aeruginosa Biofilms to Antibiotics

As shown in Fig. 1, base LIG electrodes with a 3-electrode design are first fabricated using our previously reported method (optimized parameters: power: 12.6%, speed: 5.5%, and PPI: 1000) on polyimide (Kapton^®, 500 HN). Two functionalization routes are explored: electroless gold deposition (E-Au/LIG: Fig. 1A) and direct laser functionalization process (L-Au/LIG: Fig. 1B). E-Au/LIG is obtained by immersing the LIG-patterned sample in gold chloride solution (HAuCl₄, 0.06% wt. in ethanol) and reduced with ethanol and ascorbic acid as previously reported. ³² L-Au/LIG is obtained by dropping the HAuCl₄ solution on the working electrode area and waiting 10 min to allow the solution to permeate through the porous structure of LIG, followed by another laser writing step to decompose the precursor thermally. The increased local temperature leads to rapid thermal decomposition and creation of gold nanostructures in the LIG scaffold (details in the Methods Section). We optimized the laser functionalization process by studying the effect of gold loading concentration (1, 10, and 100 mM of HAuCl₄) and finally chose 1 mM for building the sensors, as this concentration offers the highest sensitivity to PYO (discussed in Fig. 2) and also requires the least amount of the precursor, hence lowering the overall sensor cost.

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To characterize the electrode surface morphology and chemical composition, scanning electron microscopy (SEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) are performed. SEM image and EDS maps of L-Au/LIG are shown in the Supporting Information (SI), Fig. S1, confirming the highly porous structure of LIG. The formation of the porous structure is attributed to gas release during the rapid laser irradiation process, where the energy from the laser results in lattice vibrations, which in turn lead to localized high temperatures (>2,500 °C), breaking the C–O, C=O, and N–C bonds. Aromatic compounds are then rearranged to form graphitic structures. ³³ SEM image of E-Au/LIG is also shown in SI, Fig. S2. EDS mapping of E-Au/LIG and L-Au/LIG shown in Figs. S1 and S2 confirm the presence of a much lower amount of gold with 1 mM L-Au/LIG compared to E-Au/LIG, which is expected considering the significantly smaller Au load in L-Au/LIG compared to E-Au/LIG, which are created by immersion of LIG in 2.8 mM of the gold precursor solution. To confirm the formation of crystalline gold structures, we performed XRD and XPS. XRD profiles of LIG, L-Au/LIG, and E-Au/LIG are shown in Fig. 2A. Bare LIG shows a broad diffraction peak centered at 2θ = 25.5° of the graphite (002) plane, indicating a high degree of graphitization. For E-Au/LIG, peaks of (111), (200), and (220) planes of gold are observed. For L-Au/LIG, a slight peak of gold (111) is observed. In order to obtain a more precise signature of Au in both E-Au/LIG and L-Au/LIG samples, XPS analysis was performed (Fig. 2B). Specifically, to study the uniformity of the functionalization process, we performed XPS mapping by studying the center and edge of the WE with three different concentrations of HAuCl₄. As shown in Fig. S3, the atomic % of gold is 1.68% at the center with 1 mM HAuCl₄ solution, compared to 9.15% and 22.2% using 10 mM and 100 mM. Interestingly, with all concentrations, the amount of gold at the edges and center is similar, confirming excellent uniformity of the laser functionalization process.

In order to determine the best material to fabricate the sensor working electrode, we tested the biocompatibility of L-Au/LIG and E-Au/LIG. The results are shown in Figs. 2C, 2D. This study is needed as an ideal sensor electrode for studying cells should not be toxic to them. ³⁴ The effect of Nafion coating on the biocompatibility of electrodes is also studied. As presented in Fig. 2C, while cell density changes from ∼2 × 10⁶ CFU/ml (control sample; Kapton sheet) to ∼1.2 × 10⁶ CFU/ml and ∼1 × 10⁶ CFU/ml with bare LIG and L-Au/LIG within 2 h and further reduces at 4 h, E-Au/LIG completely eradicates the cells which shows its strong bactericidal effect. To further reduce the antibacterial effects, we coat the electrodes with Nafion, which is also a common antifouling layer for biosensors. ³⁵ Figure 2D indicates that the toxicity effect of L-Au/LIG is significantly reduced after coating with Nafion, while E-Au/LIG is still highly toxic. This could be because the large clusters of gold in E-Au/LIG (see SEM image in SI, Fig. S2) are hard to be entirely covered by Nafion compared to L-Au/LIG.

To investigate the effect of the functionalization methods on sensor sensitivity to phenazines, we tested L-Au/LIG and E-u/LIG with PYO ranging from 0.1 to 100 μM in brain-heart infusion (BHI). For L-Au/LIG, we studied 3 different concentrations of the gold precursor solution. As shown in Figs. 3A–3C, the sensor made with 1 mM of the gold precursor, shows better performance (much larger SWV peak currents) than the other two concentrations and is also better than E-Au/LIG shown in Fig. S4. The calibration curves in Fig. 3D compare 1 mM L-Au/LIG with E-Au/LIG. While the sensitivity of 1 mM L-Au/LIG is almost the same as E-Au/LIG (1.276 and 1.205 μA μM⁻¹, respectively), the excellent biocompatibility of L-Au/LIG with bacterial cells (see Figs. 2C, 2D) makes it suitable for the studies in this work. Moreover, the cost of L-Au/LIG is much lower than E-Au/LIG since the gold solution dosage used in building L-Au/LIG sensors is much less than E-Au/LIG sensors. The laser writing method used to manufacture L-Au/LIG is also easier to mass-produce, scale up, and readily amenable for rapid and selective functionalization of electrodes using additive manufacturing. We also compare the response of 1 mM and 100 mM L-Au/LIG to phenazine-1-carboxylic acid (PCA) with concentrations ranging from 0.1 to 100 μM in BHI, as shown in SI, Fig. S5. For PCA, 1 mM sample also shows better sensitivity than 100 mM. As a result, 1 mM L-Au/LIG is chosen to manufacture sensors for the biofilm studies in the next section.

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To further study the role of gold functionalization on the enhanced response to PYO, we investigate the electrochemically active surface area (ECSA) and the heterogeneous electron transfer rate constant, ${k}^{0},$ using cyclic voltammetry (CV) with 100 μM PYO in BHI with 1 mM L-Au/LIG and compare them to bare LIG. To eliminate the potential impact of the number of laser writing passes on the morphology of the electrodes and hence the electrochemical response, bare LIG is prepared using 2-pass writing for a fair comparison. The ECSA and ${k}^{0}$ are calculated by studying the relationship between the CV curves (the peak current$,\,{i}_{{peak}}$) and the peak separations ($\unicode{x02206}{E}_{{peak}}$) at different scan rates. ³⁶ ${i}_{{peak}}$ vs square root of the scan rate and the linear fitting curves are shown in Fig. S6A. Using the Randles-Sevcik equation, we extracted ECSA of L-Au/LIG with different concentrations of Au precursor (i.e. 1 mM, 10 mM, and 100 mM) and 2-pass written LIG. The extracted ECSA values are summarized in Table S1, showing that 1 mM L-Au/LIG offers the highest ECSA value (noting that all ECSA values are larger than the geometric area of the working electrode (3.14 mm²). ³⁷ ${k}^{0}$ of 1 mM L-Au/LIG calculated from Fig. S6B is 0.00447 cm s⁻¹ compared with 0.00245 cm s⁻¹ of bare LIG, showing almost a 2-fold enhancement, and hence faster kinetics towards PYO oxidation. In addition to ECSA analysis, we performed Brunauer-Emmett-Teller (BET) measurements to estimate the surface area of the electrodes. As shown in the Table S1, 1 mM L-Au/LIG yields the highest surface area (259.3 m²/g) compared to LIG and other concentrations of L-Au/LIG. The ECSA and BET results consistently show that 1 mM yields the highest surface area and porosity. The decrease of surface area of the 10 mM and 100 mM L-Au/LIG could be because the increased loading of gold creates a film-like layer and reduces the porosity.

We also studied the effect of the position of the laser beam focal plane, as previous research has shown that the defocus setting could potentially affect the sensor sensitivity. ³⁸ Fig. S7A shows the calibration curves of 1 mM L-Au/LIG printed with the Z-axis height changed from 20 to 80 mils. The sensitivity calculated in Fig. S7B suggests that the defocus length does not have a significant impact on sensitivity, noting that the height of LIG on the glass slide is 45 mils, and the highest sensitivity is also obtained within 40 ∼ 50 mils.

After optimizing the sensing material as described in previous sections, we moved forward to the real time studies with P. aeruginosa cells. The colony biofilms have been reported as a model to study the biofilms by Shepard's group and other researchers. ^6,7,39 To form the colony biofilm on agar, the LB agar (thickness of t = 2 mm and diameter of d = 6 mm) is inoculated with 5 μl suspension of P. aeruginosa (strain PA14), diluted at 1:100 from the overnight culture, which yields 2.8 × 10⁷ CFU/ml. The number of seeded cells on agar is estimated to be 1.4 × 10⁵ CFU. Fig. 4A shows time-evolution of the SWV waveforms with no antibiotics. There are three peaks in the SWV curves: the first oxidation peak around −0.3 V is associated with PYO, while the second and third are respectively associated with 5-MCA (5-methylphenazine-1-carboxylic acid) and PQS (2-heptyl-3-hydroxy-4-quinolone). ^6,31,40 The peaks are first noticed after 12 h and kept increasing within 48 h and then decayed over time. It should be noted that the peak current is higher than our previous work with electrodeposited MoS_x-LIG, and hence, shows higher sensitivity of L-Au/LIG to phenazines. ³¹ To further confirm the presence of PYO in PA14 cultures, we performed Liquid chromatography – Mass Spectrometry (LC-MS). Figure S8 shows the MS ion images of synthetic PYO in LB and PA14 culture in LB at 18 h after incubation. The ion abundance of m/z = 211.086 indicates the existence of PYO in the 18 h cultures, consistent with previous reports. ^41,42

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In the first set of experiments with antibiotics, we tested PA14 with ampicillin which is added at 50 μg ml⁻¹ to the agar slab. Ampicillin is known to be ineffective in treating P. aeruginosa. ⁴³ Based on the results shown in Fig. 4B, the growth of P. aeruginosa and phenazine production show that such a high concentration of ampicillin (above its breakpoint concentration according to CLSI guidelines) cannot inhibit the growth of P. aeruginosa biofilms. ⁴⁴ Interestingly, PYO level is much higher in the presence of ampicillin compared to the control test. This result suggests that with ampicillin treatment, the PYO is accumulated which in turn suggests that PYO production could be correlated to the antibiotic resistance of P. aeruginosa. Previous studies have also shown that PYO level goes up during antibiotic treatment. ⁴⁵ On the other hand, while PYO peak increases in the presence of ampicillin, the other two peaks are reduced compared to the control test (in particular, the third peak is almost completely diminished). This behavior demonstrates the existence of complex chemical signaling pathways in biofilms and importance of phenazines in developing antibiotic resistance.

After studying antibiotic resistant cells, we investigated the response of PA14 to gentamicin to which P. aeruginosa is susceptible. In particular, we investigated the effect of gentamicin on PA14 colony biofilms through two important studies, i.e., adding antibiotics to the agar slab from the start when the cells are still in planktonic (free-swimming) phase and adding antibiotics on mature biofilm (after 48 h). We first confirmed the susceptibility of PA14 to gentamicin by determining the minimum inhibitory concentration (MIC) in broth via the broth microdilution (BMD) method. ⁴⁶ The optical density data is shown in Fig. 5A, confirming a MIC value of 0.5 μg ml⁻¹ which is below the breakpoint concentration (hence, confirming the susceptibility of PA14 to gentamicin). In addition to broth tests, different dosages of gentamicin were mixed with agar, followed by inoculation of PA14 to determine the MIC on agar. As the results in Figs. 5B–5E suggest, gentamicin MIC in agar is 1 μg ml⁻¹, twice that in broth. Similar to ampicillin experiments, the PYO level in the subinhibitory concentration groups (Figs. 5B and 5C) increases significantly over time compared to control (Fig. 4A), which implies that PYO promotes antibiotic tolerance in P. aeruginosa. The role of phenazines in antibiotic resistance development is two-fold: (1) Phenazines regulate biofilm formation, in which cells are surrounded by an extracellular polymeric substances (EPS) matrix, which reduces the effect of antibiotics. ¹⁷ Secondly, pyocyanin upregulates the efflux pump to remove the toxicity from the periplasm and cytoplasm. ⁴⁷ It should be noted that to ensure those peak changes are not because of possible redox activity of the antibiotics, we tested E. coli K12 with gentamicin and ampicillin (Fig. S9). We also performed SWV with 50 μg ml⁻¹ ampicillin and gentamicin spiked in BHI to further confirm that the antibiotics themselves are not redox-active. As can be seen, there are no significant redox peaks, confirming that the antibiotics on their own do not contribute to the redox peaks observed with PA cells.

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Previous studies have shown that MIC will increase up to 1000-fold when P. aeruginosa forms biofilm. ¹² To better understand the production dynamics of PYO when mature biofilms are treated with antibiotics, we cultured PA14 cells on LB agar for 48 h to form biofilm, followed by adding gentamicin solution on top of the agar. Figures 6A–6D compare a series of gentamicin treatments ranging from 1–100 μg ml⁻¹. ${t}_{0}^{* }$ represents the time when gentamicin is added (corresponding to t₄₈ in experiments in Fig. 5). The PYO peak (the first SWV oxidation peak) is normalized to its value at ${t}_{0}^{* }$ and plotted in Fig. 6E to demonstrate the PYO dynamic and production rate among different doses of gentamicin. The results show that the PYO level with 1–10 μg ml⁻¹ gentamicin is significantly higher than the control group, highlighting the PYO-mediated antibiotic tolerance. On the other hand, the PYO level with 50 and 100 μg ml⁻¹ gentamicin is lower than the untreated group, suggesting that the cell population started to decrease with the increase of the gentamicin dosage.

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To better understand the relation between PYO level and bacterial population, we monitored the number of cells using the colony counting method. Two strains of P. aeruginosa were studied: PA14 and ATCC 9027, which according to our previous work, is a defective mutant and does not produce PYO on LB agar. ³¹ At different time points, LB agar with bacteria was dropped into 1 ml PBS, followed by ultrasonication to dislodge cells from agar. Fig. S10A and S10B compare the population of cells without gentamicin (control) and the biofilm treated with 1 μg ml⁻¹ gentamicin for PA14 and ATCC 9027, respectively. The samples were first incubated for 48 h to form biofilms, followed by the addition of 1 μg ml⁻¹ gentamicin. The results show that in both control and antibiotic-treated cells, the population of ATCC 9027 strain is lower than PA14. Consistent with our prior report, ATCC 9027 does not show PYO peak over time (see Fig. S11) and there was also no visible biofilm formed on agar, suggesting this strain is more susceptible to antibiotics compared with PA14. The higher antibiotic susceptibility of the PYO-lacking strain compared to PA14 is consistent with other works which report phenazines to promote antibiotic tolerance in P. aeruginosa. ^20,48,49 The sensor results show a higher antibiotic tolerance of PA14 compared to the PYO-lacking strain as well as the increased antibiotic tolerance of biofilms compared to planktonic cells. These results are consistent with prior studies where they showed that the production of PYO promotes antibiotic resistance by regulating biofilm formation and/or upregulating pathways for efflux pumps to prevent drugs from reaching the cells. ^17,47

Polyimide (PI) sheets are purchased from American Durafilm Co., Inc. (Kapton^® HN, 500 mils). Pyocyanin (CAS: 85-66-5), gold (III) chloride solution (CAS: 16903-35-8), L-ascorbic acid (CAS: 50-81-7), sodium bisulfite (CAS: 7631-90-5), sodium thiosulfate (CAS: 7772-98-7), ampicillin (CAS: 7177-48-2), gentamicin (CAS: 1405-41-0), brain-heart infusion powder, and Lennox L agar are purchased from Sigma Aldrich. Phenazine-1-carboxylic acid (CAS: 2538-68-3) is purchased from Apollo Scientific. Nafion D-521 dispersion and the anhydrous ethanol (64-17-5) are purchased from VWR. Dragon Skin™ silicone rubber is bought from SmoothOn, Inc.

The LIG electrode pattern is designed in AutoCAD^®, and then manufactured by the laser engraving machine (VSL2.30, Universal Laser Systems) using the processing parameter we previously reported. To create L-Au/LIG, after printing the LIG working electrode (WE), 5 μl of HAuCl₄ solution is dropped on it and left for 10 min to dry. Then the gold nanoparticles are formed by an additional laser scribing step while the counter electrode (CE) and reference electrode (RE) are also being written/patterned.

For E-Au/LIG, the printed 3-electrode LIG is immersed in 0.6 wt% HAuCl₄ in ethanol for 30 min, and then ascorbic acid is added to get to a final concentration of 1 mM, followed by an additional room temperature incubation for 1 h. The sensor area is separated from the contact pads using Dragon Skin silicone rubber to prevent liquid diffusion toward the contact pads. The RE is electrodeposited with silver on LIG with a constant current density of −150 μA/mm² for 5 min in a solution containing 250 mM silver nitrate, 750 mM sodium thiosulfate, and 500 mM sodium bisulfite. The device is then triple-washed in ultrapure deionized (DI) water and dried. Finally, 5 μl of 2% Nafion in ethanol is dropped on the sensor with 30 s of spin-coating at 3000 rpm. The sensors are kept in a N₂-filled desiccator prior to tests.

Scanning electron microscopy (SEM) micrographs are taken using a ThermoFisher Q250 instrument. Energy dispersive X-ray spectroscopy (EDS) measurements are taken using a 15 kV beam to ensure all relevant elements are detected. X-ray photoelectron spectroscopy (XPS) measurements are performed using a Physical Electronics VersaProbe II instrument with an Al Kα X-ray source (1.49 keV). Charge calibration is done using the sp² carbon peak at 285 eV. Spectra are processed and analyzed with the CasaXPS software, and the program's instrumental relative sensitivity factors are used to calculate atomic percentages.

The LIG powder are scratched from LIG films to perform the XRD and BET measurements. For XRD tests, the samples powders are loaded into a silicon holder, and diffraction data are collected from 10 to 70° 2θ using a Malvern Panalytical Empyrean^® diffractometer fitted with a copper (Kα1–2 = 1.540598/1.544426 Å) long-fine-focus tube operated at 45 kV and 40 mA. The incident beam path included iCore^® optics fitted with a BBHD^® optic with 0.03 radian Soller slits, a 14 mm primary beam mask, a 6 mm secondary beam mask, and a fixed 0.25° divergence slit. The diffracted beam path incorporated dCore^® optics with a 0.25° fixed anti-scatter slit and 0.04 radian Soller slits. A PIXcel^® detector is used in scanning line (1D) mode with an active length of 3.347°. Data is collected with a nominal step size of 0.026°.

The surface area measurement using the BET method is performed on a Tristar II Plus surface area/porosity analyzer (Micromeritics, GA, USA). Samples are first degassed on a Smart VacPrep (Micromeritics, GA, USA) degassing unit. The secondary degassing step is at 100 °C for 300 min. Samples are put on the instrument for an 18-point BET-specific surface area test using nitrogen as the absorbent. Equilibrium time was set at 20 s per point.

Stock solutions (2 mM PYO and 500 μM PCA) are first prepared in ethanol, then serially diluted to 100 nM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM in BHI every time before the measurements. Electrochemical data is measured using a multi-channel potentiostat. (MultiPalmSens4, PalmSens) The sensor is attached to a Zero Insertion Force (ZIF) socket and connected to the potentiostat with Dupont wires. To benchmark the sensor, 100 μl solution containing the synthetic PYO or PCA is dropped on the sensors. SWV measurements are performed with a frequency of 15 Hz and potential range from −0.6 to 0 V after 3 min of holding (to reach equilibrium after depositing the solution on the sensor). Between different testing solutions, the samples are cleaned by pipetting a drop of deionized water, allowing it to sit for at least 10 s, then removed with a clean Kimwipe tissue.

The LC-MS data was acquired on a Thermo QExactive mass spectrometer with a Thermo Vanquish LC system. Mobile phases A and B were water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively. The LC gradient was 0%–6% B in 5 min, followed by 7 min of high-organic wash and re-equilibration at 0% B. The flow rate was 0.3 ml min⁻¹. The column was a Waters Acquity UPLC CSH C18 1.7 μm (micrometers), 2.1 × 100 mm. The column was maintained at 45 °C. The mass spectrometer acquired the data in an alternating polarity mode over 100–500 m z⁻¹ with a resolving power of 70,000, AGC target of 10⁶, and a maximum ion accumulation time of 200 ms for each polarity.

P. aeruginosa strain PA14 (BEI Resources, NR-50573) and ATCC 9027 are cultured from frozen stock at −80 °C with lysogeny broth (LB, Lennox). The initial cultures are then streaked on LB agar and incubated to obtain single colonies. The overnight cultures are prepared by resuspending a single colony from the sub-streaked plate in LB and cultured overnight at 37 °C.

Next, cells from the overnight culture (∼1.4 × 10⁹ CFU/ml) are inoculated/seeded on an LB agar slab (diameter: 6 mm and thickness: 2 mm, punched using a sterile tissue puncher, Ted Pella Inc.) and placed on the sensor. The initial cell seeding density is 5,000 CFU/mm². The sensors with P. aeruginosa-seeded agar slabs are then placed in a custom-made incubator (37 °C, 90% humidity). The electrochemical measurements are performed using a script to control MultiPalmSens4 at specific time points after starting the inoculation. For studies with antibiotics added at the planktonic stage, LB agar is mixed with antibiotics (with the desired concentration as noted in the Results and Discussion Section) after sterilization in an autoclave while the liquid is still warm enough (before agar solidifies). For studies with antibiotics added at the biofilm stage, after ∼48 h of incubation of the seeded cells (on antibiotic-free LB agar) in the humidity chamber, 5 μl of antibiotics with the desired concentration is dropped over the biofilm and monitoring continues.

To estimate the population of the colonies in Fig. S8, agar samples are prepared following the same method discussed for the electrochemical measurements. At different times, the agar sample is transferred into a microcentrifuge tube containing 1 ml PBS. After vertexing and ultrasonicating for 2 min, the suspension is diluted 10-fold and 100 μl suspension is inoculated on LB agar. Inoculated plates are then incubated at 37 °C for 16–20 h and colonies are counted to determine the CFU/ml at each time point. All experiments were completed using three replicates and average values are plotted. For example, at ${t}_{0},$ 1:1000 dilution of PA14 agar slab yields 12.3 colonies on average, the final cell population is $12.3\times 10\times 1000=1.23\times {10}^{5}.$

The L-Au/LIG and E-Au/LIG samples are first patterned into squares of 10 mm x 10 mm, followed by coating with Nafion. To estimate the impact of Nafion, uncoated samples are also prepared. All samples are attached with double-sided adhesive tape to a sterile glass slide and put inside a 6-well plate. After sterilization with UV irradiation for 30 min, 25 μl of 1:10 diluted overnight culture is added to each sample. After 2 h and 4 h, the samples are transferred to a vial containing 1 ml PBS and sonicated for 1 min to transfer cells to the solution. Then the solution is diluted and spread on LB agar plates (10 cm diameter), followed by overnight incubation at 37 °C in order to quantify viable colonies.