Automated processing integrated with a microflow cytometer for pathogen detection in clinical matrices.

A spinning magnetic trap (MagTrap) for automated sample processing was integrated with a microflow cytometer capable of simultaneously detecting multiple targets to provide an automated sample-to-answer diagnosis in 40 min. After target capture on fluorescently coded magnetic microspheres, the magnetic trap automatically concentrated the fluorescently coded microspheres, separated the captured target from the sample matrix, and exposed the bound target sequentially to biotinylated tracer molecules and streptavidin-labeled phycoerythrin. The concentrated microspheres were then hydrodynamically focused in a microflow cytometer capable of 4-color analysis (two wavelengths for microsphere identification, one for light scatter to discriminate single microspheres and one for phycoerythrin bound to the target). A three-fold decrease in sample preparation time and an improved detection limit, independent of target preconcentration, was demonstrated for detection of Escherichia coli 0157:H7 using the MagTrap as compared to manual processing. Simultaneous analysis of positive and negative controls, along with the assay reagents specific for the target, was used to obtain dose-response curves, demonstrating the potential for quantification of pathogen load in buffer and serum.

A portable array biosensor for detecting multiple analytes in complex samples.

The Multi-Analyte Array Biosensor (MAAB) has been developed at the Naval Research Laboratory (NRL) with the goal of simultaneously detecting and identifying multiple target agents in complex samples with minimal user manipulation. This paper will focus on recent improvements in the biochemical and engineering aspects of this instrument. These improvements have enabled the expansion of the repertoire of analytes detected to include Salmonella typhimurium and Listeria monocytogenes, and also expanded the different sample matrices tested. Furthermore, all components of the biochemical assays could be prepared well in advance of sample testing, resulting in a "plug-and-play" methodology. Simultaneous detection of three toxins (ricin, staphylococcal enterotoxin B, and cholera toxin) was demonstrated using a novel fluidics cube module that limits the number of manipulations to only the initial sample loading. This work demonstrates the utility of the MAAB for rapid analysis of complex samples with multianalyte capability, with a minimum of operator manipulations required for either sample preparation or final analysis.

Fluidics cube for biosensor miniaturization.

To create a small, portable, fully automated biosensor, a compact means of fluid handling is required. We designed, manufactured, and tested a "fluidics cube" for such a purpose. This cube, made of thermoplastic, contains reservoirs and channels for liquid samples and reagents and operates without the use of any internal valves or meters; it is a passive fluid circuit that relies on pressure relief vents to control fluid movement. We demonstrate the ability of pressure relief vents to control fluid movement and show how to simply manufacture or modify the cube. Combined with the planar array biosensor developed at the Naval Research Laboratory, it brings us one step closer to realizing our goal of a handheld biosensor capable of analyzing multiple samples for multiple analytes.

Induction of BIM, a proapoptotic BH3-only BCL-2 family member, is critical for neuronal apoptosis.

Sympathetic neuronal death induced by nerve growth factor (NGF) deprivation requires the macromolecular synthesis-dependent translocation of BAX from the cytosol to mitochondria and its subsequent integration into the mitochondrial outer membrane, followed by BAX-mediated cytochrome c (cyt c) release. The gene products triggering this process remain unknown. Here, we report that BIM, a member of the BH3-only proapoptotic subfamily of the BCL-2 protein family, is one such molecule. NGF withdrawal induced expression of BIM(EL), an integral mitochondrial membrane protein that functions upstream of (or in parallel with) the BAX/BCL-2 and caspase checkpoints. Bim deletion conferred protection against developmental and induced neuronal apoptosis in both central and peripheral populations, but only transiently, suggesting that BIM--and perhaps other BH3-only proteins--serve partially redundant functions upstream of BAX-mediated cyt c release.

Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin.

The neurotrophic factors that influence the development and function of the parasympathetic branch of the autonomic nervous system are obscure. Recently, neurturin has been found to provide trophic support to neurons of the cranial parasympathetic ganglion. Here we show that GDNF signaling via the RET/GFR(alpha)1 complex is crucial for the development of cranial parasympathetic ganglia including the submandibular, sphenopalatine and otic ganglia. GDNF is required early for proliferation and/or migration of the neuronal precursors for the sphenopalatine and otic ganglia. Neurturin exerts its effect later and is required for further development and maintenance of these neurons. This switch in ligand dependency during development is at least partly governed by the altered expression of GFR(&agr;) receptors, as evidenced by the predominant expression of GFR(&agr;)2 in these neurons after ganglion formation.

Array biosensor for detection of biohazards.

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.

A ganglioside-based assay for cholera toxin using an array biosensor.

A rapid assay for cholera toxin (CT) has been developed using a fluorescence-based biosensor. This sensor was capable of analyzing six samples simultaneously for CT in 20 min with few manipulations required by the operator. The biochemical assays utilized a ganglioside-"capture" format: ganglioside GM1, utilized for capture of analyte, was immobilized in discrete locations on the surface of the optical waveguide. Binding of CT to immobilized GM1 was demonstrated with direct assays (using fluorescently labeled CT) and "sandwich" immunoassays (using fluorescently labeled tracer antibodies). Limits of detection for CT were 200 ng/ml in direct assays and 40 ng/ml and 1 microg/ml in sandwich-type assays performed using rabbit and goat tracer antibodies. Binding of CT to other glycolipid capture reagents was also observed. While significant CT binding was observed to loci patterned with GD1b, Gb3, and Gb4, CT did not bind significantly to immobilized GT1b at the concentrations tested. This is the first description of such a non-antibody-based recognition system in a multi-specific planar array sensor.

Simultaneous detection of six biohazardous agents using a planar waveguide array biosensor.

Recently, we demonstrated that an array biosensor could be used with cocktails of fluorescent antibodies to perform three assays simultaneously on a single substrate, and that multiple samples could be analyzed in parallel. We extend this technology to demonstrate the simultaneous analysis of six samples for six different hazardous analytes, including both bacteria and protein toxins. The level of antibody cross-reactivity is explored, revealing a possible common epitope in two of the toxins. A panel of environmental interferents was added to the samples; these interferents neither prevented the detection of the analytes nor caused false-positive responses.

Analysis of the retrograde transport of glial cell line-derived neurotrophic factor (GDNF), neurturin, and persephin suggests that in vivo signaling for the GDNF family is GFRalpha coreceptor-specific.

Neurturin (NRTN) and glial cell line-derived neurotrophic factor (GDNF) are members of a family of trophic factors with similar actions in vitro on certain neuronal classes. Retrograde transport of GDNF and NRTN was compared in peripheral sensory, sympathetic, and motor neurons to determine whether in vivo these factors are transported selectively by different neuronal populations. After sciatic nerve injections, NRTN was transported by sensory neurons of the dorsal root ganglion (DRG). Competition studies demonstrated only limited cross-competition between NRTN and GDNF, indicating selective receptor-mediated transport of these factors. By using immunohistochemistry, we identified two populations of NRTN-transporting DRG neurons: a major population of small, RET-positive, IB4-positive, non-TrkA-expressing neurons that also show the ability to transport GDNF and a minor population of calretinin-expressing neurons that fail to transport GDNF. Spinal motor neurons in the adult showed relatively less ability to transport NRTN than to transport GDNF, although NRTN prevented the cell death of neonatal motor neurons in a manner very similar to GDNF (Yan et al., 1995) and persephin (PSPN) (Milbrandt et al., 1998). Last, NRTN, like GDNF, was not transported to sympathetic neurons of the adult superior cervical ganglion (SCG) after injection into the anterior eye chamber. These data reveal a high degree of functional selectivity of GDNF family receptor-alpha (GFRalpha) coreceptor subtypes for NRTN and GDNF in vivo.

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.

Expression of neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse.

The GDNF family of neurotrophic factors currently has four members: neurturin (NRTN), glial cell line-derived neurotrophic factor (GDNF), persephin, and artemin. These proteins are potent survival factors for several populations of central and peripheral neurons. The receptors for these factors are complexes that include the Ret tyrosine kinase receptor and a GPI-linked, ligand-binding component called GDNF family receptor alpha 1-4 (GFRalpha1-4). We have used in situ hybridization to study the mRNA expression of NRTN, GDNF, Ret, GFRalpha1, and GFRalpha2 during embryonic development and in the adult mouse. GDNF receptors were prominently expressed during embryonic development in the nervous system, the urogenital system, the digestive system, the respiratory system, and in developing skin, bone, muscle, and endocrine glands. In some regions, incomplete receptor complexes were expressed suggesting that other, as yet unidentified, receptor components exist or that receptor complexes are formed in trans. NRTN and GDNF were expressed in many trigeminal targets during embryonic development including the nasal epithelium, the teeth, and the whisker follicles. NRTN and GDNF were also expressed in the developing limbs and urogenital system. In the embryo, GDNF factors and receptors were expressed at several sites of mesenchyme/epithelial induction, including the kidney, tooth, and submandibular gland. This expression pattern is consistent with the possibility that the GDNF factors function in inductive processes during embryonic development and with the recently discovered role of NRTN as a necessary trophic factor for the development of some parasympathetic neurons. In the mature animal, receptor expression was more limited than in the embryo. In the adult mouse, NRTN was most prominently expressed in the gut, prostate testicle, and oviduct; GDNF was most prominently expressed in the ovary.

Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons.

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha1 or GFR alpha2). Neurturin-deficient (NTN-/-) mice generated by homologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons. GFR alpha2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha2/Ret receptor complexes as the critical mediators of NTN function in vivo.

An array immunosensor for simultaneous detection of clinical analytes.

A fluorescence-based immunosensor has been developed for simultaneous analysis of multiple samples. A patterned array of recognition elements immobilized on the surface of a planar waveguide is used to "capture" analyte present in samples; bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This immunosensor was used to detect physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D-dimer, a marker of sepsis and thrombotic disorders, in spiked clinical samples.

Array biosensor: optical and fluidics systems.

Optical and fluidics systems have been developed as central components for an automated array biosensor. Disposable planar waveguides are patterned with immobilized capture antibodies using a physically isolated patterning (PIP) method. The PIP method enables simultaneous deposition of several antibodies and completely circumvents cross-immobilization problems encountered with other array deposition processes. A multi-channel fluidics cell allows numerous assays to be performed on the patterned waveguide. The sensing arrays are optically interrogated using a diode laser with a tailored output to optimize coupling to and maximize excitation uniformity within the waveguide. A patterned cladding is employed to optically isolate the waveguide from perturbations induced by the permanently attached flow cells. Compact optics image the evanescently excited fluorescence onto a large area, cooled CCD array. The image data is processed and automated signal analysis corrects for local background and noise variations.

Expression of neurturin, GDNF, and their receptors in the adult mouse CNS.

Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF) are the first two members of the GDNF family (GF) of neurotrophic factors. These two proteins are potent survival factors for several populations of central and peripheral neurons in mature and developing rodents. The receptor for these factors is a multicomponent complex that includes the RET (rearranged during transfection) tyrosine kinase receptor and one of two glycosyl phosphatidylinositol (GPI)-linked ligand-binding components called GDNF family receptor alphas (GFRalpha-1 and GFRalpha-2). We have used in situ hybridization to study the mRNA expression of NTN, GDNF, RET, GFRalpha-1, and GFRalpha-2 in the central nervous system (CNS) of adult mice. GF receptors are expressed in several areas in which neuronal populations known to respond to NTN and GDNF are located, including the ventral horn of the spinal cord and the compacta region of the substantia nigra. In addition, we have demonstrated receptor expression in other areas of the brain including the thalamus and hypothalamus. Neurons in these areas express GF receptors, and therefore, may respond to NTN or GDNF. NTN and GDNF are expressed in targets of neurons that express GF receptors. The pattern of GF factor and receptor expression in the adult brain suggests a role for these factors in maintaining neuronal circuits in the mature CNS.

Detection of multiple toxic agents using a planar array immunosensor.

A planar array immunosensor, equipped with a charge-coupled device (CCD) as a detector, was used to simultaneously detect 3 toxic analytes. Wells approximately 2 mm in diameter were formed on glass slides using a photoactivated optical adhesive. Antibodies against staphylococcal enterotoxin B (SEB), ricin, and Yersinia pestis were covalently attached to the bottoms of the circular wells to form the sensing surface. Rectangular wells containing chicken immunoglobulin were used as alignment markers and to generate control signals. After removing the optical adhesive, the slides were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction lens array was used to focus the sensing surface onto the CCD. Solutions of toxins were then placed on the slide. After rinsing, Cy5-labeled antibodies were introduced. The identity and amount of toxin bound at each location on the slide were determined by quantitative image analysis. Concentrations as low as 25 ng/mL of ricin, 15 ng/mL of pestis F1 antigen, and 5 ng/mL of SEB could be routinely measured.

GFRalpha3 is an orphan member of the GDNF/neurturin/persephin receptor family.

GDNF, neurturin, and persephin are transforming growth factor beta-related neurotrophic factors known collectively as the GDNF family (GF). GDNF and neurturin signal through a multicomponent receptor complex containing a signaling component (the Ret receptor tyrosine kinase) and either of two glycosyl-phosphatidylinositol-linked binding components (GDNF family receptor alpha components 1 and 2, GFRalpha1 or GFRalpha2), whereas the receptor for persephin is unknown. Herein we describe a third member of the GF coreceptor family called GFRalpha3 that is encoded by a gene located on human chromosome 5q31.2-32. GFRalpha3 is not expressed in the central nervous system of the developing or adult animal but is highly expressed in several developing and adult sensory and sympathetic ganglia of the peripheral nervous system. GFRalpha3 is also expressed at high levels in developing, but not adult, peripheral nerve. GFRalpha3 is a glycoprotein that is glycosyl-phosphatidylinositol-linked to the cell surface like GFRalpha1 and GFRalpha2. Fibroblasts expressing Ret and GFRalpha3 do not respond to any of the known members of the GDNF family, suggesting that GFRalpha3 interacts with an unknown ligand or requires a different or additional signaling protein to function.

Persephin, a novel neurotrophic factor related to GDNF and neurturin.

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

Postnatal development of terminals and synapses in laminae I and II of the rat medullary dorsal horn.

To better understand developing orofacial nociceptive circuits and to provide a baseline for evaluating injury-induced plasticity, the ultrastructure of the superficial laminae in the rat medullary dorsal horn was examined at birth and at postnatal days 1, 4, 17, and 90. Quantitative features of terminals and synapses were studied with stereological methods. In laminae I and II: 1) Axon terminal density increased significantly from birth to day 4 and again from day 4 to day 90. 2) The density of degenerating profiles increased significantly from birth to day 1 and from birth to day 4 and then decreased from day 4 to day 90. 3) Degenerating profiles were most dense on day 1 and declined steadily thereafter; by day 90, such profiles were rare. 4) Cavitation was by far the most common form of degeneration seen at early postnatal ages. 5) Growth cone-like profiles were most dense at birth and declined steadily during the first 2 postnatal weeks; by day 90, such profiles were absent. 6) Terminals with flat synaptic vesicles were rarely seen before day 90, when they accounted for 7% of the terminal population. 7) The density of synapses increased continuously from birth until day 90. These data suggest that, as in the spinal cord, medullary dorsal horn circuits are very immature at birth. Adult-like quantitative features are not attained until after day 17. Moreover, whereas degenerating profiles are prevalent during early postnatal development, and they have features that resemble naturally occurring degeneration, the total numbers of terminals and synapses continue to increase dramatically and gradually during a protracted postnatal period (to postnatal day 17).