Electrochemical signal detection can be readily integrated in biosensors and is thus an attractiv... more Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.
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.
We present the first demonstration of a novel bacterial biofilm treatment technique showing a 56%... more We present the first demonstration of a novel bacterial biofilm treatment technique showing a 56% average decrease in bacterial cell viability compared to traditional antibiotic treatments in a Micro-BOAT platform. Integrated linear array charge-coupled devices achieve spatially realized optical density monitoring, correlating to both average biomass and localized biofilm morphology. For on-chip demonstration of biofilm treatment, a unique bioelectric effect using a superpositioned direct and alternating current electric field is applied in the presence of antibiotics. Use of the platform demonstrated successful real-time monitoring of biofilm treatment and validated an on-chip bioelectric effect showing a decrease in both bacterial cell viability and overall biomass.
Applications in Biological Systems and Biotechnology, 2010
Page 154. 4 Biomedical Implications of the Porosity of Microbial Biofilms H. Ben-Yoav Department ... more Page 154. 4 Biomedical Implications of the Porosity of Microbial Biofilms H. Ben-Yoav Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv, Israel N. Cohen ...
A novel micro-fluidic whole cell biosensor for toxicity analysis based on bioluminescence detecti... more A novel micro-fluidic whole cell biosensor for toxicity analysis based on bioluminescence detection was developed. The optical part of the biochip was modelled and simulated to optimize the total light collection efficiency and the system response. The optimization elucidated some of the optical aspects of the biochip. A study evaluating the bioluminescence reaction kinetics was performed and revealed the interdependence between the detected bioluminescence and the physical parameters of the introduced toxin.
This work demonstrates the first utilization of virus molecules as nano-scale biotemplates assemb... more This work demonstrates the first utilization of virus molecules as nano-scale biotemplates assembled on an electrochemical biosensor, allowing for an 8-times increased signal and an improved biosensing performance of 9.5-fold. The versatile and inexpensive biological Tobacco mosaic virus was integrated as a high aspect ratio, low footprint, low-cost, easy to genetically functionalize, nanostructured three-dimensional scaffold for the synthesis of novel multifunctional electrodes. The biotemplated scaffold allows for an increased surface area resulting in higher electrochemical currents, better signal-to-noise ratio and improved sensitivity when incorporated into miniaturized biosensors.
2010 IEEE 26-th Convention of Electrical and Electronics Engineers in Israel, 2010
In this paper we propose a new approach of signal conditioning circuits. The circuit is able to h... more In this paper we propose a new approach of signal conditioning circuits. The circuit is able to handle both electrochemical and bioluminescent sensors, mainly electrochemical sensors. The integrated circuit will convert electrical signal into impulses, making the frequency the carrier of the information. In this way we avoid amplifying stages, reducing circuit complexity and area on chip. The biggest obstacles that we manage to overcome ware the major difference between the currents provided by the sensors and the sense of the currents. We still need to build an interface capable to work not only with one sensor but with 6X6 or 8X8 sensors.
A lab-on-chip consisting of a unique integration of whole-cell sensors, a MOEMS (Micro-Opto-Elect... more A lab-on-chip consisting of a unique integration of whole-cell sensors, a MOEMS (Micro-Opto-Electro-Mechanical-System) modulator, and solid-state photo-detectors was implemented for the first time. Whole-cell sensors were genetically engineered to express a bioluminescent reporter (lux) as a function of the lac promoter. The MOEMS modulator was designed to overcome the inherent low frequency noise of solid-state photo- detectors by means of a previously reported modulation technique, named IHOS (Integrated Heterodyne Optical System). The bio-reporter signals were modulated prior to photo-detection, increasing the SNR of solid-state photo-detectors at least by three orders of magnitude. Experiments were performed using isopropyl-beta-d-thiogalactopyranoside (IPTG) as a preliminary step towards testing environmental toxicity. The inducer was used to trigger the expression response of the whole-cell sensors testing the sensitivity of the lab-on-chip. Low intensity bio-reporter optical signals were measured after the whole-cell sensors were exposed to IPTG concentrations of 0.1, 0.05, and 0.02 mM. The experimental results reveal the potential of this technology for future implementation as an inexpensive massive method for rapid environmental toxicity detection.
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.
A new soluble and enzymatically active hybrid of silver and the enzyme glucose oxidase was recent... more A new soluble and enzymatically active hybrid of silver and the enzyme glucose oxidase was recently developed in our lab. We hypothesized that this hybrid carries potential as new antibacterial agent to combat bio lms: by hybrid penetration into the bio lm and scavenging of glucose traces, hydrogen peroxide will be formed by the enzyme, subsequently releasing silver ions from the hybrid’s silver “shell” by local chemical oxidation. These in situ released silver ions are expected effectively to kill bacterial cells located within their immediate vicinity. We designed and established a working ow system for in vitro bio lm growth and comparison of the ef cacy of the antibacterial activity of several forms of silver and the hybrid on E. coli bio lms. Results obtained demonstrated the feasibility of the working hypothesis, thus paving the way for subsequent in vivo studies.
Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental... more Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental mon- itoring applications. The advantages of these devices are that they can be produced as a single-chip, low-power, rugged, inexpensive component, and can be deployed in a variety of non-laboratory settings. However, such biosensors encounter inherent problems in overall system light collection efficiency. The light emitted from the bioluminescent microbial cells is isotropic and passes through various media before it reaches the photon detectors. We studied the bioluminescence distribution and propagation in microbial whole cell biochips. Optical emission and detection were modeled and simulated using an optical ray tracing method. Light emission, transfer and detection were simulated and optimized with respect to two fundamental system parameters: system geometry and bacterial concentration. Optimiza- tion elucidated some of the optical aspects of the biochip, e.g. detector radius values between 300 and 750 m, and bacterial fixation radius values between 800 and 1200 m. Understanding theses aspects may establish a basis for future optimization of similar chips.
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 biosen- sor 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 electrochemi- cal 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 geno- toxicants, 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.
The use of on-chip cellular activity monitoring for biological/chemical sensing is promising for ... more The use of on-chip cellular activity monitoring for biological/chemical sensing is promising for environmental, medical and pharmaceutical applications. The miniaturization revolution in microelectronics is harnessed to provide on-chip detection of cellular activity, opening new horizons for miniature, fast, low cost and portable screening and monitoring devices. In this chapter we survey different on-chip cellular activity detection technologies based on electrochemical, bio-impedance and optical detection. Both prokaryotic and eukaryotic cell-on-chip technologies are mentioned and reviewed.
Electrochemical signal detection can be readily integrated in biosensors and is thus an attractiv... more Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alter- native to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl -d- galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the stan- dard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.
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 micro- system-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 whole- cell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the bio- logical component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the micro- electronics, 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 in- teraction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that con- verts 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.
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.
Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assa... more Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotox- icity assays based upon the use of genetically engi- neered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induc- tion of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu-test, has been fully validated and ISO- and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual geno- toxicity testing devices.
A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong... more A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bio- reporter responded in a dose-dependent manner to three model DNA-damaging agents—hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)—detected 30–60 min after exposure. Detection thresholds were 0.15 μM for MMC, 7.5 μM for nalidixic acid, and approximately 50 μM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensi- tivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2- aminoanthracene following metabolic activation with an S9 mammalian liver fraction.
Recent advances in the convergence of the biological, chemical, physical, and engineering science... more Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.
Electrochemical signal detection can be readily integrated in biosensors and is thus an attractiv... more Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.
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.
We present the first demonstration of a novel bacterial biofilm treatment technique showing a 56%... more We present the first demonstration of a novel bacterial biofilm treatment technique showing a 56% average decrease in bacterial cell viability compared to traditional antibiotic treatments in a Micro-BOAT platform. Integrated linear array charge-coupled devices achieve spatially realized optical density monitoring, correlating to both average biomass and localized biofilm morphology. For on-chip demonstration of biofilm treatment, a unique bioelectric effect using a superpositioned direct and alternating current electric field is applied in the presence of antibiotics. Use of the platform demonstrated successful real-time monitoring of biofilm treatment and validated an on-chip bioelectric effect showing a decrease in both bacterial cell viability and overall biomass.
Applications in Biological Systems and Biotechnology, 2010
Page 154. 4 Biomedical Implications of the Porosity of Microbial Biofilms H. Ben-Yoav Department ... more Page 154. 4 Biomedical Implications of the Porosity of Microbial Biofilms H. Ben-Yoav Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv, Israel N. Cohen ...
A novel micro-fluidic whole cell biosensor for toxicity analysis based on bioluminescence detecti... more A novel micro-fluidic whole cell biosensor for toxicity analysis based on bioluminescence detection was developed. The optical part of the biochip was modelled and simulated to optimize the total light collection efficiency and the system response. The optimization elucidated some of the optical aspects of the biochip. A study evaluating the bioluminescence reaction kinetics was performed and revealed the interdependence between the detected bioluminescence and the physical parameters of the introduced toxin.
This work demonstrates the first utilization of virus molecules as nano-scale biotemplates assemb... more This work demonstrates the first utilization of virus molecules as nano-scale biotemplates assembled on an electrochemical biosensor, allowing for an 8-times increased signal and an improved biosensing performance of 9.5-fold. The versatile and inexpensive biological Tobacco mosaic virus was integrated as a high aspect ratio, low footprint, low-cost, easy to genetically functionalize, nanostructured three-dimensional scaffold for the synthesis of novel multifunctional electrodes. The biotemplated scaffold allows for an increased surface area resulting in higher electrochemical currents, better signal-to-noise ratio and improved sensitivity when incorporated into miniaturized biosensors.
2010 IEEE 26-th Convention of Electrical and Electronics Engineers in Israel, 2010
In this paper we propose a new approach of signal conditioning circuits. The circuit is able to h... more In this paper we propose a new approach of signal conditioning circuits. The circuit is able to handle both electrochemical and bioluminescent sensors, mainly electrochemical sensors. The integrated circuit will convert electrical signal into impulses, making the frequency the carrier of the information. In this way we avoid amplifying stages, reducing circuit complexity and area on chip. The biggest obstacles that we manage to overcome ware the major difference between the currents provided by the sensors and the sense of the currents. We still need to build an interface capable to work not only with one sensor but with 6X6 or 8X8 sensors.
A lab-on-chip consisting of a unique integration of whole-cell sensors, a MOEMS (Micro-Opto-Elect... more A lab-on-chip consisting of a unique integration of whole-cell sensors, a MOEMS (Micro-Opto-Electro-Mechanical-System) modulator, and solid-state photo-detectors was implemented for the first time. Whole-cell sensors were genetically engineered to express a bioluminescent reporter (lux) as a function of the lac promoter. The MOEMS modulator was designed to overcome the inherent low frequency noise of solid-state photo- detectors by means of a previously reported modulation technique, named IHOS (Integrated Heterodyne Optical System). The bio-reporter signals were modulated prior to photo-detection, increasing the SNR of solid-state photo-detectors at least by three orders of magnitude. Experiments were performed using isopropyl-beta-d-thiogalactopyranoside (IPTG) as a preliminary step towards testing environmental toxicity. The inducer was used to trigger the expression response of the whole-cell sensors testing the sensitivity of the lab-on-chip. Low intensity bio-reporter optical signals were measured after the whole-cell sensors were exposed to IPTG concentrations of 0.1, 0.05, and 0.02 mM. The experimental results reveal the potential of this technology for future implementation as an inexpensive massive method for rapid environmental toxicity detection.
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.
A new soluble and enzymatically active hybrid of silver and the enzyme glucose oxidase was recent... more A new soluble and enzymatically active hybrid of silver and the enzyme glucose oxidase was recently developed in our lab. We hypothesized that this hybrid carries potential as new antibacterial agent to combat bio lms: by hybrid penetration into the bio lm and scavenging of glucose traces, hydrogen peroxide will be formed by the enzyme, subsequently releasing silver ions from the hybrid’s silver “shell” by local chemical oxidation. These in situ released silver ions are expected effectively to kill bacterial cells located within their immediate vicinity. We designed and established a working ow system for in vitro bio lm growth and comparison of the ef cacy of the antibacterial activity of several forms of silver and the hybrid on E. coli bio lms. Results obtained demonstrated the feasibility of the working hypothesis, thus paving the way for subsequent in vivo studies.
Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental... more Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental mon- itoring applications. The advantages of these devices are that they can be produced as a single-chip, low-power, rugged, inexpensive component, and can be deployed in a variety of non-laboratory settings. However, such biosensors encounter inherent problems in overall system light collection efficiency. The light emitted from the bioluminescent microbial cells is isotropic and passes through various media before it reaches the photon detectors. We studied the bioluminescence distribution and propagation in microbial whole cell biochips. Optical emission and detection were modeled and simulated using an optical ray tracing method. Light emission, transfer and detection were simulated and optimized with respect to two fundamental system parameters: system geometry and bacterial concentration. Optimiza- tion elucidated some of the optical aspects of the biochip, e.g. detector radius values between 300 and 750 m, and bacterial fixation radius values between 800 and 1200 m. Understanding theses aspects may establish a basis for future optimization of similar chips.
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 biosen- sor 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 electrochemi- cal 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 geno- toxicants, 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.
The use of on-chip cellular activity monitoring for biological/chemical sensing is promising for ... more The use of on-chip cellular activity monitoring for biological/chemical sensing is promising for environmental, medical and pharmaceutical applications. The miniaturization revolution in microelectronics is harnessed to provide on-chip detection of cellular activity, opening new horizons for miniature, fast, low cost and portable screening and monitoring devices. In this chapter we survey different on-chip cellular activity detection technologies based on electrochemical, bio-impedance and optical detection. Both prokaryotic and eukaryotic cell-on-chip technologies are mentioned and reviewed.
Electrochemical signal detection can be readily integrated in biosensors and is thus an attractiv... more Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alter- native to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl -d- galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the stan- dard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.
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 micro- system-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 whole- cell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the bio- logical component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the micro- electronics, 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 in- teraction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that con- verts 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.
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.
Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assa... more Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotox- icity assays based upon the use of genetically engi- neered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induc- tion of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu-test, has been fully validated and ISO- and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual geno- toxicity testing devices.
A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong... more A bacterial genotoxicity reporter strain was con- structed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bio- reporter responded in a dose-dependent manner to three model DNA-damaging agents—hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)—detected 30–60 min after exposure. Detection thresholds were 0.15 μM for MMC, 7.5 μM for nalidixic acid, and approximately 50 μM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensi- tivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2- aminoanthracene following metabolic activation with an S9 mammalian liver fraction.
Recent advances in the convergence of the biological, chemical, physical, and engineering science... more Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.
Uploads
Papers by Hadar Ben-Yoav