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ScienceDirect Procedia Engineering 120 (2015) 376 – 379
EUROSENSORS 2015
Development of an automated biosensor for rapid detection and quantification of E. coli in water Jörg Ettenauer*, Karen Zuser, Karlheinz Kellner, Thomas Posnicek, Martin Brandl Danube University Krems, Center for Integrated Sensor Systems, Dr.-Karl-Dorrek Street 30, 3500 Krems, Austria
Abstract Escherichia coli are an indicator organism for fecal contamination and can represent a serious health hazard for humans. Common water quality tests are time consuming routine analysis. Our aim was the development of an automated biosensor for a fast identification and quantification of E. coli contaminations in ground-, surface- and drinking water. We applied an electrochemical assay to detect E. coli using their β-galactosidase activity. First, the enzyme production was stimulated in the cells and further, specifically released through a T2 bacteriophage mediated cell lysis. The enzyme concentration was monitored by potentiometric measurements. By using this methodological approach we were able to specifically detect at least 2 colonyforming units (CFU) of E. coli within 8 hours. The incorporation of this method into an automatic biosensor device would ensure the quality of our water. Furthermore, a continuous monitoring could be achieved and online data transfer ensures fast counteractions in water treatment plants. For future applications we developed a low-cost potentiostat with an easy-to-use PC software for environmental measurements and evaluated it with 3 different devices. ©2015 2015The The Authors. Published by Elsevier Ltd. © Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of EUROSENSORS 2015. Peer-review under responsibility of the organizing committee of EUROSENSORS 2015 Keywords: biosensor; microbe detection; EcoStat; Escherichia coli; potentiostat; quantification; water.
1. Introduction and Methodology Water is life-essential for all organisms on earth. Guaranteeing clean drinking water is a major issue for human health. Escherichia coli are the most prominent representatives of our normal bacterial gut flora. Most strains are
* Corresponding author. Tel.: +43-(0)2732-893-2635; fax: +43-(0)2732-893-4600. E-mail address:
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1877-7058 © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of EUROSENSORS 2015
doi:10.1016/j.proeng.2015.08.643
Jörg Ettenauer et al. / Procedia Engineering 120 (2015) 376 – 379
harmless, whereas some serotypes can cause serious diseases. Therefore, the identification and quantification of fecal contaminations with E. coli are essential. Nowadays, the classical cultivation methodology takes about 3-5 days to identify this indicator organism. The goal of our project was to develop a rapid and sensitive detection method that can be integrated into a standalone biosensor with on-line data transfer. Furthermore, the analysis should be done within one working day and allow a pre-testing and monitoring of water samples. Therefore, we evaluated several methods for their capability of being incorporated into a biosensor that autonomously performs quality measurements. In our studies we used an indirect assay for the detection of E. coli, relying on the studies of Neufeld et al. (2003) [1]. Different concentrations of Escherichia coli (E. coli) ATCC® 11303™ were grown in the presence of isopropylβ-D-thiogalactopyranosid (IPTG) for 4 hours, thus inducing the production of the β-galactosidase enzyme (β-Gal). Afterwards, T2 bacteriophages (ATCC® 11303-B2™; 104 pfu/ml; plaque forming units/ml; in LB medium supplemented with 10 mM MgSO4) were added to the cultures to specifically infect and lyse the E. coli cells within 3h. A filtration step separated the target enzyme from residual cell debris. The filtrate containing β-galactosidase was incubated with its specific substrate p-aminophenyl-β-D-galactopyranoside (β-PAPG) for 40 minutes. β-Gal mediated the cleavage of β-PAPG to PAP (4-aminophenyl). The cleaved product (PAP) was oxidized at a voltage of 220 mV [2] using carbon/graphite paste with Ag/AgCl paste screen-printed electrodes and a potentiostat. The measured currents of cyclic voltammetry (range: 0-500 mV; scan speed: 50 mV/s) were further compared with each other and with the cell counts of the original cell suspensions cultured on LB agar plates. The resulting current was directly proportional to the amount of β-Gal present in the sample and thus to the amount of bacteria [1,2]. Additionally, for investigating the mechanisms and resulting currents of the above described oxidation reaction of β-PAPG to PAP we preliminary built our own potentiostat according to Rowe et al. (2011) [3]. We tried to improve this open-source instrument, named CheapStat [4], and, therefore, we designed a new, low-cost, digitally controlled potentiostat [3]. Furthermore, we compared it with three other instruments (CheapStat [4]; Reference 600™, Gamry Instruments, USA; VersaStat4, Ametek - Princeton Applied Research, USA) by measuring a ferricyanide solution (100 μl of 0.5 mM ferricyanide solution, pH 7.5) with screen-printed carbon/gold electrodes, SPE (Gwent Electronic Materials Ltd, UK). The resulting data were graphically visualized in Microsoft Excel in order to compare the different instruments. 2. Results and Discussion For studying electro-analytical experiments a potentiostat with a three electrode system can be used. In these electronic devices the potential of a working electrode is maintained at a constant level with respect to the reference electrode by balancing the current at an auxiliary or counter electrode. Many professional potentiostats often exceed the practical needs as well as the financial possibilities of the users. Therefore, many laboratories have started to design their own potentiostats [4,5]. We designed the “EcoStat” system to quantify analyte concentrations in environmental samples [3]. This newly developed instrument was especially designed to work with screen-printed electrodes for cyclic voltammetry measurements. In order to investigate the accuracy of our self-built device we compared EcoStat with the open-source CheapStat [4] as well as with two commercially available high-end instruments (VersaStat4 and Reference 600). All possible software filters were switched off in order to compare the measurement data. However, hardware based filters might still have been working. First, we performed some technical experiments with dummy cells [3] and further tested the potentiostat with a ferricyanide solution on SPE. The data were graphically visualized in Microsoft Excel in order to compare the different instruments. The results of the measurements of a ferricyanide solution are shown in Fig. 1. With our self-built EcoStat we were able to obtain the typical duck shape graph with similar current values compared to the other instruments (Fig. 1a). The tree measurements showed little deviations, but again a lowered noise compared to the CheapStat instrument was observed. Generally, the two commercial instruments showed the best results with the lowest variation between the three measurements as well as very low noise disturbances (Fig. 1b).
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a
b
Fig. 1. Cyclic voltammetry graphs showing a ferricyanide solution analysed with the four potentiostats. (a) Triplicate measurements are shown for each used instrument. (b) Mean values with standard deviations of the peaks of the curves of the triplicate measurements from (a).
The chosen methodology for the specific detection of E. coli allowed a clear differentiation of the bacterial concentrations (100-103 CFU). After 40 minutes incubation of the β-Gal enzyme together with the substrate β-PAPG it was possible to oxidize the cleaved product PAP in the samples (Fig. 2). We were able to detect 2 CFU of E. coli in the sample. Furthermore, the measured current values correlated very well with the cell numbers of the original cell suspensions cultured on LB medium (Table 1). This sensitive assay is not labor-intensive and relatively easy to perform.
Jörg Ettenauer et al. / Procedia Engineering 120 (2015) 376 – 379
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C FU Fig. 2. Cyclic voltammetry measurements data at 220 mV with low bacterial concentrations (100-103 CFU). Measurements were performed every ten minutes after substrate addition. Table 1. Plate counts after overnight incubation. Expected/Calculated CFU
Number of grown bacteria
100
2
101
12
102
34
10
3
634
3. Conclusions and future outlook Our newly self-built “EcoStat” potentiostat has several advantages compared to other inexpensive instruments. The evaluation of this device showed excellent results regarding noise and current measurement accuracy. Furthermore, in combination with the easy-to-use PC software it represents a good alternative for performing electrochemical analysis in environmental applications. Combining this sensitive instrument with the presented methodology we were able to differentiate between different concentrations of E. coli. Furthermore, it was possible to verify 2 CFU of this fecal indicator organism. A prototype of such a stand-alone biosensor is currently under development. It could perform an autonomous contamination monitoring, and thus, would substantially increase the quality and safety of our drinking water. Acknowledgements The authors thank the province of Lower Austria and the European Regional Development Fund for financial support (project ID: WST3-T-91/026-2012). References [1] T. Neufeld, A. Schwartz-Mittelmann, D. Biran, E.Z. Ron, J. Rishpon: Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria, Anal Chem 75 (2003) 580-585. [2] M. Másson, Z. Liu, T. Haruyama, E. Kobatake, Y. Ikariyama, et al.: Immunosesning with amperometric detection, using galactosidase as label and p-aminophenyl-β-D-galactopyranoside as substrate, Anal Chem Acta 304 (1995) 353-359. [3] K.H. Kellner, T. Posnicek, J. Ettenauer, K. Zuser, M. Brandl: A new, low-cost potentiostat for environmental measurements with an easy-touse PC interface, Procedia Engineering, Eurosensors (2015) accepted. [4] A.A. Rowe, A.J. Bonham, R.J. White, M.P. Zimmer, R.J. Yadgar, et al.: CheapStat: an open-source, “do-it-yourself” potentiostat for analytical and educational applications, PlosOne 6(9) (2011) e23783, doi:10.1371/journal.pone.0023783. [5] R.W. Shideler, U. Bertocci: A low-noise potentiostat for the study of small amplitude signals in electrochemistry, J Res Natl Stand (1980) 85(3) 211-217.
