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On-line and in situ biosensors for monitoring environmental pollution
Biotechnology Advances 22 (2003) 27 – 33 www.elsevier.com/locate/biotechadv
On-line and in situ biosensors for monitoring environmental pollution Yossi Paitan, Dvora Biran, Israel Biran, Nelia Shechter, Reuven Babai, Judith Rishpon, Eliora Z. Ron * Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
Abstract Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals. D 2003 Elsevier Inc. All rights reserved. Keywords: Pollution; Bioremediation; Biosensors; Genotoxins; Reporter gene
1. Introduction Whole-cell biosensors employ promoters of genes fused to a reporter gene in order to detect changes in gene expression. One such type of changes is the cellular response to alterations in the environment, especially alterations that bring about physiological stress. Monitoring of environmental changes has been facilitated using reporter genes fused to
* Corresponding author. Tel.: +972-3-640-9379; fax: +972-3-641-4138. E-mail address: [email protected] (E.Z. Ron). 0734-9750/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2003.08.014
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promoters that respond to specific environmental pollutants—promoter-based biosensors. Promoters are sensitive and specific control elements present in the 5Vend of genes, upstream to the translated open reading frame. Promoters are the site action of control elements—repressors or activators. Promoters can respond to temperature, ionic strength or specific compounds, such as metabolites or environmental stress agents. Therefore, it is possible to monitor environmental factors by measuring promoter activity. The potential for development of bacterial biosensors based on promoter fusion to a reporter gene has been demonstrated for many analytes using several types of reporter genes, as reviewed by Kohler et al. (2000) and by Daunert et al. (2001). Such biosensors can be engineered by placing a reporter gene (such as those encoding h-galactosidase (lacZ), or alkaline phosphatase (AP, phoA), bacterial or firefly luciferase (lux or luc) or green fluorescent protein (GFP) under a transcriptional control mediated by the analyte. The result is that in the presence of the specific analyte, there is an increased production of the reporter protein, which is detectable and measurable. The bioluminescence reporter gene (lux) coding for the enzyme luciferase is the predominant gene used for the construction of biosensors for environmental stress, including aromatic compounds. In this case, the light emitted by the sensing bacteria is proportional to the concentrations of the pollutants. For several purposes, the gene coding for the green fluorescence protein (GFP) was also used. The reporter gene lacZ coding for the enzyme h-galactosidase which produces a color reaction with appropriate substrates was also used for the construction of various sensors for heavy metals and organic compounds (Klein et al., 1997; Kohler et al., 2000; Scott et al., 1997).
2. Electrochemical monitoring of gene expression We have recently reported the development of a novel technology for on-line and in situ monitoring of gene expression employing enzymes whose activity can be monitored electrochemically as reporters (Biran et al., 1999). There are several advantages for using
Fig. 1. Scheme of several configurations of the computerized on-line electrochemical system. The electrochemical monitoring can be performed in multititer wells (middle), flasks (bottom) or fermentors (top).
Y. Paitan et al. / Biotechnology Advances 22 (2003) 27–33
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Fig. 2. Scheme of a 7-mm disposable electrochemical cell. The electrochemical cell has a print screen disposable electrode and is air-stirred.
electrochemical measurements, including sensitivity and reproducibility as well as the ability for on-line, rapid, monitoring. The system we have been using employs a compact analyzer and disposable electrodes, and enables simultaneous measurements of several samples. Since the measurement is not optical, it is also possible to perform measurements in crude or turbid solutions. The system and one electrochemical cell are shown in Figs. 1 and 2. We showed the possible use of the electrochemical monitoring in bacteria (Biran et al., 2000). Recently, the system was also applied to eukaryotes. The results presented in Fig. 3 represent monitoring of gene expression in bacteria, yeasts and mammalian tissue cultures.
3. Applications of electrochemical biosensors for environmental studies 3.1. Monitoring concentration of pollutants Using this system, we constructed a biosensor for monitoring heavy metals and hydrocarbons. As an example, we constructed a biosensor for cadmium pollution, which consists of the lacZ gene expressed under the control of the cadmium-responsive promoter zntA (Babai and Ron, 1998). This whole-cell biosensor could detect, within minutes, nanomolar concentrations of cadmium in water, seawater and soil samples, and it was used for continuous on-line and in situ monitoring (Biran et al., 2000). In addition to cadmium, this biosensor can detect the presence of a variety of heavy metals—mercury, zinc and copper. 3.2. Monitoring general toxicity We have shown the possibility of using on-line electrochemical monitoring for determining changes in concentration of specific pollutants in the environment. Another tool that is required is a way to detect general toxicity. This is essential to detect a pollutant that is not
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Fig. 3.
Y. Paitan et al. / Biotechnology Advances 22 (2003) 27–33
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Fig. 4. Monitoring genotoxin 4NQO. The assay is based on the response of E. coli to DNA-damaging agents using a strain which carries a lacZ gene fused to a gene promoter that responds to DNA damage (sfiA) (Quillardet and Hofnung, 1985). h-Galactosidase activity was measured on-line with increasing concentrations of the genotoxin 4NQO.
present among the pollutants that are included in the monitoring system. General toxicity is often caused by genotoxic agents, such as mutagens and carcinogens of various kinds. These agents induce a number of global regulatory systems, such as the general stress response or the SOS response, which detects damage to DNA. As a model system, we used detection of genotoxins in the SOS Chromotest strain (Quillardet and Hofnung, 1985). The SOS Chromotest assay is based on the response of Escherichia coli to DNA-damaging agents monitoring by the lacZ gene fused to a promoter that is controlled by the SOS response. The results shown in Fig. 4 indicate that the electrochemical monitoring system can be used to determine the presence of genotoxic agents, such as 4NQ. 3.3. Detection of target bacteria in soil and water Rapid determination and identification of pathogenic or polluting bacteria is essential for public health. An amperometric method can be used to specifically detect target Fig. 3. Electrochemical monitoring in bacteria, yeasts and tissue cultures. The activity of h-galactosidase, coded for by the reporter gene lacZ, was monitored in microtiter wells. The bacterial enzyme (top) was monitored following addition of an inducer (IPTG). For yeasts (middle) we monitored the activity of h-galactosidase in the two Saccharomyces cerevisiae strains which represent the positive and negative control of the Yeast Two Hybrid System. The positive control strain selectively expresses lacZ in the presence of galactose. The lacZ gene in the tissue culture experiment (bottom) was carried on a virus with which we infected the culture at multiplicity of infection (M.O.I.) of 1 and 2.
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bacteria. As model system, we demonstrate the electrochemical detection of enteric bacteria in general and of E. coli specifically (Mittelmann et al., 2002). Amperometric detection enabled the determination of 1000 colony-forming units (cfu)/ml within 60– 75 min. Preincubation for 5 – 6 h further increased the sensitivity more than 100-fold. In addition, the system can be even more sensitive by the combination of virulent phagetyping and cell marker enzyme activity (Burlage, 1998). This combination enabled the detection of as low as 1 cfu/100 ml bacteria within 6 –8 h (Neufeld et al., 2003). In principle, this method can be applied to many types of bacteria, by measuring the enzymatic marker released by the lytic cycle of specific phage.
4. Conclusions Biosensors based on fusion of responsive promoters to reporter genes are sensitive and specific. Several such biosensors were constructed that can detect heavy metals and hydrocarbons. The use of reporters whose activity can be monitored electrochemically offers several advantages:
monitoring is rapid and can be performed on-line and in situ high sensitivity and reproducibility monitoring in crude or turbid solutions simultaneous measurements of several samples compact analyzer and disposable electrodes compatibility with other types of reporter genes using color, light of fluorescence the sensitivity of electrochemical measurements can also be used for amperometric determination of target bacteria in water.
Acknowledgements This work was supported by the Israel Ministry of Science and Technology and by the Manja and Morris Leigh Chair for Biophysics and Biotechnology (EZR). We are grateful to the Peikovsky Valachi grant to YP.
References Babai R, Ron EZ. An Escherichia coli gene responsive to heavy metals. FEMS Microbiol Lett 1998;167:107 – 11. Biran I, Klimentiy L, Hengge-Aronis R, Ron EZ, Rishpon J. On-line monitoring of gene expression. Microbiology 1999;145(Pt. 8):2129 – 33. Biran I, Babai R, Levcov K, Rishpon J, Ron EZ. Online and in situ monitoring of environmental pollutants: electrochemical biosensing of cadmium. Environ Microbiol 2000;2:285 – 90. Burlage RS. In: LaRossa RA, editor. Methods in molecular biology/bioluminescence. Totowa, NJ: Humana Press; 1998. p. 259 – 68. Daunert S, Barret G, Feliciano JS, Shetty RS, Shrestha S, Smith WA. Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev 2001;12:2705 – 38.
Y. Paitan et al. / Biotechnology Advances 22 (2003) 27–33
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Klein J, Altenbuchner J, Mattes R. Genetically modified Escherichia coli for colorimetric detection of inorganic and organic Hg compounds. Exs 1997;80:133 – 51. Kohler S, Belkin S, Schmid RD. Reporter gene bioassays in environmental analysis. Fresenius’ J Anal Chem 2000;366:769 – 79. Mittelmann AS, Ron EZ, Rishpon J. Amperometric quantification of total coliforms and specific detection of Escherichia coli. Anal Chem 2002;74:903 – 7. Neufeld T, Schwartz-Mitelmman A, Biran D, Ron EZ, Rishpon J. Application of phage typing in amperometric identification and quantification of specific bacteria. Anal Chem 2003;75:580 – 5. Quillardet P, Hofnung M. The SOS chromotest, a colorimetric bacterial assay for genotoxins. Mutat Res 1985;147:65 – 78. Scott DL, Ramanathan S, Shi W, Rosen BP, Daunert S. Genetically engineered bacteria: electrochemical sensing systems for antimonite and arsenite. Anal Chem 1997;69:16 – 20.
On-line and in situ biosensors for monitoring environmental pollution Yossi Paitan, Dvora Biran, Israel Biran, Nelia Shechter, Reuven Babai, Judith Rishpon, Eliora Z. Ron * Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
Abstract Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals. D 2003 Elsevier Inc. All rights reserved. Keywords: Pollution; Bioremediation; Biosensors; Genotoxins; Reporter gene
1. Introduction Whole-cell biosensors employ promoters of genes fused to a reporter gene in order to detect changes in gene expression. One such type of changes is the cellular response to alterations in the environment, especially alterations that bring about physiological stress. Monitoring of environmental changes has been facilitated using reporter genes fused to
* Corresponding author. Tel.: +972-3-640-9379; fax: +972-3-641-4138. E-mail address: [email protected] (E.Z. Ron). 0734-9750/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2003.08.014
28
Y. Paitan et al. / Biotechnology Advances 22 (2003) 27–33
promoters that respond to specific environmental pollutants—promoter-based biosensors. Promoters are sensitive and specific control elements present in the 5Vend of genes, upstream to the translated open reading frame. Promoters are the site action of control elements—repressors or activators. Promoters can respond to temperature, ionic strength or specific compounds, such as metabolites or environmental stress agents. Therefore, it is possible to monitor environmental factors by measuring promoter activity. The potential for development of bacterial biosensors based on promoter fusion to a reporter gene has been demonstrated for many analytes using several types of reporter genes, as reviewed by Kohler et al. (2000) and by Daunert et al. (2001). Such biosensors can be engineered by placing a reporter gene (such as those encoding h-galactosidase (lacZ), or alkaline phosphatase (AP, phoA), bacterial or firefly luciferase (lux or luc) or green fluorescent protein (GFP) under a transcriptional control mediated by the analyte. The result is that in the presence of the specific analyte, there is an increased production of the reporter protein, which is detectable and measurable. The bioluminescence reporter gene (lux) coding for the enzyme luciferase is the predominant gene used for the construction of biosensors for environmental stress, including aromatic compounds. In this case, the light emitted by the sensing bacteria is proportional to the concentrations of the pollutants. For several purposes, the gene coding for the green fluorescence protein (GFP) was also used. The reporter gene lacZ coding for the enzyme h-galactosidase which produces a color reaction with appropriate substrates was also used for the construction of various sensors for heavy metals and organic compounds (Klein et al., 1997; Kohler et al., 2000; Scott et al., 1997).
2. Electrochemical monitoring of gene expression We have recently reported the development of a novel technology for on-line and in situ monitoring of gene expression employing enzymes whose activity can be monitored electrochemically as reporters (Biran et al., 1999). There are several advantages for using
Fig. 1. Scheme of several configurations of the computerized on-line electrochemical system. The electrochemical monitoring can be performed in multititer wells (middle), flasks (bottom) or fermentors (top).
Y. Paitan et al. / Biotechnology Advances 22 (2003) 27–33
29
Fig. 2. Scheme of a 7-mm disposable electrochemical cell. The electrochemical cell has a print screen disposable electrode and is air-stirred.
electrochemical measurements, including sensitivity and reproducibility as well as the ability for on-line, rapid, monitoring. The system we have been using employs a compact analyzer and disposable electrodes, and enables simultaneous measurements of several samples. Since the measurement is not optical, it is also possible to perform measurements in crude or turbid solutions. The system and one electrochemical cell are shown in Figs. 1 and 2. We showed the possible use of the electrochemical monitoring in bacteria (Biran et al., 2000). Recently, the system was also applied to eukaryotes. The results presented in Fig. 3 represent monitoring of gene expression in bacteria, yeasts and mammalian tissue cultures.
3. Applications of electrochemical biosensors for environmental studies 3.1. Monitoring concentration of pollutants Using this system, we constructed a biosensor for monitoring heavy metals and hydrocarbons. As an example, we constructed a biosensor for cadmium pollution, which consists of the lacZ gene expressed under the control of the cadmium-responsive promoter zntA (Babai and Ron, 1998). This whole-cell biosensor could detect, within minutes, nanomolar concentrations of cadmium in water, seawater and soil samples, and it was used for continuous on-line and in situ monitoring (Biran et al., 2000). In addition to cadmium, this biosensor can detect the presence of a variety of heavy metals—mercury, zinc and copper. 3.2. Monitoring general toxicity We have shown the possibility of using on-line electrochemical monitoring for determining changes in concentration of specific pollutants in the environment. Another tool that is required is a way to detect general toxicity. This is essential to detect a pollutant that is not
30
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Fig. 3.
Y. Paitan et al. / Biotechnology Advances 22 (2003) 27–33
31
Fig. 4. Monitoring genotoxin 4NQO. The assay is based on the response of E. coli to DNA-damaging agents using a strain which carries a lacZ gene fused to a gene promoter that responds to DNA damage (sfiA) (Quillardet and Hofnung, 1985). h-Galactosidase activity was measured on-line with increasing concentrations of the genotoxin 4NQO.
present among the pollutants that are included in the monitoring system. General toxicity is often caused by genotoxic agents, such as mutagens and carcinogens of various kinds. These agents induce a number of global regulatory systems, such as the general stress response or the SOS response, which detects damage to DNA. As a model system, we used detection of genotoxins in the SOS Chromotest strain (Quillardet and Hofnung, 1985). The SOS Chromotest assay is based on the response of Escherichia coli to DNA-damaging agents monitoring by the lacZ gene fused to a promoter that is controlled by the SOS response. The results shown in Fig. 4 indicate that the electrochemical monitoring system can be used to determine the presence of genotoxic agents, such as 4NQ. 3.3. Detection of target bacteria in soil and water Rapid determination and identification of pathogenic or polluting bacteria is essential for public health. An amperometric method can be used to specifically detect target Fig. 3. Electrochemical monitoring in bacteria, yeasts and tissue cultures. The activity of h-galactosidase, coded for by the reporter gene lacZ, was monitored in microtiter wells. The bacterial enzyme (top) was monitored following addition of an inducer (IPTG). For yeasts (middle) we monitored the activity of h-galactosidase in the two Saccharomyces cerevisiae strains which represent the positive and negative control of the Yeast Two Hybrid System. The positive control strain selectively expresses lacZ in the presence of galactose. The lacZ gene in the tissue culture experiment (bottom) was carried on a virus with which we infected the culture at multiplicity of infection (M.O.I.) of 1 and 2.
32
Y. Paitan et al. / Biotechnology Advances 22 (2003) 27–33
bacteria. As model system, we demonstrate the electrochemical detection of enteric bacteria in general and of E. coli specifically (Mittelmann et al., 2002). Amperometric detection enabled the determination of 1000 colony-forming units (cfu)/ml within 60– 75 min. Preincubation for 5 – 6 h further increased the sensitivity more than 100-fold. In addition, the system can be even more sensitive by the combination of virulent phagetyping and cell marker enzyme activity (Burlage, 1998). This combination enabled the detection of as low as 1 cfu/100 ml bacteria within 6 –8 h (Neufeld et al., 2003). In principle, this method can be applied to many types of bacteria, by measuring the enzymatic marker released by the lytic cycle of specific phage.
4. Conclusions Biosensors based on fusion of responsive promoters to reporter genes are sensitive and specific. Several such biosensors were constructed that can detect heavy metals and hydrocarbons. The use of reporters whose activity can be monitored electrochemically offers several advantages:
monitoring is rapid and can be performed on-line and in situ high sensitivity and reproducibility monitoring in crude or turbid solutions simultaneous measurements of several samples compact analyzer and disposable electrodes compatibility with other types of reporter genes using color, light of fluorescence the sensitivity of electrochemical measurements can also be used for amperometric determination of target bacteria in water.
Acknowledgements This work was supported by the Israel Ministry of Science and Technology and by the Manja and Morris Leigh Chair for Biophysics and Biotechnology (EZR). We are grateful to the Peikovsky Valachi grant to YP.
References Babai R, Ron EZ. An Escherichia coli gene responsive to heavy metals. FEMS Microbiol Lett 1998;167:107 – 11. Biran I, Klimentiy L, Hengge-Aronis R, Ron EZ, Rishpon J. On-line monitoring of gene expression. Microbiology 1999;145(Pt. 8):2129 – 33. Biran I, Babai R, Levcov K, Rishpon J, Ron EZ. Online and in situ monitoring of environmental pollutants: electrochemical biosensing of cadmium. Environ Microbiol 2000;2:285 – 90. Burlage RS. In: LaRossa RA, editor. Methods in molecular biology/bioluminescence. Totowa, NJ: Humana Press; 1998. p. 259 – 68. Daunert S, Barret G, Feliciano JS, Shetty RS, Shrestha S, Smith WA. Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev 2001;12:2705 – 38.
Y. Paitan et al. / Biotechnology Advances 22 (2003) 27–33
33
Klein J, Altenbuchner J, Mattes R. Genetically modified Escherichia coli for colorimetric detection of inorganic and organic Hg compounds. Exs 1997;80:133 – 51. Kohler S, Belkin S, Schmid RD. Reporter gene bioassays in environmental analysis. Fresenius’ J Anal Chem 2000;366:769 – 79. Mittelmann AS, Ron EZ, Rishpon J. Amperometric quantification of total coliforms and specific detection of Escherichia coli. Anal Chem 2002;74:903 – 7. Neufeld T, Schwartz-Mitelmman A, Biran D, Ron EZ, Rishpon J. Application of phage typing in amperometric identification and quantification of specific bacteria. Anal Chem 2003;75:580 – 5. Quillardet P, Hofnung M. The SOS chromotest, a colorimetric bacterial assay for genotoxins. Mutat Res 1985;147:65 – 78. Scott DL, Ramanathan S, Shi W, Rosen BP, Daunert S. Genetically engineered bacteria: electrochemical sensing systems for antimonite and arsenite. Anal Chem 1997;69:16 – 20.