Hadar Ben-Yoav

Whole-cell biochips for bio-sensing: integration of live cells and inanimate surfaces

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.

Bacterial genotoxicity bioreporters.

Ever since the introduction of the Salmonella typhimurium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotoxicity assays based upon the use of genetically engineered 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 induction 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 genotoxicity testing devices.

Optical modeling of bioluminescence in whole cell biosensors.

Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental monitoring 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. Optimization elucidated some of the optical aspects of the biochip, e.g. detector radius values between 300 and 750 microm, and bacterial fixation radius values between 800 and 1200 microm. Understanding theses aspects may establish a basis for future optimization of similar chips.

Evaluation of chrono-amperometric signal detection for the analysis of genotoxicity by a whole cell biosensor

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.

Towards toxicity detection using a lab-on-chip based on the integration of MOEMS and whole-cell sensors

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 0.02mM. The experimental results reveal the potential of this technology for future implementation as an inexpensive massive method for rapid environmental toxicity detection.

Optical and Electrical Interfacing Technologies for Living Cell Bio-Chips

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 biological 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 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.

Enzymatically attenuated in situ release of silver ions to combat bacterial biofilms: a feasibility study

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 biofilms: by hybrid penetration into the biofilm 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 flow system for in vitro biofilm growth and comparison of the efficacy of the antibacterial activity of several forms of silver and the hybrid on E. coli biofilms. Results obtained demonstrated the feasibility of the working hypothesis, thus paving the way for subsequent in vivo studies.

VLSI universal signal conditioning circuit for electrochemical and bioluminescent sensors

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 6×6 or 8×8 sensors.

Integrated Polypyrrole Flexible Conductors for Biochips and MEMS Applications

Integrated polypyrrole, a conductive polymer, interconnects on polymeric substrates were microfabricated for flexible sensors and actuators applications. It allows manufacturing of moving polymeric microcomponents suitable, for example, for micro-optical-electromechanical (MOEMS) systems or implanted sensors. This generic technology allows producing “all polymer” components where the polymers serve as both the structural and the actuating materials. In this paper we present two possible novel architectures that integrate polypyrrole conductors with other structural polymers: (a) polypyrrole embedded into flexible polydimethylsiloxane (PDMS) matrix forming high aspect ratio electrodes and (b) polypyrrole deposited on planar structures. Self-aligned polypyrrole electropolymerization was developed and demonstrated for conducting polymer lines on either gold or copper seed layers. The electropolymerization process, using cyclic voltammetry from an electrolyte containing the monomer, is described, as well as the devices’ characteristics. Finally, we discuss the effect of integrating conducting polymers with metal seed layer, thus enhancing the device durability and reliability.

Electronically Directed Integration of Whole-Cell Biosensors on Bio-Chips

Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 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.