Toward Rapid Antibacterial Susceptibility Testing Using Electrochemical Biosensors Based on Organic-Inorganic Catalytic Complexes

Abstract

Over the past several decades, the evolution of antimicrobial resistance (AMR) in pathogenic bacteria has increasingly posed a serious risk to public health and safety.1 As such, it is important for clinicians and researchers to have access to low-cost, simple-to-use, and timely assessment of whether a specific antibiotic can inhibit bacteria or not (i.e. performing antibacterial susceptibility testing (AST)).1 Currently, clinical AST technologies rely on broth dilution (followed by measuring density of optical absorbance, OD) or the disk-diffusion methods which require at least 18-24 hours before the minimum inhibitory concentration (MIC) of a particular antibiotic can be determined.2 Toward rapid AST, phenotypic assays (based on cell metabolism, size, shape, or motion) are advantageous over gene-based techniques due to addressing a more clinically-relevant question of whether a particular antibiotic affects cell viability or not.3 However, the reported rapid phenotypic ASTs usually suffer from low sensitivity, require advanced/expensive tools, and/or utilize high concentration of indicator agents which can interfere with antibiotics and cell biology.3 Here, we report the development of scalable biosensors based on highly stable, eco-friendly catalytic crystals that are electrochemically synthesized at room temperature and at neutral pH (schematically shown in Fig. 1a). The redox-active catalytic layer (RZx) is synthesized using electrochemical reduction of resazurin molecules in an organic medium (such as Brain Heart Infusion, BHI, a commonly used bacterial culture medium). To improve material stability, the electrodes are coated with an ultrathin layer of ion-permeable polymer (Nafion®). The optical and scanning electron microscopy (SEM) images shown in Fig. 1b confirm the formation of dense films on pyrolytic graphite sheets (PGS; a flexible substrate). Characterization of elemental composition using X-ray Photoelectron Spectroscopy (XPS) indicate that the synthesized crystals are organic and are composed of resazurin and/or its reduction products. The developed redox-active electrode acts as the working electrode (WE) in a three-electrode electrochemical measurement setup. Cyclic voltammetry (CV) is utilized to study the effect of ampicillin on E. coli K12. When bacterial cells metabolize (e.g. due to inadequate antibiotic concentration, ρAnt , or resistance of cells to the treatment), they can reduce their environment and donate electrons to the RZx electrode. As such, the oxidation potential measured using CV method depends on the viability state of bacterial cells, which itself is affected by ρAnt and antibiotic incubation time, tAnt. Fig. 1c plots time-evolution of the differential oxidation potential (ΔV) over a course of two hours following addition of ampicillin at various concentrations. Our results suggest that there is a threshold of ~ ΔVt h = 50 mV for the treatment to be bactericidal, i.e. if within the detection time (~ 60 minutes) the signal is still below ΔVt h, the cells are susceptible to treatment. Susceptibility results and MIC values are verified with OD measurements at 600 nm (Fig. 1d). Importantly, while OD measurements can only identify MIC by including longer incubation times (i.e. overnight data), the RZx-based electrochemical sensor is able to identify MIC within 60 minutes. The operation principles of the proposed rapid AST technique can be applied to screening various antibiotics beyond ampicillin. The developed catalytic electrodes provide time-resolved quantitative antibiotic profiling, require minimal sample preparation and small sample volume, and do not require labeling or redox indicators suspended in the culture medium. Being an electrochemical sensor, the device is compatible with integrated circuit (IC) technology and can be interfaced with wireless communication chips to realize fast, portable, and cost-effective AST platforms with small footprint which are highly desired for resource-limited regions.