Robust Covalent Coupling Scheme for the Development of FRET Aptasensor based on Amino-Silane-Modified Graphene Oxide.

In recent years, numerous aptamers have been physisorbed on graphene oxide (GO) to develop fluorescence resonance energy transfer-based aptasensors using the fluorescence quenching property of GO. However, physisorbed aptasensors show poor signal reversibility and reproducibility as well as nonspecific probe displacement, and thereby are not suitable for many analytical applications. To overcome these problems when working with complex biological samples, we developed a facile and robust covalent surface functionalization technique for GO-based fluorescent aptasensors using a well-studied adenosine triphosphate binding aptamer (ABA). In the scheme, GO is first modified with amino-silane, and further with glutaraldehyde to create available carbonyl groups for the covalent attachment of a fluorophore and an amino dual modified ABA. The surface modification method was characterized by ζ-potential, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy (FTIR). The linearity, sensitivity, selectivity, and reversibility of the resulting GO-based covalent aptasensor was determined and systematically compared with the physisorbed aptasensor. Although both sensors showed similar performance in terms of sensitivity and linearity, better selectivity and higher resistance to nonspecific probe displacement was achieved with the developed covalent ABA sensor. The surface modification technique developed here is independent of the aptamer sequence, and therefore could be used universally for different analytical applications simply by changing the aptamer sequence for the target biomolecule.

Real-time characterization of uptake kinetics of glioblastoma vs. astrocytes in 2D cell culture using microelectrode array.

Extracellular measurement of uptake/release kinetics and associated concentration dependencies provides mechanistic insight into the underlying biochemical processes. Due to the recognized importance of preserving the natural diffusion processes within the local microenvironment, measurement approaches which provide uptake rate and local surface concentration of adherent cells in static media are needed. This paper reports a microelectrode array device and a methodology to measure uptake kinetics as a function of cell surface concentration in adherent 2D cell cultures in static fluids. The microelectrode array simultaneously measures local concentrations at five positions near the cell surface in order to map the time-dependent concentration profile which in turn enables determination of surface concentrations and uptake rates, via extrapolation to the cell plane. Hydrogen peroxide uptake by human astrocytes (normal) and glioblastoma multiforme (GBM43, cancer) was quantified for initial concentrations of 20 to 500 µM over time intervals of 4000 s. For both cell types, the overall uptake rate versus surface concentration relationships exhibited non-linear kinetics, well-described by a combination of linear and Michaelis-Menten mechanisms and in agreement with the literature. The GBM43 cells showed a higher uptake rate over the full range of concentrations, primarily due to a larger linear component. Diffusion-reaction models using the non-linear parameters and standard first-order relationships are compared. In comparison to results from typical volumetric measurements, the ability to extract both uptake rate and surface concentration in static media provides kinetic parameters that are better suited for developing reaction-diffusion models to adequately describe behavior in more complex culture/tissue geometries. The results also highlight the need for characterization of the uptake rate over a wider range of cell surface concentrations in order to evaluate the potential therapeutic role of hydrogen peroxide in cancerous cells.

Presence of stromal cells in a bioengineered tumor microenvironment alters glioblastoma migration and response to STAT3 inhibition.

Despite the increasingly recognized importance of the tumor microenvironment (TME) as a regulator of tumor progression, only few in vitro models have been developed to systematically study the effects of TME on tumor behavior in a controlled manner. Here we developed a three-dimensional (3D) in vitro model that recapitulates the physical and compositional characteristics of Glioblastoma (GBM) extracellular matrix (ECM) and incorporates brain stromal cells such as astrocytes and endothelial cell precursors. The model was used to evaluate the effect of TME components on migration and survival of various patient-derived GBM cell lines (GBM10, GBM43 and GBAM1) in the context of STAT3 inhibition. Migration analysis of GBM within the 3D in vitro model demonstrated that the presence of astrocytes significantly increases the migration of GBM, while presence of endothelial precursors has varied effects on the migration of different GBM cell lines. Given the role of the tumor microenvironment as a regulator of STAT3 activity, we tested the effect of the STAT3 inhibitor SH-4-54 on GBM migration and survival. SH-4-54 inhibited STAT3 activity and reduced 3D migration and survival of GBM43 but had no effect on GBM10. SH-4-54 treatment drastically reduced the viability of the stem-like line GBAM1 in liquid culture, but its effect lessened in presence of a 3D ECM and stromal cells. Our results highlight the interplay between the ECM and stromal cells in the microenvironment with the cancer cells and indicate that the impact of these relationships may differ for GBM cells of varying genetic and clinical histories.

Integration of a Genetically Encoded Calcium Molecular Sensor into Photopolymerizable Hydrogels for Micro-Optrode-Based Sensing.

Genetically encoded molecular-protein sensors (GEMS) are engineered to sense and quantify a wide range of biological substances and events in cells, in vitro and even in vivo with high spatial and temporal resolution. Here, we aim to stably incorporate these proteins into a photopatternable matrix, while preserving their functionality, to extend the application of these proteins as spatially addressable optical biosensors. For this reason, we examined the fabrication of 3D hydrogel microtips doped with a genetically encoded fluorescent biosensor, GCaMP3, at the end of an optical fiber. Stable incorporation parameters of GCaMP3 into a photo-cross-linkable monomer matrix were investigated through a series of characterization and optimization experiments. Different precursor-solution formulations and irradiation parameters of in situ photopolymerization were tested to determine the factors affecting protein stability and sensor reproducibility during photoencapsulation. The microstructure and performance of hydrogel microtips were controlled by varying UV irradiation intensity as well as the molecular weight and concentration of the photocurable monomer, PEGDA (polyethylene glycol diacrylate), in precursor solution. Protein-doped hydrogel micro-optrodes (microtip sensors) were fabricated successfully and reproducibly at the distal end of optical fiber. Under optimized conditions, the bioactivity of GCaMP3 within a hydrogel matrix of micro-optrodes remained similar to that of the protein-free matrix in buffer. The limit of detection of protein optrodes for free calcium was also determined to be 4.3 nM. The hydrogel formulation and fabrication process demonstrated here using microtip optrodes can be easily adapted to other conformation-dependent protein biosensors and can be used in sensing applications.

Extracellular Matrix Properties Regulate the Migratory Response of Glioblastoma Stem Cells in Three-Dimensional Culture.

Diffuse infiltration across brain tissue is a hallmark of glioblastoma and the main cause of unsuccessful total resection that leads to tumor reappearance. A subpopulation termed glioblastoma stem cells (GSCs) has been directly related to aggressive invasion; nonetheless, their migratory characteristics and regulation by the microenvironment are still unknown. In this study, we developed a composite matrix of hyaluronan (HA) structurally supported by a collagen-oligomer fibril network to simulate the brain tumor extracellular matrix (ECM) composition. Matrigel-coated microfibers were embedded within the matrix to create a tunable dual niche microenvironment that resembles the vascular network of the brain. This model was compared with the most commonly used in vitro three-dimensional (3D) culture formats, Matrigel and collagen type-I monomer matrices, to study how the mechanical and compositional properties of the ECM alter the migration characteristics of GSC neurospheres. The migration mode, distance, velocity, and morphology of the GSCs were monitored over a 72-h period. The cells altered their migration mode depending on the matrix composition, showing migration by expansive growth in Matrigel matrices, multicellular extension along rigid interfaces (as Matrigel glass and coated microfibers), and mesenchymal single-cell migration in collagen matrices. Velocity and distance of migration within each composition varied according to matrix mechanical properties. In the dual niche system, the presence of HA reduced velocity and number of migratory cells; however, cells that came in contact with the pseudovessels exhibited collective migration by an extensive strand and reached higher velocities than cells migrating individually across the 3D matrix. Our results show that GSCs adopt varied migration mechanisms to invade multiple ECM microenvironments, and the migration characteristics exhibited are highly influenced by the matrix physical properties. Moreover, GSC neurospheres exhibit concomitant single and collective migration as a function of the microenvironment topography to reach the most productive migration strategy.

Glial cells, but not neurons, exhibit a controllable response to a localized inflammatory microenvironment in vitro.

The ability to design long-lasting intracortical implants hinges on understanding the factors leading to the loss of neuronal density and the formation of the glial scar. In this study, we modify a common in vitro mixed cortical culture model using lipopolysaccharide (LPS) to examine the responses of microglia, astrocytes, and neurons to microwire segments. We also use dip-coated polyethylene glycol (PEG), which we have previously shown can modulate impedance changes to neural microelectrodes, to control the cellular responses. We find that microglia, as expected, exhibit an elevated response to LPS-coated microwire for distances of up to 150 µm, and that this elevated response can be mitigated by co-depositing PEG with LPS. Astrocytes exhibit a more complex, distance-dependent response, whereas neurons do not appear to be affected by the type or magnitude of glial response within this in vitro model. The discrepancy between our in vitro responses and typically observed in vivo responses suggest the importance of using a systems approach to understand the responses of the various brain cell types in a chronic in vivo setting, as well as the necessity of studying the roles of cell types not native to the brain. Our results further indicate that the loss of neuronal density observed in vivo is not a necessary consequence of elevated glial activation.

Resistive and reactive changes to the impedance of intracortical microelectrodes can be mitigated with polyethylene glycol under acute in vitro and in vivo settings.

The reactive response of brain tissue to implantable intracortical microelectrodes is thought to negatively affect their recordable signal quality and impedance, resulting in unreliable longitudinal performance. The relationship between the progression of the reactive tissue into a glial scar and the decline in device performance is unclear. We show that exposure to a model protein solution in vitro and acute implantation result in both resistive and capacitive changes to electrode impedance, rather than purely resistive changes. We also show that applying 4000 MW polyethylene glycol (PEG) prevents impedance increases in vitro, and reduces the percent change in impedance in vivo following implantation. Our results highlight the importance of considering the contributions of non-cellular components to the decline in neural microelectrode performance, and present a proof of concept for using a simple dip-coated PEG film to modulate changes in microelectrode impedance.

Mouse and human islets survive and function after coating by biosilicification.

Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9-15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice.

Islet ß-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model.

Type 1 diabetes is preceded by islet ß-cell dysfunction, but the mechanisms leading to ß-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in ß-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation. Prediabetic NOD mice were relatively glucose intolerant and had defective insulin secretion with elevated proinsulin:insulin ratios compared with control strains. Isolated islets from NOD mice displayed age-dependent increases in parameters of ER stress, morphologic alterations in ER structure by electron microscopy, and activation of nuclear factor-κB (NF-κB) target genes. Upon exposure to a mixture of proinflammatory cytokines that mimics the microenvironment of type 1 diabetes, MIN6 ß-cells displayed evidence for polyribosomal runoff, a finding consistent with the translational initiation blockade characteristic of ER stress. We conclude that ß-cells of prediabetic NOD mice display dysfunction and overt ER stress that may be driven by NF-κB signaling, and strategies that attenuate pathways leading to ER stress may preserve ß-cell function in type 1 diabetes.

A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors.

This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 µA mM(-1) cm(-2)), linear range (0.0037-12 mM), detection limit (3.7 µM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H(2)O(2) response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.

Cell-mediated deposition of porous silica on bacterial biofilms.

Living hybrid materials that respond dynamically to their surrounding environment have important applications in bioreactors. Silica based sol-gels represent appealing matrix materials as they form a mesoporous biocompatible glass lattice that allows for nutrient diffusion while firmly encapsulating living cells. Despite progress in sol-gel cellular encapsulation technologies, current techniques typically form bulk materials and are unable to generate regular silica membranes over complex geometries for large-scale applications. We have developed a novel biomimetic encapsulation technique whereby endogenous extracellular matrix molecules facilitate formation of a cell surface specific biomineral layer. In this study, monoculture Pseudomonas aeruginosa and Nitrosomonas europaea biofilms are exposed to silica precursors under different acid conditions. Scanning electron microscopy (SEM) imaging and electron dispersive X-ray (EDX) elemental analysis revealed the presence of a thin silica layer covering the biofilm surface. Cell survival was confirmed 30 min, 30 days, and 90 days after encapsulation using confocal imaging with a membrane integrity assay and physiological flux measurements of oxygen, glucose, and NH 4⁺. No statistical difference in viability, oxygen flux, or substrate flux was observed after encapsulation in silica glass. Shear induced biofilm detachment was assessed using a particle counter. Encapsulation significantly reduced detachment rate of the biofilms for over 30 days. The results of this study indicate that the thin regular silica membrane permits the diffusion of nutrients and cellular products, supporting continued cellular viability after biomineralization. This technique offers a means of controllably encapsulating biofilms over large surfaces and complex geometries. The generic deposition mechanism employed to form the silica matrix can be translated to a wide range of biological material and represents a platform encapsulation technology.

Optical nanosensor architecture for cell-signaling molecules using DNA aptamer-coated carbon nanotubes.

We report a novel optical biosensor platform using near-infrared fluorescent single-walled carbon nanotubes (SWNTs) functionalized with target-recognizing aptamer DNA for noninvasively detecting cell-signaling molecules in real time. Photoluminescence (PL) emission of aptamer-coated SWNTs is modulated upon selectively binding to target molecules, which is exploited to detect insulin using an insulin-binding aptamer (IBA) as a molecular recognition element. We find that nanotube PL quenches upon insulin recognition via a photoinduced charge transfer mechanism with a quenching rate of k(q) = 5.85 × 10(14) M(-1) s(-1) and a diffusion-reaction rate of k(r) = 0.129 s(-1). Circular dichroism spectra reveal for the first time that IBA strands retain a four-stranded, parallel guanine quadruplex conformation on the nanotubes, ensuring target selectivity. We demonstrate that these IBA-functionalized SWNT sensors incorporated in a collagen extracellular matrix (ECM) can be regenerated by removing bound analytes through enzymatic proteolysis. As proof-of-concept, we show that the SWNT sensors embedded in the ECM promptly detect insulin secreted by cultured pancreatic INS-1 cells stimulated by glucose influx and report a gradient contour of insulin secretion profile. This novel design enables new types of label-free assays and noninvasive, in situ, real-time detection schemes for cell-signaling molecules.

Oscillatory glucose flux in INS 1 pancreatic ß cells: a self-referencing microbiosensor study.

Signaling and insulin secretion in ß cells have been reported to demonstrate oscillatory modes, with abnormal oscillations associated with type 2 diabetes. We investigated cellular glucose influx in ß cells with a self-referencing (SR) microbiosensor based on nanomaterials with enhanced performance. Dose-response analyses with glucose and metabolic inhibition studies were used to study oscillatory patterns and transporter kinetics. For the first time, we report a stable and regular oscillatory uptake of glucose (averaged period 2.9±0.6 min), which corresponds well with an oscillator model. This oscillatory behavior is part of the feedback control pathway involving oxygen, cytosolic Ca(2+)/ATP, and insulin secretion (periodicity approximately 3 min). Glucose stimulation experiments show that the net Michaelis-Menten constant (6.1±1.5 mM) is in between GLUT2 and GLUT9. Phloretin inhibition experiments show an EC(50) value of 28±1.6 µM phloretin for class I GLUT proteins and a concentration of 40±0.6 µM phloretin caused maximum inhibition with residual nonoscillating flux, suggesting that the transporters not inhibited by phloretin are likely responsible for the remaining nonoscillatory uptake, and that impaired uptake via GLUT2 may be the cause of the oscillation loss in type 2 diabetes. Transporter studies using the SR microbiosensor will contribute to diabetes research and therapy development by exploring the nature of oscillatory transport mechanisms.

Effects of adsorbed proteins, an antifouling agent and long-duration DC voltage pulses on the impedance of silicon-based neural microelectrodes.

The successful use of implantable neural microelectrodes as neuroprosthetic devices depends on the mitigation of the reactive tissue response of the brain. One of the factors affecting the ultimate severity of the reactive tissue response and the in vivo electrical properties of the microelectrodes is the initial adsorption of proteins onto the surface of the implanted microelectrodes. In this study we quantify the increase in microelectrode impedance magnitude at physiological frequencies following electrode immersion in a 10% bovine serum albumin (BSA) solution. We also demonstrate the efficacy of a common antifouling molecule, poly(ethylene glycol) (PEG), in preventing a significant increase in microelectrode impedance. In addition, we show the feasibility of using long-duration DC voltage pulses to remove adsorbed proteins from the microelectrode surface.

In vitro biocompatibility studies of antibacterial quaternary polymers.

Quaternized copolymers of 4-vinylpyridine and poly(ethylene glycol) methyl ether methacrylate are known to have antibacterial properties and have displayed biocompatibility in red blood cell hemolysis assays. The results from hemolysis assays have shown substantial promise, but the technique is rudimentary and only a first step toward the determination of biocompatibility. The present paper further explores the biocompatibility of these copolymers through comprehensive cell viability assays performed on Caco-2 human epithelial cells cultivated in vitro. We have shown that these copolymers are biocompatible at concentrations above their minimum bactericidal concentrations, leading to selectivity values that compare well with other microbicidal products.

Magnetic insertion system for flexible electrode implantation.

Chronic recording electrodes are a vital tool for brain research and neural prostheses. Despite decades of advances in recording technology, probe structures and implantation methods have changed little over time. Then as now, compressive insertion methods require probes to be constructed from hard, stiff materials, such as silicon, and contain a large diameter shank to penetrate the brain, particularly for deeper structures. The chronic presence of these probes results in an electrically isolating glial scar, degrading signal quality over time. This work demonstrates a new magnetic tension-based insertion mechanism that allows for the use of soft, flexible, and thinner probe materials, overcoming the materials limitations of modern electrodes. Probes are constructed from a sharp magnetic tip attached to a flexible tether. A pulsed magnetic field is generated in a coil surrounding a glass pipette containing the electrode. The applied field pulls the electrode tip forward, accelerating the probe into the neural tissue with a penetration depth that is calibrated against the charge voltage. Mathematical modeling and agar gel insertion testing demonstrate that the electrode can be implanted to a predictable depth given system specific parameters. Trial rodent implantations resulted in discernible single-unit activity on one of the probes. The current prototype demonstrates the feasibility of a tension based, magnetically driven implantation system and opens the door to a wide variety of new minimally invasive probe materials and configurations.

Calibration of neurotransmitter release from neural cells for therapeutic implants.

In this work we quantified the in vitro calibration relationships between high frequency electrical stimulation and GABA and glutamate release in both mature retinoic acid differentiated P19 neurons and immortalized embryonic cortical cells engineered to express glutamic acid decarboxylase, GAD65. Extracellular glutamate and GABA was quantified by 2D gas chromatography and time of flight mass spectrometry after stimulation at varying amplitudes and frequencies. Amplitude sweeps resulted in a linear calibration for P19 neurons; the level of neurotransmitter varied over one order of magnitude from ~ 200 pg/neuron to ~ 1.2 ng/neuron for glutamate and ~ 1 ng/neuron to ~ 2 ng/neuron for GABA, depending on the stimulation amplitude. Frequency sweeps resulted in a peak release at 250 Hz for glutamate and 400 Hz for GABA in P19 cells. The GABA transporter inhibitor, nipecotic acid, increased extracellular GABA levels and decrease glutamate. In contrast the embryonic cortical cells had a strongly nonlinear dependency of release on stimulation amplitude, and a weak dependence on frequency. These cells had roughly equal extracellular glutamate and GABA levels after stimulation despite the expression of GAD65. In addition glutamate and GABA levels were insensitive to nipecotic acid. These results demonstrate an ability to calibrate and tune neurotransmitter release from neural cells using high frequency stimulation parameters.

Thin-film silica sol-gel coatings for neural microelectrodes.

The reactive tissue response of the brain to chronically implanted materials remains a formidable obstacle to stable recording from implanted microelectrodes. One approach to mitigate this response is to apply a bioactive coating in the form of an ultra-porous silica sol-gel, which can be engineered to improve biocompatibility and to enable local drug delivery. The first step in establishing the feasibility of such a coating is to investigate the effects of the coating on electrode properties. In this paper, we describe a method to apply a thin-film silica sol-gel coating to silicon-based microelectrodes, and discuss the resultant changes in the electrode properties. Fluorescently labeled coatings were used to confirm coating adherence to the electrode. Cyclic voltammetry and impedance spectroscopy were used to evaluate electrical property changes. The silica sol-gel was found to successfully adhere to the electrodes as a thin coating. The voltammograms revealed a slight increase in charge carrying capacity of the electrodes following coating. Impedance spectrograms showed a mild increase in impedance at high frequencies but a more pronounced decrease in impedance at mid to low frequencies. These results demonstrate the feasibility of applying silica sol-gel coatings to silicon-based microelectrodes and are encouraging for the continued investigation of their use in mitigating the reactive tissue response.

Compartmentalized nanocomposite for dynamic nitric oxide release.

Nitric oxide (NO) is an important cell-signaling molecule whose role in a variety of cellular processes such as differentiation and apoptosis depends strongly on its concentration and flux levels. This work describes and characterizes a novel nitric oxide releasing nanocomposite, capable of photostimulated NO flux that can by dynamically modulated in within a range of biological levels. This material mimics the common compartmentalization strategies used by living cells to achieve its novel features. The material is constructed by encapsulating a photosensitive nitric oxide donor within lipid vesicles with an average diameter of 150 nm. The vesicles are then doped into the interstitial liquid phase of a solid porous silica matrix, which has previously demonstrated biological compatibility and capabilities as a growth surface for mammalian cells. Stimulation by a light source produces a step increase in NO concentration within seconds. The NO flux at the surface of the material is measured to be 14 pmol-cm(-2) sec(-1) using a NO selective self-referencing amperometric microsensor. The NO concentration profile decreases with distance perpendicular to the surface as expected for diffusion from a surface through an aqueous environment. A pattern of one minute light pulses produced uniform pulses of increased NO concentration of one minute duration. A linear relationship exists between NO surface concentration and photon flux, and this relationship can be used to tune the material response.

A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins.

Cell-based biosensors (CBBs) are becoming important tools for biosecurity applications and rapid diagnostics in food microbiology for their unique capability of detecting physiologically hazardous materials. A multi-well plate-based biosensor containing B-cell hybridoma, Ped-2E9, encapsulated in type I collagen matrix, was developed for rapid detection of viable cells of pathogenic Listeria, the toxin listeriolysin O, and the enterotoxin from Bacillus species. This sensor measures the alkaline phosphatase release from infected Ped-2E9 cells colorimetrically. Pathogenic L. monocytogenes cells and toxin preparations from L. monocytogenes or B. cereus showed cytotoxicity ranging from 24 to 98% at 3-6 h postinfection. In contrast, nonpathogenic L. innocua (F4247) and B. subtilis induced minimal cytotoxicity, ranging only 0.4-7.6%. Laser scanning cytometry and cryo-nano scanning electron microscopy confirmed the live or dead status of the infected Ped-2E9 cells in gel matrix. This paper presents the first example of a cell-based sensing system using collagen-encapsulated mammalian cells for rapid detection of pathogenic bacteria or toxin, and demonstrates a potential for onsite use as a portable detection system.