Nadezhda V. Kulagina

A portable microelectrode array recording system incorporating cultured neuronal networks for neurotoxin detection.

Cultured neuronal networks, which have the capacity to respond to a wide range of neuroactive compounds, have been suggested to be useful for both screening known analytes and unknown compounds for acute neuropharmacologic effects. Extracellular recording from cultured neuronal networks provides a means for extracting physiologically relevant activity, i.e. action potential firing, in a noninvasive manner conducive for long-term measurements. Previous work from our laboratory described prototype portable systems capable of high signal-to-noise extracellular recordings from cardiac myocytes. The present work describes a portable system tailored to monitoring neuronal extracellular potentials that readily incorporates standardized microelectrode arrays developed by and in use at the University of North Texas. This system utilizes low noise amplifier and filter boards, a two-stage thermal control system with integrated fluidics and a graphical user interface for data acquisition and control implemented on a personal computer. Wherever possible, off-the-shelf components have been utilized for system design and fabrication. During use with cultured neuronal networks, the system typically exhibits input referred noise levels of only 4-6 microVRMS, such that extracellular potentials exceeding 40 microV can be readily resolved. A flow rate of up to 1 ml/min was achieved while the cell recording chamber temperature was maintained within a range of 36-37 degrees C. To demonstrate the capability of this system to resolve small extracellular potentials, pharmacological experiments with cultured neuronal networks have been performed using ion channel blockers, tetrodotoxin and tityustoxin. The implications of the experiments for neurotoxin detection are discussed.

Nonantibody-based recognition: alternative molecules for detection of pathogens.

Immunoassays have been well established for many years as the cornerstone of detection technologies. These assays are sensitive, selective and, in general, highly resistant to interference from complex sample matrices when compared with nucleic acid-based tests. However, both antibody- and nucleic acid-based detection systems require a priori knowledge of the target and development of specific reagents; multiplexed assays can become increasingly problematic when attempting to detect a plethora of different targets, the identities of which are unknown. In an effort to circumvent many of the limitations inherent in these conventional assays, other recognition reagents are being explored as alternatives, or indeed as adjuncts, to antibodies for pathogen and toxin detection. This article will review a number of different recognition systems ranging in complexity from small molecules, such as nucleic-acid aptamers, carbohydrates and peptides, to systems as highly complicated as whole cells and organisms. All of these alternative systems have tremendous potential to achieve superior sensitivity, selectivity, and stability, but are also subject to their own limitations, which are also discussed. In short, while in its infancy, this field holds great promise for the development of rapid, fieldable assays that are highly complementary to existing antibody- and nucleic acid-based technologies.

Methods for characterizing interspike intervals and identifying bursts in neuronal activity

Neurons produce complex patterns of electrical spikes, which are often clustered in bursts. The patterns of spikes and bursts can change substantially when neurons are exposed to toxins and chemical agents. For that reason, characterization of these patterns is important for the development of neuron-based biosensors for environmental threat exposure. Here, we develop a quantitative approach to describe the distribution of interspike intervals, based on plotting histograms of the logarithm of the interspike interval. This approach provides a method for automatically classifying spikes into bursts, which does not depend on assumptions about the burst parameters. Furthermore, the approach provides a sensitive technique for detecting changes in spike and burst patterns induced by pharmacological exposure. Hence, it is suitable for use both as a research tool and for deployment in a neuron-based biosensor.

Antimicrobial Peptides: New Recognition Molecules for Detecting Botulinum Toxins

Many organisms secrete antimicrobial peptides (AMPs) for protection against harmful microbes. The present study describes detection of botulinum neurotoxoids A, B and E using AMPs as recognition elements in an array biosensor. While AMP affinities were similar to those for anti-botulinum antibodies, differences in binding patterns were observed and can potentially be used for identification of toxoid serotype. Furthermore, some AMPs also demonstrated superior detection sensitivity compared to antibodies: toxoid A could be detected at 3.5 LD50 of the active toxin in a 75-min assay, whereas toxoids B and E were detected at 14 and 80 LD50 for their respective toxins.

Sensitivity of the Neuronal Network Biosensor to Environmental Threats

It is widely acknowledged that there is a critical need for broad-spectrum environmental threat detection. While cells/tissue-based biosensors have been discussed for many years as a means of meeting this critical need, these kinds of systems have met with logistic concerns, in particular with regard to stability. Our group has been working with cultured neuronal networks, which have the capacity to respond to a wide range of neuroactive compounds and are sufficiently robust to be shipped to end users. The basis of operation involves extracellular recording using thin-film microelectrode arrays where spontaneous bioelectrical activity, that is, spike firing, can be monitored in a noninvasive manner conducive for potentially long-term measurements. This work describes the current status of our efforts for the fabrication of a portable biosensor that incorporates cultured neuronal networks grown over standardized microelectrode arrays. Based on our protocol for aqueous phase sample introduction under constant flow conditions, minimal variation in mean spike rate is observed, consistent with temporal stability, such that changes of>10% are readily distinguished. To demonstrate the capability of this system, changes are reported in mean spike rate and network synchronization resulting from exposure to different model environmental threats, cadmium and strychnine. The sensitivity of this assay approach and implications of the experimental findings for environmental threat detection are discussed.

Blind Laboratory Trials for Multiple Pathogens in Spiked Food Matrices

Abstract Previously developed assays for Salmonella typhimurium and staphylococcal enterotoxin B (SEB) were combined into a single multiplexed test and integrated into a fully automated prototype of the NRL Array Biosensor. Tests were performed on 216 blind samples of water, apple juice, and milk spiked with SEB (1–10,000 pg/ml) and S. typhimurium (5×103−5×107 colony‐forming units/ml). SEB and S. typhimurium were routinely detected in both water and apple juice at 100 pg/ml and 5×105 colony‐forming units/ml respectively. Inclusion of milk as a sample matrix decreased the sensitivity of the assays by an order of magnitude.