A Flexible Electrochemical Biosensor Based on Modified Laser-Induced Graphene for Selective Detection of Phenazines
Authors: Zhou Keren , Butler Derrick , Yang Jian , Ebrahimi Aida 
Published: July 2021

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) is the main cause of various acute infections, especially in patients with severely burned and chronic infections. Moreover, P. aeruginosa can be found in lungs of patients with cystic fibrosis and in implanted devices.1 Biofilm of P. aeruginosa usually shows increased tolerance to antimicrobial agents.2,3 Among main virulence factors secreted by P. aeruginosa are pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA) which belong to a group of quorum sensing molecules called phenazines. These phenazines also play an important role in the formation and growth of P. aeruginosa biofilms. As specific biomarkers of P. aeruginosa, detection of PYO and PCA can enable diagnosis of P. aeruginosa infection. Existing methods for detection of phenazines include high performance liquid chromatography-mass spectrometry (HPLC-MS),4 fluorescence measurements5, and electrochemical methods.6–8 But bulky and expensive instruments and the complex sample preparation process limit the broad application of the MS method. In addition, neither MS and fluorescence detection method can achieve in-situ monitoring of biofilm formation and growth. Compared to these methods, electrochemical sensors are low-cost, user-friendly, and compact. They also show the potential for in vivo monitoring.9

In this work, we have developed an all-in-one electrochemical sensor for selective identification of PYO and PCA down to 1 µM and 10 µM respectively in bacterial culture medium (brain heart infusion, BHI) using square wave voltammetry (SWV). The sensor working electrode (WE) is comprised of platinum (Pt) nanocrystals electrodeposited on laser-induced graphene (LIG). The counter (CE) and pseudo-reference electrode (RE) are based on LIG and electrodeposited silver (Ag) on LIG, respectively. We studied the effect of various electrodeposition conditions to optimize the material stability and sensor response. The fabrication process is facile, scalable, and user-friendly. The schematic of the fabrication process is shown in Figure 1a. The inset shows a picture of a fabricated sensor on polyimide. Figure 1b and Figure 1c show the SWV results for different concentrations of PYO and PCA in BHI. The oxidation peak for PYO and PCA are located at -0.15 V and -0.25 V, respectively, which enable selective identification of these phenazines. We studied the effect of SWV frequency on the sensor signal and found that 15 Hz and 10 Hz are the optimum frequency with the highest ratio of peak current to the baseline current for PYO and PCA, respectively. Compared to other electrochemical sensors based on gold7,10 or carbon,11 our sensor is flexible and integrates all three electrodes in a compact and scalable design.
In conclusion, we successfully developed a printable flexible sensor based on LIG, modified using electrodeposition of platinum and silver to create a portable and compact three-electrode electrochemical device for selective detection of two phenazines (PYO and PCA) directly in bacterial culture broth. A limit of detection of 1 µM was achieved for PYO and 10 µM for PCA. To achieve a high sensitivity, the SWV frequency was optimized. The ability to identify both phenazines can advance our knowledge on how the interplay between these molecules affect biofilm phenotypes. Future works include improving the sensitivity through electrode engineering, growing P. aeruginosa biofilms on the sensors, and in situ monitoring of the pyocyanin level during biofilm growth. The flexibility of this sensor also promises its application in wearable technology or medical devices (e.g. for smart catheters, smart wound dressings, etc.).

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Figure 1