Neuronal Network Biosensor for Environmental Threat Detection

Introduction: A critical technology void exists in our ability to detect a broad range of environmental threats ranging from toxic industrial chemicals and materials through chemical agents. NRL has developed a biosensor system that combines microelectrode array technology with neuronal cell culture techniques to yield physiologically relevant information about a broad set of known and unanticipated threats. Extracellular monitoring of bioelectrical activity from cultured networks grown over microelectrode arrays provides a noninvasive approach that allows long-term monitoring. The biosensor system condenses a large amount of laboratory instrumentation to a portable instrument format that requires only minimal training to operate. We have successfully demonstrated the use of this system in recent field exercises in which detection of toxins in both potable water samples and in sea water was performed. A strong correlation between biosensor sensitivity and lethal dosages of a range of environmental threats is presented, and challenges for the future are discussed.

Neurons on Microelectrode Arrays: The sensing element of the neuronal network biosensor contains mouse spinal cord or frontal cortex neurons grown on substrate-integrated microelectrode arrays (MEAs). The MEAs consist of transparent patterns of indium-tin oxide conductors, 10 mm wide, which are photoetched and passivated with a polysiloxane resin. Laser de-insulation of the resin results in 64 recording sites over an area of 1 mm.2 After depositing biological attachment factors on the surface, embryonic neurons are placed over the surface and allowed to grow into mature interconnected networks1 (Fig. 6(a)). Neurons communicate with one another via action potentials or spikes; changes in spike rate are the fundamental means by which information is transmitted in the nervous system. While spikes are typically monitored by piercing cells with glass or metal microelectrodes (inevitably a destructive process), the MEAs allow noninvasive recording of extracellular spike activity (Fig. 6(b)). Neuroactive compounds, which include many environmental threats, act on neuronal receptors to modulate the spike dynamics. This information is readily monitored by the MEAs, and changes in such activity can be used as a means of detecting neuroactive compounds.

FIGURE 6
Spinal cord neuronal network serving as a biological component for the biosensor system. (A) The dual fluorescence image highlights neuronal features with immunocytochemical techniques where the neuronal axons are shown in red and the cell bodies and dendrites are shown in green. On the microelectrode array substrate, several of the 64 recording sites, which are 10 mm in diameter, fluoresce yellow. (B) Representative extracellular recordings from spinal cord cultures.

NRL, in collaboration with the University of North Texas, has recently developed a robust method to store and transport living networks, such that living networks can be shipped across the United States via commercial carrier. This success has led to the emergence of a commercial effort (Applied Neuronal Network Dynamics, Inc.) to supply the biological component of the biosensor system. With the availability of the biological component of the sensor to many potential end-users, neuronal networks are uniquely poised to provide broad spectrum threat detection.

The NRL Portable Biosensor: Traditionally, a large quantity of complex laboratory equipment requiring both significant space and experienced personnel have been needed to make use of cultured neuronal networks. NRL has developed a portable system that condenses this equipment while reducing the expertise needed to operate it.2 The portable biosensor consists of an aluminum case (45 × 30 × 40 cm) containing pumps, valves, and temperature controllers with amplifiers and filters capable of processing microvolt-level extracellular signals (Fig. 7). Each neuronal network on an MEA is enclosed in a stainless steel recording chamber, which maintains the cultures at physiological temperature and provides a means of introducing aqueous phase samples. A high-level user interface for real-time monitoring of neuronal network activity has been developed by NRL.

FIGURE 7
Portable neuronal network biosensor. Open case view showing several internal subsystems including the stainless steel chamber containing the neuronal network.

Sensitivity to Environmental Threats: The neuronal network biosensor has been successfully used to detect a broad spectrum of environmental threats including toxins, organophosphates, and metals. Figure 8(a) shows the rapid response of the neuronal network sensor to saxitoxin, a Schedule 1 chemical agent. The biosensor has been successfully tested off-site from NRL for the blind evaluation of samples at the Eliatox-Oregon workshop held at Oregon-State University in 2002, and more recently for the evaluation of red tide toxins in sea water cultures at the National Oceanic Atmospheric Administration in Charleston, South Carolina. Overall, there is a strong positive correlation (r = 0.92) between the sensitivity of the networks versus toxicity for a wide range of environmental threats (Fig. 8(b)). In general, the networks respond to environmental threats at concentrations 2-3 orders of magnitude below that which induces toxicity.

FIGURE 8
(a) Rapid and reversible inhaibition of mean spike rate from neuronal network biosensor with exposure to the chemical agent saxitoxin at 1nM or 0.3 parts per billion. (b) Strong positive correlation between the sensitivity of the neuronal network biosensor represented as log (IC₅₀), concentration that induces a 50% of the maximal modulation, x-asix and the log (LD₅₀), mg/kg dosage of a compound that, when injected, induces lethality in 50% of the mice or rats. Data points comiled from following agents: tetrodotoxin, saxittoxin, botulinum toxin A, cadmium, mercury, arsenic, chloroquine, brevetoxins, trimethylolpropane phosphate, domoic acid, ethyl alcohol, strychnine, and cyanide.

Prospects for Future Development: The neuronal network biosensor monitors a broad range of compounds for toxicity without being specific for any one compound. Alterations in the baseline bioelectrical activity of the neuronal network provide a means of rapidly detecting environmental threats. Future challenges involve the development and validation of statistically robust threat detection algorithms, exploration of cell preservation techniques to enhance shelf-life of the biological component, and the implementation of neural stem cell technology to reduce or eliminate reliance on animal-derived tissue for biosensor use. This biosensor technology can be readily miniaturized and implemented for environmental monitoring, municipal water source security, and homeland defense applications.

[Sponsored by DARPA and ONR]