David A. Stenger

Development and application of cell-based biosensors

AbstractBiosensors incorporate a biological sensing element that converts a change in an immediate environment to signals conducive for processing. Biosensors have been implemented for a number of applications ranging from environmental pollutant detection to defense monitoring. Biosensors have two intriguing characteristics: (1) they have a naturally evolved selectivity to biological or biologically active analytes; and (2) biosensors have the capacity to respond to analytes in a physiologically relevant manner. In this paper, molecular biosensors, based on antibodies, enzymes, ion channels, or nucleic acids, are briefly reviewed. Moreover, cell-based biosensors are reviewed and discussed. Cell-based biosensors have been implemented using microorganisms, particularly for environmental monitoring of pollutants. Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner that can offer insight into the physiological effect of an analyte. Several approaches for transduction of cellular signals are discussed; these approaches include measures of cell metabolism, impedance, intracellular potentials, and extracellular potentials. Among these approaches, networks of excitable cells cultured on microelectrode arrays are uniquely poised to provide rapid, functional classification of an analyte and ultimately constitute a potentially effective cell-based biosensor technology. Three challenges that constitute barriers to increased cell-based biosensor applications are presented: analytical methods, reproducibility, and cell sources. Possible future solutions to these challenges are discussed.

Detection of physiologically active compounds using cell-based biosensors

Cell-based biosensors are portable devices that contain living biological cells that monitor physiological changes induced by exposure to environmental perturbations such as toxicants, pathogens or other agents. Methods of detecting physiological changes include extracellular electrical recordings, optical measurements, and, in the future, functional genomics and proteomics. Several technical developments are occurring that will increase the feasibility of cell-based biosensors for field applications; these developments include stem cell and 3D culture technologies. Possible scenarios for the use of cell-based biosensors include broad-range detectors of unknown threat agents and functional assessment of identified agents.

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth‐regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell‐derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF‐deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth‐stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2‐bis‐(O‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G‐proteins, the protein kinase C inhibitor H7 and the mitogen‐activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth‐regulatory signal during neurogenesis via pathways involving pertussis toxin‐sensitive G‐proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection

ABSTRACT DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, φ29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and φ29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses

ABSTRACT The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B1, B2, C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.

Using a Resequencing Microarray as a Multiple Respiratory Pathogen Detection Assay

ABSTRACT Simultaneous testing for detection of infectious pathogens that cause similar symptoms (e.g., acute respiratory infections) is invaluable for patient treatment, outbreak prevention, and efficient use of antibiotic and antiviral agents. In addition, such testing may provide information regarding possible coinfections or induced secondary infections, such as virally induced bacterial infections. Furthermore, in many cases, detection of a pathogen requires more than genus/species-level resolution, since harmful agents (e.g., avian influenza virus) are grouped with other, relatively benign common agents, and for every pathogen, finer resolution is useful to allow tracking of the location and nature of mutations leading to strain variations. In this study, a previously developed resequencing microarray that has been demonstrated to have these capabilities was further developed to provide individual detection sensitivity ranging from 101 to 103 genomic copies for more than 26 respiratory pathogens while still retaining the ability to detect and differentiate between close genetic neighbors. In addition, the study demonstrated that this system allows unambiguous and reproducible sequence-based strain identification of the mixed pathogens. Successful proof-of-concept experiments using clinical specimens show that this approach is potentially very useful for both diagnostics and epidemic surveillance.

Survival and neurite outgrowth of rat cortical neurons in three-dimensional agarose and collagen gel matrices.

To better understand interactions between neurons and extracellular matrix equivalents, embryonic day-18 rat cortical neurons were immobilized and maintained in culture for up to 24 days in agarose and type I collagen gels. Using live/dead staining, neuronal cultures in low density collagen gel lasted at least 3 weeks. At 14 days, over 50% of immobilized cells in collagen gel were found viable while in low density agarose gel no cells survived. In situ cell death detection showed that most, if not all, dead cells in either of the gels underwent apoptosis. The collagen-trapped neurons exhibited normal neuronal polarity and developed long neurites, estimated at over 500 microm. The results suggest that collagen, because it is a major extracellular matrix constituent, suppresses apoptosis and provides a suitable substrate for neuronal survival and differentiation.

Portable cell-based biosensor system for toxin detection

Abstract A portable cell-based biosensor has been developed and characterized. The prototype system relies on extracellular recording from excitable cells cultured over an array of platinized gold microelectrodes. Extracellular potentials were bandpass filtered between 80 Hz to 2.8 kHz and amplified with a selectable gain of either 1000 or 5000. The input-referred noise level of the system was only 8.7 μV RMS in the laboratory setting, reaching only 10.6 μV RMS in an outdoor environment, more than sufficient for measurement of extracellular potentials from excitable cells. The system also incorporates a feedback control system for temperature regulation and a 36-channel multiplexer for selection of up to four output channels for simultaneous display. Wherever possible, low-cost ‘off-the-shelf’ components were utilized in this prototype biosensor design. Using this system, extracellular recordings from chick myocardiocytes were performed under both laboratory and outdoor conditions.

Characterization of acute neurotoxic effects of trimethylolpropane phosphate via neuronal network biosensors

We have utilized cultured neuronal networks grown on microelectrode arrays to demonstrate rapid, reliable detection of a toxic compound, trimethylolpropane phosphate (TMPP). Initial experiments, which were performed blind, demonstrated rapid classification of the compound as a convulsant, a finding consistent with previous whole animal neurobehavioral studies. TMPP (2-200 microM) reorganized network spike activity into synchronous, quasi-periodic burst episodes. Integrated burst amplitudes invariably increased, reflecting higher spike frequencies within each burst. The variability of network burst parameters, quantified as coefficients of variation (CVs), was decreased. Mean CVs for burst duration, interburst interval, and burst rate were lowered by 42+/-13, 58+/-5.5, and 62+/-1.8%, respectively (mean+/-SEM, n=8 cultures, 197 channels). These changes in network activity paralleled the effects induced by bicuculline, a known disinhibitory and seizure-inducing drug, and confirmed classification of TMPP as a potential epileptogenic compound. Simple pharmacological tests permit exploration of mechanisms underlying observed activity shifts. The EC(50) for GABA inhibition of network activity was increased from 2.8 to 7.0 microM by 20 microM TMPP and to 20.5 microM by 200 microM TMPP. Parallel dose-response curves suggest that TMPP acts by a competitive antagonism of GABA inhibition, and are consistent with reported patch-clamp analysis of TMPP-induced reduction of inhibitory postsynaptic current amplitudes. The potency of TMPP in generating epileptiform activity in vitro was comparable to concentrations reported for in vivo studies. TMPP and bicuculline produced both increases and decreases in burst rate depending on native spontaneous bursting levels. These results demonstrate a need for multivariate analysis of network activity changes to yield accurate predictions of compound effects.

Rational pattern design for in vitro cellular networks using surface photochemistry

The ability to create patterns of specific silane monolayers by deep ultraviolet lithography has been previously demonstrated, and preliminary attempts have been made to use these patterns to control adhesion and outgrowth of neurons and other types of mammalian cells. Here we report characterization of the mechanisms involved in these photoinitiated processes and their utility in various strategies for creating patterns for biologically relevant systems. We have divided the mechanisms into three general classes. The first is surface photolysis of the silane monolayer, which appears to proceed by a purely photochemical mechanism. The second mechanism involves direct photochemical conversion of a terminal functional group on a silane monolayer into a species with altered properties, e.g., the conversion of a thiol to a more oxidized form that inhibits the subsequent adhesion of proteins. The third is a photolytic degradation of the monolayer. The mechanisms have been probed by x‐ray photoelectron spectrosc...