Reagent-Free Electrochemical Monitoring of Bacterial Viability Using Novel Organic Crystals Deposited on Flexible Substrates
Authors: Bolotsky Adam , Muralidharan Ritvik , Zhou Keren , Butler Derrick , Root Kayla , Ebrahimi Aida 
Published: December 2020

Monitoring bacterial viability is critical in food safety, water quality, and medical applications [1]–[8]. Specifically, having a portable, low-cost, and rapid analytical tool for bacterial viability is highly desirable in academic research, healthcare, and industrial sectors. There are various methods for probing bacterial viability [7], [9], but they usually possess drawbacks in relation to point-of-care/portable application for timely diagnosis. One of the gold standard methods is colony counting on agar plates. Despite being highly sensitive, low-cost, and simple, colony counting is quite slow, requiring at least 18-24 hours incubation [7]. Another method for monitoring viability uses polymerase chain reaction to measure the DNA of cells with normal cell-wall integrity [7]. The procedure is fast (< 1 hour [10]), but unintended biases can be introduced by intact non-viable cells and/or incomplete neutralization of exposed DNA [11]. Moreover, it requires fluorescence measurement equipment and skilled personnel, and uses expensive reagents. There are also colorimetric and fluorescent viability methods that measure change of the redox state of a reagent (such as resazurin) in response to cell metabolic activity [12]–[14]. The drawback of these methods is the need to utilize reagents that may interfere with normal bacterial physiology [14], possible interactions with dissolved compounds, as well as requiring rather bulky/expensive optical equipment for readout.
In this work, we have developed low-cost, portable electrochemical sensors for in situ detection of bacterial viability without using any external redox reagent. The sensors enable monitoring bacterial metabolic activity by utilizing novel redox-active organic crystals, termed as RZx, which are deposited using a facile, environmental- and user-friendly process. We had previously demonstrated deposition of RZx on pyrolytic graphite sheets for testing the effect of the antibiotic ampicillin on growth of E. coli [15]. In this Abstract, we demonstrate that the process is scalable to deposit RZx on printed electrodes on plastic or paper substrates. Hence, flexible sensors with desirable designs can be developed for detection of various bacterial pathogens. RZx was successfully deposited on laser induced graphene (LIG) as well as screen-printed carbon electrodes on paper. LIG was fabricated directly on polyimide sheets using a 25 W desktop laser-writing system (Universal Laser system, VersaLASER VLS 2.30, 10.6 μm wavelength) by adopting the process reported elsewhere [16]. Laser power (peak power), speed, and pulse per inch (ppi) were optimized to 26%, 29%, and 1000, respectively. A schematic of LIG and RZx synthesis processes are presented in Figure 1a and 1b, respectively. Figure 1c depicts a scanning electron microscope (SEM) image of RZx on LIG; the inset shows a magnified view of an RZx crystal. The high surface coverage of the RZx crystals enhances the faradaic current used in analysis of the sensor signal. The final step in device fabrication is to spin-coat a biocompatible Nafion layer onto the RZx/LIG to minimize drift of the sensor response.
Once the sensor fabrication is complete, bacterial suspension with varying cell concentration – confirmed using optical density measurement – is deposited on the sensor surface, followed by performing cyclic voltammetry. A characteristic oxidation peak appears at , as depicted Figure 1d. As cells metabolize, shifts to more positive values. Figure 1e plots the time-resolved differential signal with respect to the initial time for viable and dead E. coli K-12 cells spiked in whole blood. As illustrated, the sensor is able to monitor viability in real time. In addition, we measured the sensor response to different initial E. coli concentrations, as shown in Figure 1f for 106 and 107 CFU/mL. Finally, to demonstrate versatility of the RZx deposition on other carbon-based electrodes, we utilized screen-printed carbon electrodes on paper. The sensors consist of printed counter electrode and AgCl paste as reference electrode (inset of Figure 1g). The data measured using the on-chip paper-based sensors is shown in Figure 1g, demonstrating a similar response to LIG-based sensors in Figure 1e (which were measured using off-chip Pt counter electrode and Ag/AgCl glass reference electrode).
The sensors successfully enabled reagent-free electrochemical monitoring of viability of both E. coli and Salmonella as model species. The flexibility, portability, and compactness of the as-fabricated sensors (on LIG or screen-printed carbon electrodes) lends itself to broader applications, such as use in smart-catheters that can detect bacterial infection early on, or portable probes for monitoring food or water quality in the field.

Figure 1