© Springer International Publishing Switzerland 2015 I. Lacković and D. Vasić (eds.), 6th European Conference of the International Federation for Medical and Biological Engineering, 333 IFMBE Proceedings 45, DOI: 10.1007/978-3-319-11128-5_83 Multiparametric Biosensor for Detection and Monitoring of Bacterial Biofilm Adhesion and Growth Sheila Becerro 1,2 , Jacobo Paredes 1,2 , and Sergio Arana 1,2 1 CIC microGUNE, Goiru Kalea 9, 20500 Arrasate-Mondragon, Spain 2 CEIT-IK4 and Tecnun (University of Navarra), Paseo de Manuel Lardizábal 15, 20018 Donostia-San Sebastián, Spain Abstract— Bacterial biofilms cause numerous problems in a wide variety of sectors as the medical environment, the food and water industry or the naval sector. The detection of the infection is usually made in advanced states when treatment is hampered by the antibiotic resistance of biofilms. During the first phases, there are not evidential indicators that warn about the presence of pathogens. Therefore, it is necessary to find new methods for the early detection of biofilm development so as to improve the efficiency of treatments. For this purpose, this paper focuses on the design and development of interdigitated microelectrode based biosensors that allow the detection of bacterial adhesion since the first steps of biofilm generation through electrochemistry and two supplementary physicochemical parameters: temperature and pH. The designed biosensor has been proved as a suitable tool for biofilm detection since the first steps of development by means of differential pulse voltammetry and temperature measurements. Keywords— interdigitated microelectrode, biofilm, electrochemical detection, temperature sensor, pH monitoring. I. INTRODUCTION Bacterial biofilms are complex and heterogeneous biological structures formed by a single or several species embedded into a polysaccharide extracellular matrix [1]. They are generated after the adhesion of bacteria onto any kind of surface and their subsequent immobilization, growth and reproduction. During the growth phase, bacteria produce extracellular polymeric substances (EPS) that extend developing a complex framework of molecular fibers which supply the system with unique features. In this way, complex and tridimensional communities are formed. Moreover, the extracellular matrix increases the antimicrobial resistance of the adhered microorganisms by blocking the access of antibiotics trough it [2]. Besides, once bacterial colonization completed, single cells are able to scatter from the colony and pervade adjoining regions. Biofilms are able to develop in any surface with a combination of nutrients and humidity. For this reason, bacterial adhesion can be found in metals, plastics, medical implants, human tissues, natural materials over or under the land or in any other imaginable place. Hence, biofilms entail a serious problem in a wide variety of areas. For example, the surface of implanted devices is a common origin of bacterial adhesion and biofilm formation with more than 80% of the diagnosed infections associated to biofilms [3]. In this work, electrochemical techniques have been used as suitable tools for biofilm real-time monitoring. Bacteria cells are composed of a wide range of macromolecules with electrochemical active groups that can react with the free electrons of the surface [4]. By monitoring this behavior with electromechanical techniques, it might be possible to study them and hence, to detect bacterial presence since the first steps. Moreover, temperature and pH can be used so as to obtain additional information. For this purpose, a complex multiparametric chip composed of three different sensors that are able to monitor biofilm growth in real-time has been designed and fabricated. Differential pulse voltammetry, temperature and pH are measured at the same time so as to detect bacterial adhesion and to study the behavior of microorganisms during biofilm formation. II. MATERIALS AND METHODS A. Chemicals and Reagents Electrochemical detection of bacterial biofilms was conducted on the culturing medium Tryptic Soy Broth, TSB (BBL ™ , ref: 211768) enriched with 5% glucose (Dextrose from Difco ™ , ref: 215530). Phospate buffered saline, PBS (0.01 M, pH 7.4) purchased from Sigma-Aldrich (ref: P5368-10PAK), saline 4,5 % solution from Panreac (PA- ACS-ISO ref: 131659) and Brain Heart Infusion, BHI (BactoTM, ref: 237500) were used for microorganisms culture. All solutions and media were prepared with deionized water (Merck Millipore) and media were also sterilized at 121ºC for 1 hour in the autoclave. Biosensors were cleaned with deionized water, ethanol at 99, 5% (Panreac, ref: 131659) and 10% Hellmanex II dissolution (Hellma Analytics, ref: 9-307-010-507).