< A new bacterial bioassay for toxicity of particulate air pollution is presented. < Bioassay res... more < A new bacterial bioassay for toxicity of particulate air pollution is presented. < Bioassay responses indicating toxicity were observed for several PM samples. < These responses suggest oxidative stress, respiration inhibition and Fe deficiency. < A metal chelating treatment of the samples relieved the bioassay's responses. < Bioavailability analysis suggested that Cr was related to these toxic responses. a b s t r a c t Numerous studies have demonstrated that elevated concentrations of suspended atmospheric particulate matter (PM) are associated with adverse health effects. In order to minimize the adverse public health effects of atmospheric PM by exposure management, there is a need for a greater understanding of the toxic mechanisms and the components that are responsible for the toxic effects. The aim of this study was to utilize bioassay techniques to investigate these aspects. For this purpose a reporter panel of 9 genetically engineered bacterial (Escherichia coli) strains was composed. Each panel member was designed to report on a different stress condition with a measurable light signal produced by the luciferase enzyme. Toxic mechanisms and components were studied using six anthropogenic PM source samples, including two vehicle combustion particles, three coal fly ash (CFA) samples and an urban dust sample. The most prominent outcome of the panel exposure results were broad panel responses observed for two of the CFA samples, indicating oxidative stress, respiration inhibition and iron deficiency. These responses were relieved when the samples were treated with EDTA, a non-specific metal chelator, suggesting the involvement of metals in the observed effects. Bioavailability analysis of the samples suggests that chromium was related to the toxic responses induced by two of the CFA samples. Oxidative stress was also observed in several samples of ambient atmospheric aerosols and excess metal toxicity in an urban dust sample collected in a parking lot. The reporter panel approach, as demonstrated in this study, has the potential of providing novel insights as to the mechanisms of atmospheric PM toxicity. Furthermore, combining the panel's results with bioavailability data can enlighten about the role of different PM components in the observed toxicity.
This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The bi... more This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosensor presented here is based on bacterial cells that are genetically "tailored" to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemical active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 m and 300 m. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different genotoxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10 g/ml for NA using 30 min for induction and 0.31 M for IQ using 120 min for induction, both 3 min after the addition of the substrate material.
... Michael Stieber ... As a direct consequence, the biodegradation of these compounds has been e... more ... Michael Stieber ... As a direct consequence, the biodegradation of these compounds has been extensively studied, and their degradative pathways are known to a large extent (Cer-niglia andHeitkamp, 1989; Morgan and Watkinson, 1989; Mueller et al., 1989, Heitkamp et al ...
Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made... more Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by microsystem-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such wholecell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the microelectronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.
Over the last few years, the physical dimensions of microchip devices have decreased, enhancing t... more Over the last few years, the physical dimensions of microchip devices have decreased, enhancing the interest in the integration of various devices and complex operations onto a compatible "lab on a chip" system with desirable characteristics and capabilities. This work presents a novel μ-fluidics whole cell biosensor for water toxicity analysis. The biosensor is based on bacterial cells genetically "tailored" to generate an electrochemical bio-signal in the presence of toxic materials. The μ-chip was electrochemically characterized, and demonstrated the potential toxicity analysis with a model toxicant. A novel concept of bacterial biosensors deposition by means of electrophoretic force was examined for the first time. Preliminary results demonstrated the ability to detect electrochemical signal generated by the deposited bacterial cells indicating the potential use for patterning of bacterial cells on solid-state surfaces for the use in bio-sensing.
The electrode geometry and material have a significant effect on the electrochemical biochip tran... more The electrode geometry and material have a significant effect on the electrochemical biochip transduction of chemical signals into electrical current or voltage. In this work we focus on the working electrode aiming to improve the signal level of live cell sensors integrated on solid-state microchips. We present here a model and measurements describing the effect of the electrode material and dimensions on the biochip performance. The research hypothesis was that the electrode transduction efficiency increases as its effective area increases. Therefore, we investigated two methods to increase the electrode effective area: 3D structures and a polymer modified electrode. An electrochemical microchip was fabricated with a working electrode that was further modified, resulting in two structure types: 3D metallic pillar-based and polypyrrole-coated. The electrochemical performance of both modified electrodes was characterized and their utilization as working electrodes in whole-cell biochips for toxicity sensing was studied. Bio- detection efficiency analysis demonstrated higher biosensing performance for the metallic (e.g. Cu/Au) pillar-based microchip than for the polypyrrole-modified and the non-modified microchips. Therefore, we conclude that the enhanced signal of the modified geometry electrode is most probably due to the increased effective surface area and the improved charge transfer efficiency.
In parallel to the continuous development of increasingly more sophisticated physical and chemica... more In parallel to the continuous development of increasingly more sophisticated physical and chemical analytical technologies for the detection of environmental pollutants, there is a progressively more urgent need also for bioassays which report not only on the presence of a chemical but also on its bioavailability and its biological effects. As a partial fulfillment of that need, there has been a rapid development of biosensors based on genetically engineered bacteria. Such microorganisms typically combine a promoter-operator, which acts as the sensing element, with reporter gene(s) coding for easily detectable proteins. These sensors have the ability to detect global parameters such as stress conditions, toxicity or DNA-damaging agents as well as specific organic and inorganic compounds. The systems described in this review, designed to detect different groups of target chemicals, vary greatly in their detection limits, specificity, response times and more. These variations reflect ...
This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited on... more This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited onto solid surfaces that are a part of a micro-machined system. The development of such novel hybrid functional sensors depends on the cell deposition methods; in this work new approach integrating live bacterial cells on a bio-chip using electrophoretic deposition is presented. The bio-material deposition technique was characterized under various driving potential and chamber configurations. The deposited bio-mass included genetically engineered bacterial cells generating electrochemically active byproduct upon exposure to toxic materials in the aqueous solution. In this paper we present the deposition apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of the new deposition method.
The ever-growing use of pharmaceutical compounds, including antibacterial substances, poses a sub... more The ever-growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy-based non-antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.
The last decade has witnessed a significant increase in interest in whole-cell biosensors for div... more The last decade has witnessed a significant increase in interest in whole-cell biosensors for diverse applications, as well as a rapid and continuous expansion of array technologies. The combination of these two disciplines has yielded the notion of whole-cell array biosensors. We present a potential manifestation of this idea by describing the printing of a whole-cell bacterial bioreporters array. Exploiting natural bacterial tendency to adhere to positively charged abiotic surfaces, we describe immobilization and patterning of bacterial ''spots'' in the nanolitre volume range by a non-contact robotic printer. We show that the printed Escherichia coli-based sensor bacteria are immobilized on the surface, and retain their viability and biosensing activity for at least 2 months when kept at 4 C. Immobilization efficiency was improved by manipulating the bacterial genetics (overproducing curli protein), the growth and the printing media (osmotic stress and osmoprotectants) and by a chemical modification of the inanimate surface (self-assembled layers of 3-aminopropyl-triethoxysilane). We suggest that the methodology presented herein may be applicable to the manufacturing of whole-cell sensor arrays for diverse high throughput applications.
We describe a flow-through biosensor for online continuous water toxicity monitoring. At the hear... more We describe a flow-through biosensor for online continuous water toxicity monitoring. At the heart of the device are disposable modular biochips incorporating agar-immobilized bioluminescent recombinant reporter bacteria, the responses of which are probed by single-photon avalanche diode detectors. To demonstrate the biosensor capabilities, we equipped it with biochips harboring both inducible and constitutive reporter strains and exposed it to a continuous water flow for up to 10 days. During these periods we challenged the biosensor with 2-h pulses of water spiked with model compounds representing different classes of potential water pollutants, as well as with a sample of industrial wastewater. The biosensor reporter panel detected all simulated contamination events within 0.5-2.5 h, and its response was indicative of the nature of the contaminating chemicals. We believe that a biosensor of the proposed design can be integrated into future water safety and security networks, as part of an early warning system against accidental or intentional water pollution by toxic chemicals.
30 The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface... more 30 The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface between the terrestrial biosphere and solar radiation. It is estimated to host up to 10 26 microbial cells that may intercept part of the photon flux impinging on the leaves. Based on 454pyrosequencing generated metagenome data, we report on the existence 35 of diverse microbial rhodopsins in five distinct phyllospheres from tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Our findings, for the first time describing microbial rhodopsins from nonaquatic habitats, point toward the potential coexistence of microbial 40 rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.
An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate v... more An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%. Environ. Mol. Mutagen. 00:000-000, 2012. V V C 2012 Wiley Periodicals, Inc.
This paper presents an integrated whole-cell biochip system where functioning cells are deposited... more This paper presents an integrated whole-cell biochip system where functioning cells are deposited on the solid micro-machined surfaces while specially designed indium tin oxide electrodes that can be used to apply controllable electric fields during various stages; for example during cell deposition. The electrodes can be used also for sensing currents associated with the sensing mechanisms of electrochemical whole-cell biosensors. In this work a new approach integrating live bacterial cells on a biochip using electrophoretic deposition is presented. The biomaterial deposition technique was characterized under various driving potentials and chamber configurations. An analytical model of the electrophoretic deposition kinetics was developed and presented here. The deposited biomass included genetically engineered bacterial cells that may respond to toxic material exposure by expressing proteins that react with specific analytes generating electrochemically active byproducts. In this study the effect of external electric fields on the whole-cell biochips has been successfully developed and tested. The research hypothesis was that by applying electric fields on bacterial whole-cells, their permeability to the penetration of external analytes can be increased. This effect was tested and the results are shown here. The effect of prolonged and short external electric fields on the bioelectrochemical signal generated by sessile bacterial whole-cells in response to the presence of toxins was studied. It was demonstrated that relatively short 10 ms external DC electric pulse improves the performance of bacterial biosensors by 15% relative to un-biased biosensors. The application of prolonged 1 h external alternating electric fields deteriorated the whole-cell performance in the presence of toxins. In this paper we present the electrode apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of this new concept.
Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in in... more Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.
< A new bacterial bioassay for toxicity of particulate air pollution is presented. < Bioassay res... more < A new bacterial bioassay for toxicity of particulate air pollution is presented. < Bioassay responses indicating toxicity were observed for several PM samples. < These responses suggest oxidative stress, respiration inhibition and Fe deficiency. < A metal chelating treatment of the samples relieved the bioassay's responses. < Bioavailability analysis suggested that Cr was related to these toxic responses. a b s t r a c t Numerous studies have demonstrated that elevated concentrations of suspended atmospheric particulate matter (PM) are associated with adverse health effects. In order to minimize the adverse public health effects of atmospheric PM by exposure management, there is a need for a greater understanding of the toxic mechanisms and the components that are responsible for the toxic effects. The aim of this study was to utilize bioassay techniques to investigate these aspects. For this purpose a reporter panel of 9 genetically engineered bacterial (Escherichia coli) strains was composed. Each panel member was designed to report on a different stress condition with a measurable light signal produced by the luciferase enzyme. Toxic mechanisms and components were studied using six anthropogenic PM source samples, including two vehicle combustion particles, three coal fly ash (CFA) samples and an urban dust sample. The most prominent outcome of the panel exposure results were broad panel responses observed for two of the CFA samples, indicating oxidative stress, respiration inhibition and iron deficiency. These responses were relieved when the samples were treated with EDTA, a non-specific metal chelator, suggesting the involvement of metals in the observed effects. Bioavailability analysis of the samples suggests that chromium was related to the toxic responses induced by two of the CFA samples. Oxidative stress was also observed in several samples of ambient atmospheric aerosols and excess metal toxicity in an urban dust sample collected in a parking lot. The reporter panel approach, as demonstrated in this study, has the potential of providing novel insights as to the mechanisms of atmospheric PM toxicity. Furthermore, combining the panel's results with bioavailability data can enlighten about the role of different PM components in the observed toxicity.
This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The bi... more This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosensor presented here is based on bacterial cells that are genetically "tailored" to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemical active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 m and 300 m. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different genotoxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10 g/ml for NA using 30 min for induction and 0.31 M for IQ using 120 min for induction, both 3 min after the addition of the substrate material.
... Michael Stieber ... As a direct consequence, the biodegradation of these compounds has been e... more ... Michael Stieber ... As a direct consequence, the biodegradation of these compounds has been extensively studied, and their degradative pathways are known to a large extent (Cer-niglia andHeitkamp, 1989; Morgan and Watkinson, 1989; Mueller et al., 1989, Heitkamp et al ...
Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made... more Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by microsystem-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such wholecell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the microelectronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.
Over the last few years, the physical dimensions of microchip devices have decreased, enhancing t... more Over the last few years, the physical dimensions of microchip devices have decreased, enhancing the interest in the integration of various devices and complex operations onto a compatible "lab on a chip" system with desirable characteristics and capabilities. This work presents a novel μ-fluidics whole cell biosensor for water toxicity analysis. The biosensor is based on bacterial cells genetically "tailored" to generate an electrochemical bio-signal in the presence of toxic materials. The μ-chip was electrochemically characterized, and demonstrated the potential toxicity analysis with a model toxicant. A novel concept of bacterial biosensors deposition by means of electrophoretic force was examined for the first time. Preliminary results demonstrated the ability to detect electrochemical signal generated by the deposited bacterial cells indicating the potential use for patterning of bacterial cells on solid-state surfaces for the use in bio-sensing.
The electrode geometry and material have a significant effect on the electrochemical biochip tran... more The electrode geometry and material have a significant effect on the electrochemical biochip transduction of chemical signals into electrical current or voltage. In this work we focus on the working electrode aiming to improve the signal level of live cell sensors integrated on solid-state microchips. We present here a model and measurements describing the effect of the electrode material and dimensions on the biochip performance. The research hypothesis was that the electrode transduction efficiency increases as its effective area increases. Therefore, we investigated two methods to increase the electrode effective area: 3D structures and a polymer modified electrode. An electrochemical microchip was fabricated with a working electrode that was further modified, resulting in two structure types: 3D metallic pillar-based and polypyrrole-coated. The electrochemical performance of both modified electrodes was characterized and their utilization as working electrodes in whole-cell biochips for toxicity sensing was studied. Bio- detection efficiency analysis demonstrated higher biosensing performance for the metallic (e.g. Cu/Au) pillar-based microchip than for the polypyrrole-modified and the non-modified microchips. Therefore, we conclude that the enhanced signal of the modified geometry electrode is most probably due to the increased effective surface area and the improved charge transfer efficiency.
In parallel to the continuous development of increasingly more sophisticated physical and chemica... more In parallel to the continuous development of increasingly more sophisticated physical and chemical analytical technologies for the detection of environmental pollutants, there is a progressively more urgent need also for bioassays which report not only on the presence of a chemical but also on its bioavailability and its biological effects. As a partial fulfillment of that need, there has been a rapid development of biosensors based on genetically engineered bacteria. Such microorganisms typically combine a promoter-operator, which acts as the sensing element, with reporter gene(s) coding for easily detectable proteins. These sensors have the ability to detect global parameters such as stress conditions, toxicity or DNA-damaging agents as well as specific organic and inorganic compounds. The systems described in this review, designed to detect different groups of target chemicals, vary greatly in their detection limits, specificity, response times and more. These variations reflect ...
This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited on... more This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited onto solid surfaces that are a part of a micro-machined system. The development of such novel hybrid functional sensors depends on the cell deposition methods; in this work new approach integrating live bacterial cells on a bio-chip using electrophoretic deposition is presented. The bio-material deposition technique was characterized under various driving potential and chamber configurations. The deposited bio-mass included genetically engineered bacterial cells generating electrochemically active byproduct upon exposure to toxic materials in the aqueous solution. In this paper we present the deposition apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of the new deposition method.
The ever-growing use of pharmaceutical compounds, including antibacterial substances, poses a sub... more The ever-growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy-based non-antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.
The last decade has witnessed a significant increase in interest in whole-cell biosensors for div... more The last decade has witnessed a significant increase in interest in whole-cell biosensors for diverse applications, as well as a rapid and continuous expansion of array technologies. The combination of these two disciplines has yielded the notion of whole-cell array biosensors. We present a potential manifestation of this idea by describing the printing of a whole-cell bacterial bioreporters array. Exploiting natural bacterial tendency to adhere to positively charged abiotic surfaces, we describe immobilization and patterning of bacterial ''spots'' in the nanolitre volume range by a non-contact robotic printer. We show that the printed Escherichia coli-based sensor bacteria are immobilized on the surface, and retain their viability and biosensing activity for at least 2 months when kept at 4 C. Immobilization efficiency was improved by manipulating the bacterial genetics (overproducing curli protein), the growth and the printing media (osmotic stress and osmoprotectants) and by a chemical modification of the inanimate surface (self-assembled layers of 3-aminopropyl-triethoxysilane). We suggest that the methodology presented herein may be applicable to the manufacturing of whole-cell sensor arrays for diverse high throughput applications.
We describe a flow-through biosensor for online continuous water toxicity monitoring. At the hear... more We describe a flow-through biosensor for online continuous water toxicity monitoring. At the heart of the device are disposable modular biochips incorporating agar-immobilized bioluminescent recombinant reporter bacteria, the responses of which are probed by single-photon avalanche diode detectors. To demonstrate the biosensor capabilities, we equipped it with biochips harboring both inducible and constitutive reporter strains and exposed it to a continuous water flow for up to 10 days. During these periods we challenged the biosensor with 2-h pulses of water spiked with model compounds representing different classes of potential water pollutants, as well as with a sample of industrial wastewater. The biosensor reporter panel detected all simulated contamination events within 0.5-2.5 h, and its response was indicative of the nature of the contaminating chemicals. We believe that a biosensor of the proposed design can be integrated into future water safety and security networks, as part of an early warning system against accidental or intentional water pollution by toxic chemicals.
30 The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface... more 30 The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface between the terrestrial biosphere and solar radiation. It is estimated to host up to 10 26 microbial cells that may intercept part of the photon flux impinging on the leaves. Based on 454pyrosequencing generated metagenome data, we report on the existence 35 of diverse microbial rhodopsins in five distinct phyllospheres from tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Our findings, for the first time describing microbial rhodopsins from nonaquatic habitats, point toward the potential coexistence of microbial 40 rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.
An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate v... more An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%. Environ. Mol. Mutagen. 00:000-000, 2012. V V C 2012 Wiley Periodicals, Inc.
This paper presents an integrated whole-cell biochip system where functioning cells are deposited... more This paper presents an integrated whole-cell biochip system where functioning cells are deposited on the solid micro-machined surfaces while specially designed indium tin oxide electrodes that can be used to apply controllable electric fields during various stages; for example during cell deposition. The electrodes can be used also for sensing currents associated with the sensing mechanisms of electrochemical whole-cell biosensors. In this work a new approach integrating live bacterial cells on a biochip using electrophoretic deposition is presented. The biomaterial deposition technique was characterized under various driving potentials and chamber configurations. An analytical model of the electrophoretic deposition kinetics was developed and presented here. The deposited biomass included genetically engineered bacterial cells that may respond to toxic material exposure by expressing proteins that react with specific analytes generating electrochemically active byproducts. In this study the effect of external electric fields on the whole-cell biochips has been successfully developed and tested. The research hypothesis was that by applying electric fields on bacterial whole-cells, their permeability to the penetration of external analytes can be increased. This effect was tested and the results are shown here. The effect of prolonged and short external electric fields on the bioelectrochemical signal generated by sessile bacterial whole-cells in response to the presence of toxins was studied. It was demonstrated that relatively short 10 ms external DC electric pulse improves the performance of bacterial biosensors by 15% relative to un-biased biosensors. The application of prolonged 1 h external alternating electric fields deteriorated the whole-cell performance in the presence of toxins. In this paper we present the electrode apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of this new concept.
Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in in... more Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.
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Papers by S. Belkin