Citation: Zamzami, M.; Alamoudi, S.; Ahmad, A.; Choudhry, H.; Khan, M.I.; Hosawi, S.; Rabbani. G.; Shalaan, E.-S.; Arkook, B. Direct Identification of Label-Free Gram-Negative Bacteria with Bioreceptor-Free Concentric Interdigitated Electrodes. Biosensors 2023, 13, 179. https:// doi.org/10.3390/bios13020179 Received: 22 November 2022 Revised: 5 January 2023 Accepted: 17 January 2023 Published: 23 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biosensors Article Direct Identification of Label-Free Gram-Negative Bacteria with Bioreceptor-Free Concentric Interdigitated Electrodes Mazin Zamzami 1,2, * , Samer Alamoudi 1 , Abrar Ahmad 1 , Hani Choudhry 1,2 , Mohammad Imran Khan 1,2 , Salman Hosawi 1,2 , Gulam Rabbani 3 , El-Sayed Shalaan 4 and Bassim Arkook 4,5 * 1 Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia 2 Center for Artificial Intelligence in Precision Medicines, King Abdulaziz University, Jeddah 21589, Saudi Arabia 3 Department of Medical Biotechnology, Yeungnam University, 280 Daehak-ro, Gyeongsan, Gyeongbuk 38541, Republic of Korea 4 Department of Physics, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia 5 Department of Physics and Astronomy, University of California, Riverside, CA 92521, USA * Correspondence: mzamzami@kau.edu.sa (M.Z.); barkook@kau.edu.sa (B.A.) Abstract: This investigation demonstrates an electrochemical method for directly identifying un- labeled Gram-negative bacteria without other additives or labeling agents. After incubation, the bacterial cell surface is linked to the interdigitated electrode through electroadsorption. Next, these cells are exposed to a potential difference between the two electrodes. The design geometry of an elec- trode has a significant effect on the electrochemical detection of Gram-negative bacteria. Therefore, electrode design geometry is a crucial factor that needs to be considered when designing electrodes for electrochemical sensing. They provide the area for the reaction and are responsible for transferring electrons from one electrode to another. This work aims to study the available design in the commer- cial market to determine the most suitable electrode geometry with a high detection sensitivity that can be used to identify and quantify bacterial cells in normal saline solutions. To work on detecting bacterial cells without the biorecognition element, we have to consider the microelectrode’s design, which makes it very susceptible to bacteria size. The concentration–dilution technique measures the effect of the concentration on label-free Gram-negative bacteria in a normal saline solution without needing bio-recognized elements for a fast screening evaluation. This method’s limit of detection (LOD) cannot measure concentrations less than 10 2 CFU/mL and cannot distinguish between live and dead cells. Nevertheless, this approach exhibited excellent detection performance under optimal experimental conditions and took only a few hours. Keywords: electrochemical biosensors; bioreceptor-free; biorecognition-element-free; concentric interdigitated electrodes; label-free; Gram-negative bacteria; Escherichia coli 1. Introduction Pathogens are organisms that cause disease in humans. They threaten human health, and the number of these pathogens is expanding [1]. The main types of pathogens are bacteria, viruses, and parasites. Bacteria and viruses are present on all surfaces and in the air. Therefore, they can easily be transferred to the hands of an individual who touches an infected surface or object. In that case, they have a good chance of contracting the infection themselves—one of the most typical pathogens is Escherichia coli (E. coli)[2–4]. Therefore, rapid and sensitive detection of bacterial pathogens is a strong demand and is needed for today’s global food market and healthy communities. Furthermore, the required technology can be adjusted for various target organisms. The traditional techniques used to quantify pathogens in cell culture are time-consuming and require skilled operators to perform the sample preparations. In addition, the operator must be well-trained, experienced, and knowledgeable [5–7]. Pathogens can be recognized Biosensors 2023, 13, 179. https://doi.org/10.3390/bios13020179 https://www.mdpi.com/journal/biosensors