Electrochemical Sensors Based on Graphene and Nanobrush Structures for Point of Use Detection of Foodborne Pathogens

Abstract

Recurrent foodborne pathogen outbreaks result in significant economic and public health burden. Thus, there is a critical need for the development of real-time and reliable detection methods to prevent disease outbreaks and replace current highly technical and time-consuming methods. This study aimed to develop different electrochemical biosensor platforms with enhanced limit of detection (LOD) and sensitivity for quantification of bacteria, Listeria monocytogenes and Salmonella enterica serovar Typhimurium, two of the most common foodborne bacteria. Two sensing platforms included stimuli-responsive polymer nanobrushes embedded with platinum nanoparticles fabricated using a simultaneous one-step sonoelectrodeposition method and subsequently functionalized with aptamers as biorecognition agents. Two pH-sensitive polymers, chitosan and alginate, were tested as well as different polymers preparation, grafting parameters, polymer response to pH changes (actuation) and aptamer concentrations. The optimized electrodeposition of chitosan/platinum (CHI/Pt) onto electrodes increased (p < 0.05) the average electroactive surface area (ESA) by 11 times compared to the bare electrode, while the alginate-thiomer/platinum (ALG-thiomer/Pt) deposition increased (p < 0.05) ESA by 7 times. Actuation protocol for the polymer brushes, i.e., bacteria capture with brushes on extended conformation and electrochemical signal transduction on collapsed state, led to the improved sensing performance for the detection of L. monocytogenes with a low LOD in chicken broth of 3.3 ± 0.9 CFU/mL and 4.4 ± 0.8 CFU/mL for the CHI/Pt-aptamer and ALG-thiomer/Pt- aptamer sensors, respectively. A third sensing platform consisting of laser-induced graphene with nickel oxide nanoparticles (LIG-NiO) was fabricated by a two-step process using polyimide film and nickel acetate solution as substrates. The sensor was functionalized with Salmonella-specific antibodies and its performance investigated towards bacteria sensing in PBS alone and with interferent (Escherichia coli) and in chicken broth. The results showed that the immunosensor was highly specific and sensitive for the detection of Salmonella Typhimurium with a LOD of 1.28 ± 0.21 CFU/mL in chicken broth. All the three biosensors tested had a detection time of approximately 17 minutes (sample exposure plus testing) and a wide detection range from 10 to 10⁶ CFU/mL of the target bacteria, which covers the relevant levels for food safety analysis. In addition to enable bacteria detection in very low levels with a time frame of minutes, the devices studied present other advantages such as easy fabrication and no need for labeling or bacteria concentration.