Sensors and Actuators B 158 (2011) 366–371 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical j o ur nal homep a ge: www.elsevier.com/locate/snb Bacterial biofilm-based water toxicity sensor Hadar Ben-Yoav a,∗ , Tal Amzel a , Alva Biran b , Marek Sternheim c , Shimshon Belkin b , Amihay Freeman d , Yosi Shacham-Diamand a a Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel-Aviv 69978, Israel b Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel c The Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel-Aviv 69978, Israel d Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Tel-Aviv 69978, Israel a r t i c l e i n f o Article history: Received 25 April 2011 Received in revised form 3 June 2011 Accepted 7 June 2011 Available online 14 June 2011 Keywords: Whole-cell biosensor Biochip Microbial biofilm Toxicity bioassay Bioelectrochemistry Alkaline phosphatase a b s t r a c t Cell-based toxicity bioassays harbor the potential for efficient detection and monitoring of hazardous materials. However, their use in the field has been limited by harsh and unstable environmental condi- tions that shorten shelf-life, introduce significant noise, and reduce the signal and signal-to-noise ratio; such conditions may thus decrease the probability of correct decisions, increasing both false positive and false negative outcomes. Therefore, there is a need for a stable cell-on-chip integration that offers long- term storage and resilience to environmental factors. The use of intact microbial biofilms as biological elements in a whole-cell biosensor, and their integration into specialized biochips, holds promise for enhancing sensor stability as well as providing an innovative platform for biofilm research. We report here for the first time on the integration of a bacterial biofilm as the sensing element of a whole-cell biosensor, as a means to stabilize and preserve reproducibility, viability and functionality of the bacte- rial sensor cells. We have employed a genetically engineered Escherichia coli sensor strain, tailored to respond to the presence of genotoxic (DNA damaging) agents by the induction of a reporter enzyme, alkaline phosphatase, and tested its functionality in colorimetric and electrochemical assays. Three dif- ferent bacterial integration forms were examined: planktonic cells, electronically deposited sessile cells, and biofilms. For all integration forms, a clear dose-dependent positive response to the presence of the model toxicant nalidixic acid was observed, with biofilms displaying higher current density and detec- tion sensitivity than planktonic and sessile cells. We present the electrode apparatus and methods and biochip characterization of such chips, e.g. signal vs. time and induction factor, and discuss the advantage and potential problems of the new biofilm-biochip technology. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Integration of diverse chemical and biological processes onto a microchip, often referred to as a micro total analysis system (micro- TAS) or “lab on a chip”, is currently generating major interest due to the potential for functional integration with other technolo- gies and miniaturization, leading to portability, high throughput usage, and low cost mass production. Over the last few years, microchips’ footprint decreased and their complexity increased, due to advances in micro- and nano-fabrication and cross dis- ciplinary micro and nano system technology. Biochips integrate diverse biological components on a chip; in a subset of this field, microbial cells are integrated into micro-environmental systems and their reactions to the tested samples are monitored on-chip [1–4]. In these bio-micro-electro-mechanical-systems (bio-MEMS) ∗ Corresponding author. Tel.: +972 3 6406827, fax: +972 3 6423508. E-mail addresses: benyoav@post.tau.ac.il, benyoav@umd.edu (H. Ben-Yoav). sensors, live microbial cells convert a chemical, physical or a bio- logical signal into an electrical one [5]. Microbial sensors can be genetically engineered [6] to detect very complex series of reac- tions that can exist only in an intact, functioning cell [7]. Microbial biosensors have been proposed for usage in diverse applications including monitoring glucose [8], microbial growth rate [9] and response to biocides [10]. They are also used for environmental monitoring including the detection of toxicity, genotoxicity, and the presence of specific groups of chemicals [11–15]. Adaptation of whole-cell biochip technology into a field-compatible format would provide real-time information about contamination sources and allow field use and on-site analysis. It will also reduce the costs involved in sample transfer and maintenance, thus signifi- cantly reducing the overall response time. Towards this aim, we propose to integrate the sensor cells into the hardware platform in the form of a permanent biofilm, generating an environmentally stable whole-cell biochip. As the development of technologies for the construction of low profile biochips is a relatively new concept, it should be thoroughly 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.06.037