A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

University of Maryland

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.

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

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.

Optical modeling of bioluminescence in whole cell biosensors.

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.

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

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.

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

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.

Bacterial genotoxicity bioreporters.

A bacterial genotoxicity reporter strain was constructed 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 bioreporter 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 sensitivity 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.

Microbial genotoxicity bioreporters based on sulA activation.

DNA hybridization detection in microfluidic devices can reduce sample volumes, processing times, and can be integrated with other measurements. However, as device footprints decrease and their complexity increase, the signal-to-noise ratio in these systems also decreases and the sensitivity is thereby compromised. Device miniaturization produces distinct properties and phenomena with greater influence at the micro-scale than at the macro-scale. Here, a diffusion-restriction model was applied to a miniaturized biochip nanovolume reactor to accurately characterize DNA hybridization events that contribute to shifts in both charge transfer resistance and diffusional resistance. These effects are shown to play a significant role in electrochemical impedance spectroscopy (EIS) analyses at these length scales. Our highly functional microfluidic biosensor enables the detection of ssDNA targets selectively, with a calculated detection limit of 3.8 nM, and cross-reactivity of 13% following 20 min incubation with the target. This new biosensing approach can be further modeled and tested elucidating diffusion behavior in miniaturized devices and improving the performance of biosensors.

A microfluidic-based electrochemical biochip for label-free diffusion-restricted DNA hybridization analysis.

Microelectronic devices that contain biological components are typically used to interrogate biology rather than control biological function. Patterned assemblies of proteins and cells have, however, been used for in vitro metabolic engineering, where coordinated biochemical pathways allow cell metabolism to be characterized and potentially controlled on a chip. Such devices form part of technologies that attempt to recreate animal and human physiological functions on a chip and could be used to revolutionize drug development. These ambitious goals will, however, require new biofabrication methodologies that help connect microelectronics and biological systems and yield new approaches to device assembly and communication. Here, we report the electrically mediated assembly, interrogation and control of a multi-domain fusion protein that produces a bacterial signalling molecule. The biological system can be electrically tuned using a natural redox molecule, and its biochemical response is shown to provide the signalling cues to drive bacterial population behaviour. We show that the biochemical output of the system correlates with the electrical input charge, which suggests that electrical inputs could be used to control complex on-chip biological processes.

Hadar Ben-Yoav

MEMS Sensors and Actuators Laboratory (MSAL),
Department of Electrical and Computer Engineering,
Institute for Systems Research,
MEMS Sensors and Actuators Laboratory (MSAL), Department of Electrical and Computer Engineering, Institute for Systems Research

University of Maryland

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis