Rapid fingerprinting of bacterial species using nanocavities created on screen-printed electrodes modified by β-cyclodextrin

Rapid and precise identification of infectious microorganisms is important across a range of applications where microbial contamination can cause serious issues ranging from microbial resistance to corrosion. In this paper a screen-printed, polymeric β-cyclodextrin (β-CD) modified electrode, affording nanocavities for inclusion of the analytes, is shown as a disposable sensor capable of identifying bacteria by their metabolites. Three bacterial species were tested: two from the Pseudomonas genus, Pseudomonas fluorescens (P. fluorescens) and Pseudomonas aeruginosa (P. aeruginosa), and Serratia marcescens (S. marcescens), a member of the family, Enterobacteriaceae. On biofilm formation each species gave distinct, reproducible, redox fingerprints with a detection limit of 4 × 10−8 M. Square wave adsorptive stripping voltammetry (SWAdSV) was used for detection. Scanning electron microscopy (SEM) and cyclic voltammetry (CV) techniques were used to characterize the morphology and electrical conductivity of the modified electrode. In comparison to the bare screen-printed electrode, the modified electrode showed a considerably higher performance and offered an excellent sensitivity along with a relatively fast analysis time.

Characterization of the electrode.The electrochemical characterization of the bare and β-CD/SPCE was performed by CV and EIS using a 5.0 mM [Fe (CN) 6] -3/-4 in PBS.The CV responses bare and modified SPCEs in the potential range from -0.7 to +1 V at a scan rate of 100 mV/s.The presence of the polymer layer affects the electron transfer rate at the electrode surface, leading to changes in the current density and potential range of the redox reaction.The peak current at the modified SPCE in (Figure S2a) is much larger (14 µA) than that at the bare SPCE.This demonstrates that the deposition of Pβ-CD has substantially enhanced the electron transfer rate of SPCE.It is also evident that in the case of β-CD/SPCE, the peak-to-peak separation ( ) decreased from 0.67 V to 0.38 V compared to the case of the bare SPCE.Considering the ∆  small value and large current response of β-CD/SPCE, the electron transfer process is likely quick and Electrochemical impedance spectroscopy (EIS) measurements in a frequency range of 100 KHz to 0.1Hz were used to characterize interface properties of the bare and β-CD/SPCE.The Nyquist plot of electrochemical impedance spectra for bare and β-CD modifies SPCE, and Randle's equivalent circuit model were used to fit the experimental data over the whole frequency range are shown in (Figure S2b).This circuit includes charge transfer resistance (R ct ), diffusion impedance (W), solution resistance (Rs), and constant phase element (CPE) corresponding to the double-layer capacitance   The surface morphologies of the Pβ-CD/SPCE and SPCE were observed by using SEM.(Figure S3a) displays a typical image of the surface of SPCE, which shows a sheet-like structure with a lot of small particles dispersing in between.After the electrodeposition of β-CD polymer, the electrode morphology changed greatly in (Figure S3b).The surface shows rough and porous structure due to the surface coverage and thickness of cyclodextrin molecules on the electrode surface.The image reveal that the β-CD polymer is well adhered to the electrode surface and covered the entire surface area of the electrode.Bacteria can be clearly observed on the bare electrodes (Figure S3c).However, on CD modified electrodes bacteria are not clearly evident (Figure S3d).Previous publications indicate that CD cavities can cause bacteria to be partially included in the cavities [4].This could explain why we could not see the bacteria on the surface of the electrode.

Electrochemical behaviour of 1-OHPHZ, PCA and PYO (phenazine metabolites) on β-CD /SPCE
The dependence of on  in (Figure S4a) for anodic and cathodic 1-OHPHZ peaks corresponded to the respectively.These results suggest that the voltametric response in (Figure 1) comes from the surfaceconfined phenazine molecules, which are included in the cavity of β-CD attached to the surface of the carbon electrode, with a little contribution of the diffusion of the phenazine molecules [5].The dependence of anodic and cathodic peaks on the scan rate was further evaluated in (Figure S4b), where the logarithm of the phenazine peak current was plotted versus the logarithm of scan rate (at scan rates 20-160mV/s), which confirmed the following equations: Log  = 0.99log (  ) -1.05 ( 2 = 0.9992), .
Log  = 0.94log (  ) -0.9 ( 2 = 0.9988) For such a relation, the slop value of 1.0 indicate an ideal surface reaction, while the value of 0.5 indicate the diffusion-controlled process [6].Based on experimental result, the slope value of equations
[3].W is the Warburg impedance, which is related to the diffusion of the reactive species at the surface of the electrodes and is illustrated by the straight line in the Nyquist plots.At high frequency to medium frequency the charge transfer kinetics of the redox species [Fe (CN) 6] -3/-4 at electrode/electrolyte interface (R ct ) is represented by Electronic Supplementary Material (ESI) for Sensors & Diagnostics.This journal is © The Royal Society of Chemistry 2023 the diameter of the semicircle.The smaller R ct , the faster the electron transfer rate, indicating better electrochemical activity of the modified electrode due to the increased surface area of the electrode.

Figure
Figure S2.(a) CV plot and (b) Nyquist diagram of electrochemical impedance spectra for bare SPCE and β-CD modifies SPCE in 5.0 mM [Fe (CN)6]-3/-4.Randles-circuit used for fitting the experimental results (Inset of (b)),where Rs is solution resistance, CPE is constant phase element, R ct is the charge transfer and W is diffusion hindered impedance.

Figure S3 .
Figure S3.SEM image of the (a) bare SPCE, (b) the β-CD modified SPCE.And images of the electrode when placed in the Pseudomonas fluorescence culture after few days, (c) bare electrode (d) β-CD modified SPCE.

Figure S4 .
Figure S4.corresponding relationship of (a) vs.  and (b) vs. log  on β-CD/SPCE for 1-OHPHZ peaks.  log plot was 0.99 which showed that phenazine had strongly adsorbed on the surface of β-CD log   .log /SPCE.

Figure S5 :
Figure S5: the cyclic voltammogram (a), different pulse voltammogram (b) and square wave adsorptive stripping voltammogram (c) before (blue) and after (red) adding 3 µM 1-OHPHZ into PBS.A pre-accumulation time of 120 s was applied prior to all measurements.As the results shows, SWASV shows peak current 5 times larger than DPV.