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Biosensing environmental pollution
Biosensing environmental pollution Eliora Z Ron Whole-cell biosensors are finding increasing use for the detection of environmental pollution and toxicity. These biosensors are constructed through the fusion of promoters, responsive to the relevant environmental conditions, to easily monitored reporter genes. Depending on the choice of reporter gene, expression can be monitored by the production of colour, light, fluorescence or electrochemical reactions. Recent advances in this area have included the development of biosensors of compact size that enable the on-line and in situ monitoring of a large number of environmental parameters. Addresses Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel Corresponding author: Ron, Eliora Z ([email protected])
Current Opinion in Biotechnology 2007, 18:252–256 This review comes from a themed issue on Environmental biotechnology Edited by Eliora Z Ron and Philip Hugenholtz Available online 25th May 2007 0958-1669/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2007.05.005
Introduction Bioremediation (i.e. the use of plants or microorganisms to clean up environmental pollution) has seen a surge of interest in recent years. Traditionally, the efficacy of bioremediation has been determined chemically, by measuring changes in total pollutant concentrations, usually complemented by chromatographic results (gas chromatography or gas chromatography-mass spectrometry). Recently, however, attempts have been made to use biosensors, especially microbial whole-cell biosensors, to monitor the rate of pollutant elimination [1–7].
is that they detect only the biologically active pollutants, and the response is proportional to the level of toxicity. This review focuses on whole-cell, ‘man-made’ biosensors based on recombinant DNA technology. These biosensors are constructed by fusing a pollutant-responsive gene promoter to a gene coding for a protein that can be easily quantified. In one such example, an Escherichia coli K-12 strain has been adapted for the detection of aromatic hydrocarbons. The E. coli cells carry the promoter of the xylS gene (induced by aromatic hydrocarbons) from Pseudomonas putida fused to a promoterless luciferase operon (from Vibrio harveyi or firefly). The expression of luciferase is induced by the presence of aromatic compounds and can be measured by light emission. Similar biosensors were constructed that can monitor the level of additional environmental pollutants, such as octane [3,4]. Recently, new whole-cell biosensors have been reported in which gene expression is monitored by electrochemical means. These promoter-based, whole-cell biosensors are suitable for on-line and in situ monitoring of pollutants [8,10–12]. An example is depicted in Figure 1, which shows a recombinant gene construct effective in biosensing heavy metals [8,9]. In this construct, the promoter and N-terminal region of the zntA gene, involved in the efflux of heavy metals, is fused to a promoterless lacZ gene, which codes for the enzyme b-galactosidase. Bacteria carrying this gene fusion respond to the presence of heavy metals by inducing the production of b-galactosidase, an enzyme that can easily be monitored by colorimetric and electrochemical reactions. Here, I review the major types of reporters and promoters used for the construction of biosensors and discuss the advantages and disadvantages of these different systems. I go on to look at possible future directions in biosensor research in relation to monitoring environmental pollution.
The construction of biosensors Biosensors are defined as monitoring systems based on the use of biological organisms or biologically derived reactions. For example, whole organisms, such as Daphnia or small fish, are being used to monitor water toxicity and enzymes can be used as biosensors of oxygen or glucose. The main drawback of biosensors is that, because they depend on biological systems, they are less reproducible than chemical methods and the values obtained are usually relative and not absolute. However, they can be considerably more time-effective and cost-effective and constitute a good tool for monitoring changes or online processes. Another important advantage of biosensors Current Opinion in Biotechnology 2007, 18:252–256
As mentioned above, whole-cell, man-made biosensors typically consist of a promoter, responsive to one or more pollutants or toxicants, genetically fused to a promoterless reporter gene. The recombinant genes can be located on plasmids or on the chromosome. An effective biosensor depends on the correct choice of its two constituents: the promoter and the reporter gene. Choosing a promoter
Several factors need to be considered when choosing a responsive promoter, the key ones being sensitivity and specificity. As a general rule, biological systems are very www.sciencedirect.com
Biosensing environmental pollution Ron 253
Figure 1
A biosensor responsive to heavy metals. (a) The genetic construction of the biosensor. A truncated, promoterless lacZ gene (coding for b-galactosidase) was fused downstream to the promoter and N-terminal region of the zntA gene of E. coli, which encodes for a heavy metal pump. (b) The relative induction of b-galactosidase as a function of mercury concentration.
sensitive; for example, bacterial gene promoters can detect heavy metals in concentrations as low as parts per billion and hydrocarbons can even be detected as a vapour. However, promoters often respond to groups of compounds, rather than to a specific compound. For example, promoters that detect cadmium usually also detect all other heavy metals, such as mercury, zinc and copper, and some even detect lead [8]. A variety of well-characterized promoters is available for genetic manipulations. These include promoters for various heavy metals [7,13–21], hydrocarbons and organic solvents [12,22,23–26], pesticides [14,27], salicylates [28], various organophosphorous nerve agents [29,30], mutagens and genotoxins [31,32]. Promoters are also available for the evaluation of general toxicity [33–36]. If the required promoter is not available, it is always possible to identify new, suitable promoters. Today, this can be done using transcriptomic or proteomic technologies to identify genes under the control of the relevant promoters. For example, promoters responsive to the www.sciencedirect.com
presence of a certain toxin can be identified by exposing microbial cultures to the toxin and looking for increased gene expression. The expression of each gene is determined by looking at the complete set of expressed proteins (i.e. the proteome) using two-dimensional gel electrophoresis or microarrays, which can be used to determine the relative transcriptional activity from each gene. Once the responsive genes are identified, their promoters can be amplified using the polymerase chain reaction (PCR) and cloned upstream of a suitable reporter gene. Choosing a reporter gene
The reporter gene usually encodes an enzyme that catalyzes a reaction that can be easily monitored. For example, the activity of the enzyme b-galactosidase, which splits b-galactose bonds, can be determined by a colorimetric reaction; the intensity of the colour is proportional to the level of enzyme present in the reaction and serves as an indicator of the activity of the gene coding for this enzyme. If this gene is cloned downstream to a promoter that responds to heavy metals, the colour Current Opinion in Biotechnology 2007, 18:252–256
254 Environmental biotechnology
intensity will be proportional to the concentration of the heavy metal. Enzymes are useful as reporters in biosensors, as the signal is amplified by the catalytic nature of the enzymatic reaction. In addition to the widely used b-galactosidase, other enzymes such as alkaline phosphatase can also be employed [7,11,12,37,38]. The use of more than one enzyme can facilitate a differential sensing of two pollutants by one biosensor. Other systems have used the bacterial or firefly luciferase genes (lux or luc) as reporters or the gene for green fluorescent protein (gfp). The bioluminescence reporter gene (lux) coding for the enzyme luciferase is the predominant gene used for the construction of biosensors for monitoring environmental stress, mainly the presence of hydrocarbons or heavy metals. In this case, the light emitted by the sensing bacteria is proportional to the concentration of the pollutants. These light-emitting systems have the advantage that the signal molecule has a short life — once emitted, the light does not stay — in contrast to the colour produced in the enzymatic reaction of b-galactosidase or alkaline phosphatase. In addition, the light-emitting system can be used for remote sensing. However, these systems have several serious drawbacks. On the practical side, the instruments for determining light emission are big and have to be used in the absence of outside light. On the biological side, the light emission system requires considerably high energy sources and cannot function in anaerobic conditions.
In situ and on-line measurements An important aspect of biosensing environmental pollutants is the ability to monitor in situ and preferably on-line. Having biosensors that can be used in the field dispenses with the need to bring samples to the laboratory, and enables real-time assessment of the level of pollution. On-line and in situ monitoring of gene expression can be performed by employing reporter enzymes whose activity can be monitored electrochemically [8]. The electrochemical measurements are highly sensitive, reproducible and employ a compact analyzer and disposable electrodes. These systems enable the simultaneous measurement of several samples. In addition, as the measurement is not optical, it is possible to perform measurements in crude or turbid solutions — an important consideration when monitoring pollutants in water systems or even in soil. Electrochemical systems have also been used for monitoring gene expression in bacteria, yeasts and even in mammalian tissue cultures [7,8,12].
Monitoring with biological systems – when and where Biosensors are fast, cost-effective and indicate the level of ‘relevant’ toxic materials. Their main disadvantage is that they do not give absolute chemical values. Moreover, because they depend on biological systems, they can only indicate relative levels, never absolute levels. For Current Opinion in Biotechnology 2007, 18:252–256
authorities such as the Environmental Protection Agency, with the present set of regulations, the inability to determine absolute concentrations is a drawback. However, because there is no way to determine absolute concentrations of toxicants in real-time, the use of biosensors should be recommended at least for on-line, real-time determination of toxicity levels. Such a good on-line monitoring system can be complemented by absolute chemical determinations, taken periodically. Another important advantage of biosensors is that they can be used to monitor general toxicity. Chemical measurements determine only known and likely toxicants; the presence of unexpected toxicants would be missed by standard chemical measurements. This fact further supports the use of a combined system — biological monitoring on-line together with periodical chemical determinations.
Future directions for biosensor development There are several obvious directions for developing biosensors. One of the main problems with the use of biological material is the difficulty of maintaining constant activity for a long time. For environmental biosensors it is also important to enable long storage at room temperature. To meet these demands, various conservation techniques have been reported, including freeze drying, vacuum drying, continuous cultivation, and immobilization in biocompatible polymers of organic or inorganic origin [36,39,40]. However, it is clear that further optimization is required to enable the fabrication of biosensors with a long shelf-life. Another obvious development will involve the production of biosensors capable of simultaneously monitoring several environmental stresses. Such sensors might monitor general toxicity, several stress parameters (e.g. temperature and oxidative stress), as well as several toxicants and genotoxic compounds. Such biosensors use a large number of recombinant bacteria, each reporting the presence of a specific toxicant or the existence of general stress or toxicity [31,41,42–44,45]. A matter of size
Typically, the response of biosensors using the lux or gfp genes as reporters is monitored using luminometers, spectrophotometers or fluorometers. These instruments are large and have to be operated in a laboratory. Clearly, these are not suitable for on-line and on-site monitoring, for which a compact and mobile device is required. Several reporter gene systems have already been adapted to the nanoscale. The resulting biosensors can be very compact and easy to handle. An example of one such development is described by Popovtzer et al. [45], who developed a novel integrated electrochemical nanochip for toxicity detection in water. The nanochip is based on minute electrodes and the data are collected on a hand-held computer. www.sciencedirect.com
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An electrochemical microbial chip was also constructed for mutagen screening by scanning electrochemical microscopy. This system used recombinant bacteria embedded in a microcavity (5 nl) on a glass substrate using collagen gel. The bacterial system consisted of Salmonella typhimurium TA1535 carrying a plasmid with a promoter responsive to DNA damage (umuC) fused to a promoterless lacZ. This system provides the means for multisample, quick and reliable electrochemical monitoring for the presence of mutagens [31]. Mini biosensors based on luminescence have also been developed using bacteria inserted into a microwell array formed on a chip (microbial array chip) or on one end of an imaging fibre [22,31,44,46]. Single-cell biosensors represent an interesting development that has already been tested for the detection of mercury and genotoxic materials. These biosensors consist of live cell arrays fabricated by immobilizing bacterial cells on the face of an optical imaging fibre containing a high-density array of microwells. Each microwell accommodates a single bacterium genetically engineered to respond to a specific analyte (through the fusion of a responsive promoter to a reporter gene coding for a fluorescent protein). Because each fibre in the array has its own light pathway, the data are composed of thousands of individual cell responses which are monitored simultaneously with both spatial and temporal resolution. This optical imaging fibre-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and to accommodate a variety of sensing strains [41,47].
Caenorhabditis elegans to identify sensitive biomarkers for environmental monitoring and risk assessment. Using green fluorescent protein as the reporter, they tested the toxicity of cadmium, lead, chromium and arsenite on stress-related gene expression, growth, reproduction and mortality in transgenic nematodes. The results suggest that nematodes are potentially useful as biosensors for environmental toxicity. Future developments are likely to include sensing devices that enable the detection of compounds currently undetected by unicellular-bacterial biosensors.
Conclusions In conclusion, cell-based biosensors provide effective tools for detecting environmental pollution and toxicity. In the future, biosensors are likely to become smaller and more flexible and will enable the on-line and in situ monitoring of a large number of environmental parameters. Clearly, we are a long way from the times of the ‘classic’ biosensor — the canary down the mine shaft or the dog of the wine maker!
Acknowledgements This work was supported, in part, by the Manja and Morris Leigh Chair for Biophysics and Biotechnology.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Biosensing of toxicity for humans
Most available biosensors are based on bacterial systems and these systems are clearly important for indicating the presence of toxicants or stressors. However, for several reasons, single-cell bacteria are not always a good model system for measuring toxicity against multicellular humans. Firstly, bacteria are potentially more resistant to many toxicants, because they have a strong cell wall that protects against osmotic shock, membrane damage, and so on. The bacterial cell wall also presents a barrier for the entry of many hydrophobic materials, such as acridine dyes, which are potent mutagens. Secondly, some compounds only become toxic after processing by the liver, and these will be missed by the current biosensing systems. Lastly, it should be noted that unicellular organisms are bad indicators for agents that affect differentiation or the nervous system. For these reasons, it is desirable to complement the results obtained using bacterial systems with additional data, probably obtained from multicellular eukaryotic systems. An example of the development of multicellular reporter systems is presented by Roh et al. [48] who used www.sciencedirect.com
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29. Lei Y, Mulchandani P, Wang J, Chen W, Mulchandani A: Highly sensitive and selective amperometric microbial biosensor for direct determination of p-nitrophenyl-substituted organophosphate nerve agents. Environ Sci Technol 2005, 39:8853-8857. 30. Lei Y, Mulchandani P, Chen W, Wang J, Mulchandani A: Whole cell-enzyme hybrid amperometric biosensor for direct determination of organophosphorous nerve agents with p-nitrophenyl substituent. Biotechnol Bioeng 2004, 85:706-713. 31. Matsui N, Kaya T, Nagamine K, Yasukawa T, Shiku H, Matsue T: Electrochemical mutagen screening using microbial chip. Biosens Bioelectron 2006, 21:1202-1209. 32. Norman A, Hansen LH, Sorensen SJ: A flow cytometry optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments. Mutat Res 2006, 603:164-172. 33. Neufeld T, Biran D, Popovtzer R, Erez T, Ron EZ, Rishpon J: Genetically engineered pfabA pfabR bacteria: an electrochemical whole cell biosensor for detection of water toxicity. Anal Chem 2006, 78:4952-4956. 34. Sorensen SJ, Burmolle M, Hansen LH: Making bio-sense of toxicity: new developments in whole-cell biosensors. Curr Opin Biotechnol 2006, 17:11-16. 35. Belkin S, Smulski DR, Vollmer AC, Van Dyk TK, LaRossa RA: Oxidative stress detection with Escherichia coli harboring a katG’:lux fusion. Appl Environ Microbiol 1996, 62:2252-2256. 36. Choi JW, Park KW, Lee DB, Lee W, Lee WH: Cell immobilization using self-assembled synthetic oligopeptide and its application to biological toxicity detection using surface plasmon resonance. Biosens Bioelectron 2005, 20:2300-2305. 37. Belkin S: Microbial whole-cell sensing systems of environmental pollutants. Curr Opin Microbiol 2003, 6:206-212. 38. Daunert S, Barrett G, Feliciano JS, Shetty RS, Shrestha S, SmithSpencer W: Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev 2000, 100:2705-2738.
20. Fujimoto H, Wakabayashi M, Yamashiro H, Maeda I, Isoda K, Kondoh M, Kawase M, Miyasaka H, Yagi K: Whole-cell arsenite biosensor using photosynthetic bacterium Rhodovulum sulfidophilum: Rhodovulum sulfidophilum as an arsenite biosensor. Appl Microbiol Biotechnol 2006, 73:332-338.
39. Bjerketorp J, Hakansson S, Belkin S, Jansson JK: Advances in preservation methods: keeping biosensor microorganisms alive and active. Curr Opin Biotechnol 2006, 17:43-49.
21. Shetty RS, Deo SK, Liu Y, Daunert S: Fluorescence-based sensing system for copper using genetically engineered living yeast cells. Biotechnol Bioeng 2004, 88:664-670.
40. Marques S, Aranda-Olmedo I, Ramos JL: Controlling bacterial physiology for optimal expression of gene reporter constructs. Curr Opin Biotechnol 2006, 17:50-56.
22. Campbell DW, Muller C, Reardon KF: Development of a fiber optic enzymatic biosensor for 1,2-dichloroethane. Biotechnol Lett 2006, 28:883-887.
41. Kuang Y, Biran I, Walt DR: Living bacterial cell array for genotoxin monitoring. Anal Chem 2004, 76:2902-2909.
23. Leedjarv A, Ivask A, Virta M, Kahru A: Analysis of bioavailable phenols from natural samples by recombinant luminescent bacterial sensors. Chemosphere 2006, 64:1910-1919. 24. Phoenix P, Keane A, Patel A, Bergeron H, Ghoshal S, Lau PC: Characterization of a new solvent-responsive gene locus in Pseudomonas putida F1 and its functionalization as a versatile biosensor. Environ Microbiol 2003, 5:1309-1327. 25. Tizzard AC, Bergsma JH, Lloyd-Jones G: A resazurin-based biosensor for organic pollutants. Biosens Bioelectron 2006, 22:759-763. 26. Xu Z, Mulchandani A, Chen W: Detection of benzene, toluene, ethyl benzene, and xylenes (BTEX) using toluene dioxygenaseperoxidase coupling reactions. Biotechnol Prog 2003, 19:1812-1815. 27. Farre M, Goncalves C, Lacorte S, Barcelo D, Alpendurada MF: Pesticide toxicity assessment using an electrochemical biosensor with Pseudomonas putida and a bioluminescence inhibition assay with Vibrio fischeri. Anal Bioanal Chem 2002, 373:696-703. 28. Huang WE, Wang H, Zheng H, Huang L, Singer AC, Thompson I, Whiteley AS: Chromosomally located gene fusions constructed in Acinetobacter sp. ADP1 for the detection of salicylate. Environ Microbiol 2005, 7:1339-1348. Current Opinion in Biotechnology 2007, 18:252–256
42. Pedahzur R, Polyak B, Marks RS, Belkin S: Water toxicity detection by a panel of stress-responsive luminescent bacteria. J Appl Toxicol 2004, 24:343-348. 43. Gu MB, Mitchell RJ, Kim BC: Whole-cell-based biosensors for environmental biomonitoring and application. Adv Biochem Eng Biotechnol 2004, 87:269-305. 44. Lee JH, Mitchell RJ, Kim BC, Cullen DC, Gu MB: A cell array biosensor for environmental toxicity analysis. Biosens Bioelectron 2005, 21:500-507. 45. Popovtzer R, Neufeld T, Biran D, Ron EZ, Rishpon J, Shacham Diamand Y: Novel integrated electrochemical nano-biochip for toxicity detection in water. Nano Lett 2005, 5:1023-1027. 46. Lee JH, Gu MB: An integrated mini biosensor system for continuous water toxicity monitoring. Biosens Bioelectron 2005, 20:1744-1749. 47. Biran I, Rissin DM, Ron EZ, Walt DR: Optical imaging fiber-based live bacterial cell array biosensor. Anal Biochem 2003, 315:106-113. 48. Roh JY, Lee J, Choi J: Assessment of stress-related gene expression in the heavy metal-exposed nematode Caenorhabditis elegans: a potential biomarker for metalinduced toxicity monitoring and environmental risk assessment. Environ Toxicol Chem 2006, 25:2946-2956. www.sciencedirect.com
Current Opinion in Biotechnology 2007, 18:252–256 This review comes from a themed issue on Environmental biotechnology Edited by Eliora Z Ron and Philip Hugenholtz Available online 25th May 2007 0958-1669/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2007.05.005
Introduction Bioremediation (i.e. the use of plants or microorganisms to clean up environmental pollution) has seen a surge of interest in recent years. Traditionally, the efficacy of bioremediation has been determined chemically, by measuring changes in total pollutant concentrations, usually complemented by chromatographic results (gas chromatography or gas chromatography-mass spectrometry). Recently, however, attempts have been made to use biosensors, especially microbial whole-cell biosensors, to monitor the rate of pollutant elimination [1–7].
is that they detect only the biologically active pollutants, and the response is proportional to the level of toxicity. This review focuses on whole-cell, ‘man-made’ biosensors based on recombinant DNA technology. These biosensors are constructed by fusing a pollutant-responsive gene promoter to a gene coding for a protein that can be easily quantified. In one such example, an Escherichia coli K-12 strain has been adapted for the detection of aromatic hydrocarbons. The E. coli cells carry the promoter of the xylS gene (induced by aromatic hydrocarbons) from Pseudomonas putida fused to a promoterless luciferase operon (from Vibrio harveyi or firefly). The expression of luciferase is induced by the presence of aromatic compounds and can be measured by light emission. Similar biosensors were constructed that can monitor the level of additional environmental pollutants, such as octane [3,4]. Recently, new whole-cell biosensors have been reported in which gene expression is monitored by electrochemical means. These promoter-based, whole-cell biosensors are suitable for on-line and in situ monitoring of pollutants [8,10–12]. An example is depicted in Figure 1, which shows a recombinant gene construct effective in biosensing heavy metals [8,9]. In this construct, the promoter and N-terminal region of the zntA gene, involved in the efflux of heavy metals, is fused to a promoterless lacZ gene, which codes for the enzyme b-galactosidase. Bacteria carrying this gene fusion respond to the presence of heavy metals by inducing the production of b-galactosidase, an enzyme that can easily be monitored by colorimetric and electrochemical reactions. Here, I review the major types of reporters and promoters used for the construction of biosensors and discuss the advantages and disadvantages of these different systems. I go on to look at possible future directions in biosensor research in relation to monitoring environmental pollution.
The construction of biosensors Biosensors are defined as monitoring systems based on the use of biological organisms or biologically derived reactions. For example, whole organisms, such as Daphnia or small fish, are being used to monitor water toxicity and enzymes can be used as biosensors of oxygen or glucose. The main drawback of biosensors is that, because they depend on biological systems, they are less reproducible than chemical methods and the values obtained are usually relative and not absolute. However, they can be considerably more time-effective and cost-effective and constitute a good tool for monitoring changes or online processes. Another important advantage of biosensors Current Opinion in Biotechnology 2007, 18:252–256
As mentioned above, whole-cell, man-made biosensors typically consist of a promoter, responsive to one or more pollutants or toxicants, genetically fused to a promoterless reporter gene. The recombinant genes can be located on plasmids or on the chromosome. An effective biosensor depends on the correct choice of its two constituents: the promoter and the reporter gene. Choosing a promoter
Several factors need to be considered when choosing a responsive promoter, the key ones being sensitivity and specificity. As a general rule, biological systems are very www.sciencedirect.com
Biosensing environmental pollution Ron 253
Figure 1
A biosensor responsive to heavy metals. (a) The genetic construction of the biosensor. A truncated, promoterless lacZ gene (coding for b-galactosidase) was fused downstream to the promoter and N-terminal region of the zntA gene of E. coli, which encodes for a heavy metal pump. (b) The relative induction of b-galactosidase as a function of mercury concentration.
sensitive; for example, bacterial gene promoters can detect heavy metals in concentrations as low as parts per billion and hydrocarbons can even be detected as a vapour. However, promoters often respond to groups of compounds, rather than to a specific compound. For example, promoters that detect cadmium usually also detect all other heavy metals, such as mercury, zinc and copper, and some even detect lead [8]. A variety of well-characterized promoters is available for genetic manipulations. These include promoters for various heavy metals [7,13–21], hydrocarbons and organic solvents [12,22,23–26], pesticides [14,27], salicylates [28], various organophosphorous nerve agents [29,30], mutagens and genotoxins [31,32]. Promoters are also available for the evaluation of general toxicity [33–36]. If the required promoter is not available, it is always possible to identify new, suitable promoters. Today, this can be done using transcriptomic or proteomic technologies to identify genes under the control of the relevant promoters. For example, promoters responsive to the www.sciencedirect.com
presence of a certain toxin can be identified by exposing microbial cultures to the toxin and looking for increased gene expression. The expression of each gene is determined by looking at the complete set of expressed proteins (i.e. the proteome) using two-dimensional gel electrophoresis or microarrays, which can be used to determine the relative transcriptional activity from each gene. Once the responsive genes are identified, their promoters can be amplified using the polymerase chain reaction (PCR) and cloned upstream of a suitable reporter gene. Choosing a reporter gene
The reporter gene usually encodes an enzyme that catalyzes a reaction that can be easily monitored. For example, the activity of the enzyme b-galactosidase, which splits b-galactose bonds, can be determined by a colorimetric reaction; the intensity of the colour is proportional to the level of enzyme present in the reaction and serves as an indicator of the activity of the gene coding for this enzyme. If this gene is cloned downstream to a promoter that responds to heavy metals, the colour Current Opinion in Biotechnology 2007, 18:252–256
254 Environmental biotechnology
intensity will be proportional to the concentration of the heavy metal. Enzymes are useful as reporters in biosensors, as the signal is amplified by the catalytic nature of the enzymatic reaction. In addition to the widely used b-galactosidase, other enzymes such as alkaline phosphatase can also be employed [7,11,12,37,38]. The use of more than one enzyme can facilitate a differential sensing of two pollutants by one biosensor. Other systems have used the bacterial or firefly luciferase genes (lux or luc) as reporters or the gene for green fluorescent protein (gfp). The bioluminescence reporter gene (lux) coding for the enzyme luciferase is the predominant gene used for the construction of biosensors for monitoring environmental stress, mainly the presence of hydrocarbons or heavy metals. In this case, the light emitted by the sensing bacteria is proportional to the concentration of the pollutants. These light-emitting systems have the advantage that the signal molecule has a short life — once emitted, the light does not stay — in contrast to the colour produced in the enzymatic reaction of b-galactosidase or alkaline phosphatase. In addition, the light-emitting system can be used for remote sensing. However, these systems have several serious drawbacks. On the practical side, the instruments for determining light emission are big and have to be used in the absence of outside light. On the biological side, the light emission system requires considerably high energy sources and cannot function in anaerobic conditions.
In situ and on-line measurements An important aspect of biosensing environmental pollutants is the ability to monitor in situ and preferably on-line. Having biosensors that can be used in the field dispenses with the need to bring samples to the laboratory, and enables real-time assessment of the level of pollution. On-line and in situ monitoring of gene expression can be performed by employing reporter enzymes whose activity can be monitored electrochemically [8]. The electrochemical measurements are highly sensitive, reproducible and employ a compact analyzer and disposable electrodes. These systems enable the simultaneous measurement of several samples. In addition, as the measurement is not optical, it is possible to perform measurements in crude or turbid solutions — an important consideration when monitoring pollutants in water systems or even in soil. Electrochemical systems have also been used for monitoring gene expression in bacteria, yeasts and even in mammalian tissue cultures [7,8,12].
Monitoring with biological systems – when and where Biosensors are fast, cost-effective and indicate the level of ‘relevant’ toxic materials. Their main disadvantage is that they do not give absolute chemical values. Moreover, because they depend on biological systems, they can only indicate relative levels, never absolute levels. For Current Opinion in Biotechnology 2007, 18:252–256
authorities such as the Environmental Protection Agency, with the present set of regulations, the inability to determine absolute concentrations is a drawback. However, because there is no way to determine absolute concentrations of toxicants in real-time, the use of biosensors should be recommended at least for on-line, real-time determination of toxicity levels. Such a good on-line monitoring system can be complemented by absolute chemical determinations, taken periodically. Another important advantage of biosensors is that they can be used to monitor general toxicity. Chemical measurements determine only known and likely toxicants; the presence of unexpected toxicants would be missed by standard chemical measurements. This fact further supports the use of a combined system — biological monitoring on-line together with periodical chemical determinations.
Future directions for biosensor development There are several obvious directions for developing biosensors. One of the main problems with the use of biological material is the difficulty of maintaining constant activity for a long time. For environmental biosensors it is also important to enable long storage at room temperature. To meet these demands, various conservation techniques have been reported, including freeze drying, vacuum drying, continuous cultivation, and immobilization in biocompatible polymers of organic or inorganic origin [36,39,40]. However, it is clear that further optimization is required to enable the fabrication of biosensors with a long shelf-life. Another obvious development will involve the production of biosensors capable of simultaneously monitoring several environmental stresses. Such sensors might monitor general toxicity, several stress parameters (e.g. temperature and oxidative stress), as well as several toxicants and genotoxic compounds. Such biosensors use a large number of recombinant bacteria, each reporting the presence of a specific toxicant or the existence of general stress or toxicity [31,41,42–44,45]. A matter of size
Typically, the response of biosensors using the lux or gfp genes as reporters is monitored using luminometers, spectrophotometers or fluorometers. These instruments are large and have to be operated in a laboratory. Clearly, these are not suitable for on-line and on-site monitoring, for which a compact and mobile device is required. Several reporter gene systems have already been adapted to the nanoscale. The resulting biosensors can be very compact and easy to handle. An example of one such development is described by Popovtzer et al. [45], who developed a novel integrated electrochemical nanochip for toxicity detection in water. The nanochip is based on minute electrodes and the data are collected on a hand-held computer. www.sciencedirect.com
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An electrochemical microbial chip was also constructed for mutagen screening by scanning electrochemical microscopy. This system used recombinant bacteria embedded in a microcavity (5 nl) on a glass substrate using collagen gel. The bacterial system consisted of Salmonella typhimurium TA1535 carrying a plasmid with a promoter responsive to DNA damage (umuC) fused to a promoterless lacZ. This system provides the means for multisample, quick and reliable electrochemical monitoring for the presence of mutagens [31]. Mini biosensors based on luminescence have also been developed using bacteria inserted into a microwell array formed on a chip (microbial array chip) or on one end of an imaging fibre [22,31,44,46]. Single-cell biosensors represent an interesting development that has already been tested for the detection of mercury and genotoxic materials. These biosensors consist of live cell arrays fabricated by immobilizing bacterial cells on the face of an optical imaging fibre containing a high-density array of microwells. Each microwell accommodates a single bacterium genetically engineered to respond to a specific analyte (through the fusion of a responsive promoter to a reporter gene coding for a fluorescent protein). Because each fibre in the array has its own light pathway, the data are composed of thousands of individual cell responses which are monitored simultaneously with both spatial and temporal resolution. This optical imaging fibre-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and to accommodate a variety of sensing strains [41,47].
Caenorhabditis elegans to identify sensitive biomarkers for environmental monitoring and risk assessment. Using green fluorescent protein as the reporter, they tested the toxicity of cadmium, lead, chromium and arsenite on stress-related gene expression, growth, reproduction and mortality in transgenic nematodes. The results suggest that nematodes are potentially useful as biosensors for environmental toxicity. Future developments are likely to include sensing devices that enable the detection of compounds currently undetected by unicellular-bacterial biosensors.
Conclusions In conclusion, cell-based biosensors provide effective tools for detecting environmental pollution and toxicity. In the future, biosensors are likely to become smaller and more flexible and will enable the on-line and in situ monitoring of a large number of environmental parameters. Clearly, we are a long way from the times of the ‘classic’ biosensor — the canary down the mine shaft or the dog of the wine maker!
Acknowledgements This work was supported, in part, by the Manja and Morris Leigh Chair for Biophysics and Biotechnology.
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Most available biosensors are based on bacterial systems and these systems are clearly important for indicating the presence of toxicants or stressors. However, for several reasons, single-cell bacteria are not always a good model system for measuring toxicity against multicellular humans. Firstly, bacteria are potentially more resistant to many toxicants, because they have a strong cell wall that protects against osmotic shock, membrane damage, and so on. The bacterial cell wall also presents a barrier for the entry of many hydrophobic materials, such as acridine dyes, which are potent mutagens. Secondly, some compounds only become toxic after processing by the liver, and these will be missed by the current biosensing systems. Lastly, it should be noted that unicellular organisms are bad indicators for agents that affect differentiation or the nervous system. For these reasons, it is desirable to complement the results obtained using bacterial systems with additional data, probably obtained from multicellular eukaryotic systems. An example of the development of multicellular reporter systems is presented by Roh et al. [48] who used www.sciencedirect.com
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