Identification and Quantitation of Aqueous Single- and Multianalyte Solutions of the Isomers Ethylbenzene, m-, p-, and o-Xylene Using a Single Specifically Tailored Sensor Coating and Estimation Theory-Based Signal Processing.

The isomer-specific detection and quantitation of m-, p-, and o-xylene and ethylbenzene, dissolved singly and as mixtures in aqueous solutions at concentrations from 100 to 1200 ppb by volume, is reported for a specifically designed polymer-plasticizer coating on a shear-horizontal surface acoustic wave (SH-SAW) device. The polystyrene-ditridecyl phthalate-blend coating was designed utilizing Hansen solubility parameters and considering the dipole moment and polarizability of the analytical targets and coating components to optimize the affinity of the sensor coating for the four chemical isomers. The two key coating sorption properties, sensitivity and response time constant, are determined by the (slightly different) dipole moments and polarizabilities of the four target analytes: as analyte dipole moment decreases, coating sensitivity increases; as analyte polarizability decreases, coating response time lengthens. Using the measured sensitivities and time constants for the targets, sensor signals were processed with exponentially weighted recursive-least-squares estimation (EW-RLSE) to identify (with near 100% accuracy) and quantify (with ± 5-7% accuracy) the isomers. This impressive performance was achieved by combining the specifically tailored, high-sensitivity coating and an SH-SAW platform (yielding a detection limit of 5 ppb for the analytes) and using the EW-RLS estimator, which estimates unknown parameters accurately even in the presence of measurement noise and for analytes with only minor differences in response. Identification of the xylene isomers is important for applications including environmental monitoring and chemical manufacturing.

Application-Specific Adaptable Coatings for Sensors: Using a Single Polymer-Plasticizer Pair to Detect Aromatic Hydrocarbons, Mixtures, and Interferents in Water with Single Sensors and Arrays.

A relatively simple design procedure is presented for new, adaptable chemical sensor coatings made from a single polymer-plasticizer pair to detect single or a mixture of chemical compounds (e.g., BTEX, the small aromatic hydrocarbon family). Affinity between coating components and target analytes, expressed through Hansen solubility parameters and relative energy difference values, describes the sensitivity of the resultant coatings to each analyte. While analyte affinity is paramount for plasticizer selection, for the aqueous-phase sensing application described here, it must be traded off with the permanence in the host polymer, i.e., resistance to leaching into the ambient aqueous phase; deleterious effects including coating creep must also be minimized. By varying the polymer:plasticizer mixing ratio, the physical and chemical properties of the resultant coatings can be tuned across a range of sensing properties, in particular the differential response magnitude and rate, for multiple analytes. Together with the measurement of multiple sensor response parameters (relative sensitivity and response time constant) for each coating, this approach allows for identification and quantification of target analytes not previously separable using commercial off-the-shelf (COTS) polymer sensor coatings. Sensing results using a five-sensor array based on five different mixing ratios of a single plasticizer polymer pair (plasticizer: ditridecyl phthalate; polymer: polystyrene) demonstrate unique identification of mixtures of BTEX analytes, including differentiation of the chemical isomers ethylbenzene and total xylene (or "xylenes"), something not previously feasible for separation-free liquid-phase sensing with commercially available polymer coatings. Ultimately, the response of a single optimized sensor coating identified and quantified the components of various mixtures, including identification of likely interferents, using a customized estimation-theory-based multivariate signal-processing technique.

Quantitative Detection of Complex Mixtures using a Single Chemical Sensor: Analysis of Response Transients using Multi-Stage Estimation.

Most chemical sensors are only partially selective to any specific target analyte(s), making identification and quantification of analyte mixtures challenging, a problem often addressed using arrays of partially selective sensors. This work presents and experimentally verifies a signal-processing technique based on estimation theory for online identification and quantification of multiple analytes using only the response data collected from a single polymer-coated sensor device. The demonstrated technique, based on multiple stages of exponentially weighted recursive least-squares estimation (EW-RLSE), first determines which of the analytes included in the sensor response model are absent from the mixture being analyzed; these are then eliminated from the model prior to executing the final stage of EW-RLSE, in which the sample's constituent analytes are more accurately quantified. The overall method is based on a sensor response model with specific parameters describing each coating-analyte pair and requires no initial assumptions regarding the concentrations of the analytes in a given sample. The technique was tested using the measured responses of polymer-coated shear-horizontal surface acoustic wave devices to multi-analyte mixtures of benzene, toluene, ethylbenzene, xylenes, and 1,2,4-trimethylbenzene in water. The results demonstrate how this method accurately identifies and quantifies the analytes present in a sample using the measured response of just a single sensor device. This effective, simple, lower-cost alternative to sensor arrays needs no arduous training protocol, just measurement of the response characteristics of each individual target analyte and the likely interferents and/or classes thereof.

Obtaining Chemical Selectivity from a Single, Nonselective Sensing Film: Two-Stage Adaptive Estimation Scheme with Multiparameter Measurement to Quantify Mixture Components and Interferents.

A new approach is reported to detect and quantify the members of a group of small-aromatic-molecule target analytes: benzene, toluene, ethylbenzene, and xylenes (BTEX), dissolved in water, in the presence of interferents, using only the data collected from a single polymer-coated SH-SAW (shear horizontal surface acoustic wave) device and a two-stage adaptive estimation scheme. This technique is composed of exponentially weighted recursive least-squares estimation (EW-RLSE) and a bank of Kalman filters (BKFs) and does not require any prior knowledge of the initial concentration range of the target analytes. The proposed approach utilizes the transient sensor response to sorption and/or desorption of the analytes as well as the error range associated with the response time constants to provide more information about the analyte-specific interactions with the polymer film. The approach assumes that the sensor response to contaminated groundwater is a linear combination of the responses to the single target analytes, the interferents that interact with the selected polymer sensor coatings, and measurement noise. The proposed technique was tested using actual sensor responses to contaminated groundwater samples containing multiple BTEX compounds with concentrations ranging from 10 to 2000 parts per billion, as well as common interferents including ethanol, 1,2,4-trimethylbenzene, naphthalene, n-heptane, and MTBE (methyl tert-butyl ether). Estimated concentration values, accurate to ±10% for benzene/toluene and ±15% for ethylbenzene/xylenes, are obtained in near-real time. The utilization of sorption and/or desorption data enables detection and quantification of BTEX compounds with improved accuracy, high tolerance to measurement noise, and improved chemical selectivity.

Investigation of Polymer-Plasticizer Blends as SH-SAW Sensor Coatings for Detection of Benzene in Water with High Sensitivity and Long-Term Stability.

We report the first-ever direct detection of benzene in water at concentrations below 100 ppb (parts per billion) using acoustic wave (specifically, shear-horizontal surface acoustic wave, SH-SAW) sensors with plasticized polymer coatings. Two polymers and two plasticizers were studied as materials for sensor coatings. For each polymer-plasticizer combination, the influence of the mixing ratio of the blend on the sensitivity to benzene was measured and compared to commercially available polymers that were used for BTEX (benzene, toluene, ethylbenzene, and xylene) detection in previous work. After optimizing the coating parameters, the highest sensitivity and lowest detection limit for benzene were found for a 1.25 µm thick sensor coating of 17.5%-by-weight diisooctyl azelate-polystyrene on the tested acoustic wave device. The calculated detection limit was 45 ppb, with actual sensor responses to concentrations down to 65 ppb measured directly. Among the sensor coatings that showed good sensitivity to benzene, the best long-term stability was found for a 1.0 µm thick coating of 23% diisononyl cyclohexane-1,2-dicarboxylate-polystyrene, which was studied here because it is known to show no detectable leaching in water. The present work demonstrates that, by varying type of plasticizer, mixing ratio, and coating thickness, the mechanical and chemical properties of the coatings can be conveniently tailored to maximize analyte sorption and partial chemical selectivity for a given class of analytes as well as to minimize acoustic-wave attenuation in contact with an aqueous phase at the operating frequency of the sensor device.

Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling.

In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.

Analysis of binary mixtures of aqueous aromatic hydrocarbons with low-phase-noise shear-horizontal surface acoustic wave sensors using multielectrode transducer designs.

The present work investigates a compact sensor system that provides rapid, real-time, in situ measurements of the identities and concentrations of aromatic hydrocarbons at parts-per-billion concentrations in water through the combined use of kinetic and thermodynamic response parameters. The system uses shear-horizontal surface acoustic wave (SH-SAW) sensors operating directly in the liquid phase. The 103 MHz SAW sensors are coated with thin sorbent polymer films to provide the appropriate limits of detection as well as partial selectivity for the analytes of interest, the BTEX compounds (benzene, toluene, ethylbenzene, and xylenes), which are common indicators of fuel and oil accidental releases in groundwater. Particular emphasis is placed on benzene, a known carcinogen and the most challenging BTEX analyte with regard to both regulated levels and its solubility properties. To demonstrate the identification and quantification of individual compounds in multicomponent aqueous samples, responses to binary mixtures of benzene with toluene as well as ethylbenzene were characterized at concentrations below 1 ppm (1 mg/L). The use of both thermodynamic and kinetic (i.e., steady-state and transient) responses from a single polymer-coated SH-SAW sensor enabled identification and quantification of the two BTEX compounds in binary mixtures in aqueous solution. The signal-to-noise ratio was improved, resulting in lower limits of detection and improved identification at low concentrations, by designing and implementing a type of multielectrode transducer pattern, not previously reported for chemical sensor applications. The design significantly reduces signal distortion and root-mean-square (RMS) phase noise by minimizing acoustic wave reflections from electrode edges, thus enabling limits of detection for BTEX analytes of 9-83 ppb (calculated from RMS noise); concentrations of benzene in water as low as ~100 ppb were measured directly. Reliable quantification of BTEX analytes in binary mixtures is demonstrated in the sub-parts-per-million concentration range.

Identification and quantification of aqueous aromatic hydrocarbons using SH-surface acoustic wave sensors.

A need exists for compact sensor systems capable of in situ monitoring of groundwater for accidental releases of fuel and oil. The work reported here addresses this need, using shear horizontal surface acoustic wave (SH-SAW) sensors, which function effectively in liquid environments. To achieve enhanced sensitivity and partial selectivity for hydrocarbons, the devices are coated with thin chemically sensitive polymer films. Various polymer materials are investigated with the goal of identifying a set of coatings suitable for a sensor array. The system is tested with compounds indicative of fuel and oil releases, in particular, the BTEX compounds (benzene, toluene, ethylbenzene, and xylenes), in the low milligrams/liters to high micrograms/liters concentration range. Particular emphasis is placed on detection of benzene, a known carcinogen. It was observed that within the above concentration range, responses to multiple analytes in a mixture are additive, and there is a characteristic response time for each coating/analyte pair, which is largely independent of concentration. With the use of both the steady-state and transient-response information of SH-SAW sensor devices coated with three different polymer materials, poly(ethyl acrylate), poly(epichlorohydrin), and poly(isobutylene), a response pattern was obtained for benzene that is easily distinguishable from those of the other BTEX compounds. The time courses of the responses to binary analyte mixtures were modeled accurately using dual-exponential fits, yielding a characteristic concentration-independent time constant for each analyte/coating pair. Benzene concentration was quantified in the aqueous phase in the presence of the other BTEX compounds.

Local axonal function of STAT3 rescues axon degeneration in the pmn model of motoneuron disease.

Axonal maintenance, plasticity, and regeneration are influenced by signals from neighboring cells, in particular Schwann cells of the peripheral nervous system. Schwann cells produce neurotrophic factors, but the mechanisms by which ciliary neurotrophic factor (CNTF) and other neurotrophic molecules modify the axonal cytoskeleton are not well understood. In this paper, we show that activated signal transducer and activator of transcription-3 (STAT3), an intracellular mediator of the effects of CNTF and other neurotrophic cytokines, acts locally in axons of motoneurons to modify the tubulin cytoskeleton. Specifically, we show that activated STAT3 interacted with stathmin and inhibited its microtubule-destabilizing activity. Thus, ectopic CNTF-mediated activation of STAT3 restored axon elongation and maintenance in motoneurons from progressive motor neuronopathy mutant mice, a mouse model of motoneuron disease. This mechanism could also be relevant for other neurodegenerative diseases and provide a target for new therapies for axonal degeneration.

Direct detection of DNA conformation in hybridization processes.

DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays.

Downregulation of genes with a function in axon outgrowth and synapse formation in motor neurones of the VEGFdelta/delta mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGFdelta/delta mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. RESULTS: Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGFdelta/delta mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGFdelta/delta mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGFdelta/delta mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGFdelta/delta mice, axon outgrowth is significantly reduced compared to wild-type littermates. CONCLUSIONS: Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.

Novel role for vascular endothelial growth factor (VEGF) receptor-1 and its ligand VEGF-B in motor neuron degeneration.

Although vascular endothelial growth factor-B (VEGF-B) is a homolog of the angiogenic factor VEGF, it has only minimal angiogenic activity, raising the question of whether this factor has other (more relevant) biological properties. Intrigued by the possibility that VEGF family members affect neuronal cells, we explored whether VEGF-B might have a role in the nervous system. Here, we document that the 60 kDa VEGF-B isoform, VEGF-B(186), is a neuroprotective factor. VEGF-B(186) protected cultured primary motor neurons against degeneration. Mice lacking VEGF-B also developed a more severe form of motor neuron degeneration when intercrossed with mutant SOD1 mice. The in vitro and in vivo effects of VEGF-B(186) were dependent on the tyrosine kinase activities of its receptor, Flt1, in motor neurons. When delivered intracerebroventricularly, VEGF-B(186) prolonged the survival of mutant SOD1 rats. Compared with a similar dose of VEGF, VEGF-B(186) was safer and did not cause vessel growth or blood-brain barrier leakiness. The neuroprotective activity of VEGF-B, in combination with its negligible angiogenic/permeability activity, offers attractive opportunities for the treatment of neurodegenerative diseases.

Probing mechanical properties of liposomes using acoustic sensors.

Acoustic devices were employed to characterize variations in the mechanical properties (density and viscoelasticity) of liposomes composed of 1-oleoyl-2-palmitoyl- sn-glycero-3-phosphocholine (POPC) and cholesterol. Liposome properties were modified in three ways. In some experiments, the POPC/cholesterol ratio was varied prior to deposition on the device surface. Alternatively, the ratio was changed in situ via either insertion of cholesterol or removal of cholesterol with beta-cyclodextrin. This was done for liposomes adsorbed directly on the device surface and for liposomes attached via a biotin-terminated poly(ethylene glycol) linker. The acoustic measurements make use of two simultaneous time-resolved signals: one signal is related to the velocity of the acoustic wave, while the second is related to dissipation of acoustic energy. Together, they provide information not only about the mass (or density) of the probed medium but also about its viscoelastic properties. The cholesterol-induced increase in the surface density of the lipid bilayer was indeed observed in the acoustic data, but the resulting change in signal was larger than expected from the change in surface density. In addition, increasing the bilayer resistance to stretching was found to lead to a greater dissipation of the acoustic energy. The acoustic response is assessed in terms of the possible distortions of the liposomes and the known effects of cholesterol on the mechanical properties of the lipid bilayer that encloses the aqueous core of the liposome. To aid the interpretation of the acoustic response, it is discussed how the above changes in the lipid bilayer will affect the effective viscoelastic properties of the entire liposome/solvent film on the scale of the acoustic wavelength. It was found that the acoustic device is very sensitive to the mechanical properties of lipid vesicles; the response of the acoustic device is explained, and the basic underlying mechanisms of interaction are identified.

High-efficiency gene transfer into cultured embryonic motoneurons using recombinant lentiviruses.

Primary neurons are a common tool for investigating gene function for survival and morphological and functional differentiation. Gene transfer techniques play an important role in this context. However, the efficacy of conventional gene transfer techniques, in particular for primary motoneurons is low so that it is not possible to distinguish whether the observed effects are representative for all neurons or only for the small subpopulation that expresses the transfected cDNA. In order to develop techniques that allow high gene transfer rates, we have optimized lentiviral-based gene transfer for cultured motoneurons by using a replication-defective viral vector system. These techniques result in transduction efficacies higher than 50%, as judged by EGFP expression under the control of SFFV or CMV promoters. Under the same conditions, survival and morphology of the cultured motoneurons was not altered, at least not when virus titers did not exceed a multiplicity of infection of 100. Under the same cell culture conditions, electroporation resulted in less than 5% transfected motoneurons and reduced survival. Therefore we consider this lentivirus-based gene transfer protocol as a suitable tool to study the effects of gene transfer on motoneuron survival, differentiation and function.

The temperature-sensitive ion channel TRPV2 is endogenously expressed and functional in the primary sensory cell line F-11.

In sensory neurons heat is transduced by a subfamily of TRP channels sharing sequence homology with the capsaicin-sensitive vanilloid receptor subtype 1 (TRPV1), but differing in their thermal response thresholds. To identify a neuronal cell line endogenously expressing noxious heat-transducing ion channels, we examined F-11 cells, a hybridoma derived from rat dorsal root ganglia and mouse neuroblastoma. Using RT-PCR, transcripts homologous to TRPV2 and TRPV4, but not to TRPV1 or TRPV3, were found. We isolated a full-length cDNA of 2.4 kb coding for a 757-amino acid protein corresponding to mouse TRPV2, which was further characterized by immunocytochemistry and electrophysiology. Using the whole-cell patch-clamp technique, we observed a heat-evoked increase in outward and inward currents with a threshold of 51.6 +/- 0.2 degrees C. The current-voltage relationship stimulated by a temperature of 52 degrees C was characterized by an outward rectification with a reversal potential close to -10 mV. Heat-evoked currents could be inhibited by ruthenium red. There was no activation by stimulation with capsaicin or 2-aminoethoxydiphenyl borate. Our results indicate that F-11 cells express functional noxious heat-sensitive TRPV2 channels. Thus, we propose that F-11 cells represent a valuable in vitro model to characterize the properties of TRPV2 in a native neuronal environment.

On-line monitoring of polymer deposition for tailoring the waveguide characteristics of love-wave biosensors.

The Love-wave sensor is an acoustic sensing device which is particularly suitable for sensing in a liquid environment. The superior characteristics of the device are achieved by the use of an acoustic waveguide, consisting of a thin layer deposited on the surface of the substrate material. The exact thickness and material properties of the layer will not only determine sensitivity and sensing performance of the resulting device but can also be adjusted to generate higher-order Love modes. Thus, to obtain a sensing device with the desired specifications, precise control over the process of waveguide deposition is required. This has been realized by implementation of a vapor deposition polymerization system where the transmission curve (amplitude vs frequency) of one of the sensing devices is continuously monitored during deposition. As soon as the desired device specifications are reached, the deposition can be interrupted immediately. From the recorded transmission curves, information about the sensitivity of the device can be deduced, and the formation of higher-order Love modes can be visualized. The system has been used to produce biosensors based on various Love modes. It is shown that sensors operated on higher-order Love modes have a high mass sensitivity which, together with their excellent shielding properties, makes them advantageous for biosensing in conducting buffer solutions.

A surface acoustic wave biosensor concept with low flow cell volumes for label-free detection.

Special surface acoustic wave (SAW) devices using horizontally polarized surface shear waves can be operated in water. They allow an easy detection of molecules with biological relevance (e.g., proteins) via direct detection of the adsorbed mass. The transducer structures of conventional SAW devices are usually connected to the electronics by bond wires. In consequence, flow cell volumes can hardly be designed smaller than 50 microL. A new type of SAW device that is coupled capacitively with the electronics enables the reduction of flow cell volumes down to 60 nL, which decreases sample consumption and reduces the length of the measurement cycles down to a few minutes. To create an immunosensor, the SAW devices first are coated with a thin parylene layer for creating a sensor surface that is chemically homogeneous. Then OptoDex, a dextran containing both photoactive and functional groups is immobilized photochemically. Finally, antibodies are coupled via conventional EDC/NHS chemistry. The technique has been used to monitor urease binding at anti-urease-coated SAW devices in real time and with good resolution. Because of the simple sensor handling and the economical sample use, the new SAW device is particularly suitable for the design of an array.