Design of Organic-Inorganic Hybrids and Application to Electrochemical Sensor for Detecting Food Poisoning Bacteria
Authors: Itagaki Satohiro , Tsuda Shintaro , Yamamoto Yojiro , Sadanaga Yasuhiro , Shiigi Hiroshi 
Published: December 2024

Pathogenic Escherichia coli, Salmonella enterica, and Staphylococcus aureus are well-known as causative organisms of food poisoning. The ingestion of food and drinking water contaminated with these pathogens causes various symptoms such as diarrhea, abdominal pain, and vomiting, and in severe cases, hemolytic uremic disease. Current microbiological testing methods such as the polymerase chain reaction, enzyme-linked immunosorbent assay, and colony counting require skilled experimentalists and/or a complex and time-intensive culture process. In recent years, many studies have reported the development of biosensors that apply novel materials and technologies. Biosensors based on physical or chemical signals, such as optics, bioluminescence, and piezoelectricity, have been developed for the highly sensitive and rapid detection of pathogenic bacteria. Although methods based on these principles achieve highly sensitive and specific detection, they require expensive and bulky detectors, which limits their use in the field. Therefore, in order to prevent health hazards such as food poisoning and infectious diseases, it is necessary to develop new testing methods that can quickly detect food-poisoning bacteria.
Electrochemical biosensors are particularly useful in fields where on-site inspection is required, because of their high sensitivity, rapid measurement, and ease of device miniaturization. Generally, biosensors use electrochemical reporters as labels to obtain electrochemical signals. In particular, metal nanoparticles have unique surface effects and catalytic functionality and are used in biosensor design to improve specificity, sensitivity, and simplicity. However, it is necessary to separate electrochemical cells for different labels or to fabricate a multiarray of electrodes that is selective for bacterial species when multiple bacterial species need to be detected simultaneously.
Previously, we successfully developed organic-inorganic hybrids with a densely assembled structure of gold nanoparticles in a polyaniline matrix (AuNH).1 This fabrication method exploited the fact that the reduction of aurate by aniline and the oxidation of aniline by aurate occurred in the same reaction field, enabling the production of nanometer-scaled hybrids in a single process. Similarly, silver NH (AgNH) and copper NH (CuNH) were synthesized using silver nitrate and copper sulfate as metal sources.2 The hybridization of metal nanoparticles and conductive polymers is expected to endow various functions, such as environmental stability, electrical conductivity, and electrochemical activity.3 We investigated the characteristic electrochemical properties of the hybrids in detail and attempted to use them as electrochemical labels for detecting bacteria.4

The results of cyclic voltammetry revealed that NHs exhibit redox current derived from there constituent materials, either polymers or metal nanoparticles. Also, different NHs showed similar current sensitivities at different potentials in differential pulse voltammetry. This shows that these qualifications and quantifications can be achieved simultaneously. AuNH and CuNH did not interfere with each other in terms of electrochemical signals obtained on the same electrode. Therefore, antibodies were introduced into these NHs to act as electrochemical labels to target specific bacteria. AuNH was modified with anti-E. coli O26 antibody, and CuNH was modified with anti-Staphylococcus aureus antibody. Electrochemical measurements using screen-printed electrodes dry-fixed with NH-labeled bacterial cells enabled the estimation of bacterial species and number within minutes, based on the distinct current response of the labels. The development of this analytical technology will ensure the safety of food and drug production and contribute to the realization of a safe, secure, and comfortable lifestyle.
[1] S. Itagaki, S. Tanabe, H. Ikeda, X. Shan, S. Nishii, Y. Yamamoto, Y. Sadanaga, Z. Chen, H. Shiigi, Analyst, 147, 2355 (2022).
[2] S. Tanabe, S. Itagaki, K. Matsui, S. Nishii, Y. Yamamoto, Y. Sadanaga, H. Shiigi, Anal. Chem., 94, 10984 (2022).
[3] S. Nakamura, A. Nakao, S. Itagaki, K. Matsui, S. Nishii, X. Shan, Y. Yamamoto, Y. Sadanaga, Z. Chen, H. Shiigi, Sensors and Materials, 35, 4761 (2023).
[4] S. Itagaki, A. Nakao, S. Nakamura, M. Fujita, S. Nishii, Y. Yamamoto, Y. Sadanaga, H. Shiigi, Anal. Chem., 96, 3787 (2024).