RESUMO
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.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas de Membrana , Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Processamento Alternativo , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose , Proteína 11 Semelhante a Bcl-2 , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Caspases/metabolismo , Células Cultivadas , Cicloeximida/farmacologia , Grupo dos Citocromos c/metabolismo , Citosol/metabolismo , Dactinomicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Membranas Intracelulares/metabolismo , MAP Quinase Quinase 4 , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Mutagênese , Fator de Crescimento Neural/administração & dosagem , Fator de Crescimento Neural/fisiologia , Neurônios/ultraestrutura , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/análise , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2RESUMO
Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.
Assuntos
Proteínas de Drosophila , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células 3T3/química , Células 3T3/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Sequência de Bases , Northern Blotting , Química Encefálica/fisiologia , Mapeamento Cromossômico , Expressão Gênica/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Hibridização in Situ Fluorescente , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurturina , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/análise , Ratos , Nervo Isquiático/química , Nervo Isquiático/citologia , Nervo Isquiático/lesões , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Testículo/química , Regulação para Cima/fisiologiaRESUMO
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.
Assuntos
Proteínas de Drosophila , Intestinos/inervação , Fatores de Crescimento Neural/fisiologia , Neurônios Aferentes/fisiologia , Neurônios/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Animais , Marcação de Genes , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Aparelho Lacrimal/inervação , Camundongos , Camundongos Endogâmicos , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/genética , Neurônios Aferentes/metabolismo , Neurturina , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismo , Glândulas Salivares/inervaçãoRESUMO
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.
Assuntos
Neurônios Motores/química , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores/metabolismo , Animais , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Gânglios Espinais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Mesencéfalo/citologia , Camundongos , Dados de Sequência Molecular , Neurônios Motores/fisiologia , Neurturina , Gânglio Nodoso/citologia , Reação em Cadeia da Polimerase/métodos , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento/fisiologia , Receptores do Ácido Retinoico/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Gânglio Cervical Superior/citologia , Transfecção , Gânglio Trigeminal/citologia , Ureter/citologia , Ureter/embriologiaRESUMO
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.
Assuntos
Proteínas de Drosophila , Gânglios Espinais/fisiologia , Neurônios Motores/fisiologia , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios Aferentes/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Nervo Isquiático/fisiologia , Transdução de Sinais/fisiologia , Medula Espinal/fisiologia , Animais , Animais Recém-Nascidos , Transporte Axonal , Transporte Biológico , Tamanho Celular , Gânglios Espinais/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Imuno-Histoquímica , Radioisótopos do Iodo , Masculino , Neurônios Motores/citologia , Neurônios Aferentes/citologia , Neurturina , Proteínas Proto-Oncogênicas c-ret , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologiaRESUMO
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).
Assuntos
Bulbo/crescimento & desenvolvimento , Ratos Sprague-Dawley/fisiologia , Sinapses/fisiologia , Núcleos do Trigêmeo/crescimento & desenvolvimento , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Feminino , Masculino , Bulbo/citologia , Microscopia Eletrônica , Degeneração Neural/fisiologia , Neuritos/fisiologia , Neuritos/ultraestrutura , Ratos , Córtex Somatossensorial/citologia , Sinapses/ultraestrutura , Núcleos do Trigêmeo/citologiaRESUMO
Although much is known of the central consequences of infraorbital nerve (ION) transection at birth, little is known about the effects of this lesion on the organization of the ION itself. To advance our understanding of how deafferentation alters the developing trigeminal neuraxis, 19 newborn rats were subjected to left ION section and perfused 1, 2, 4, 7, 17, or 90 days later. Left IONs were removed in the orbit proximal to the nerve injury site, and axon numbers, types, and fasciculation patterns were assessed with light and electron microscopic methods. Complete axon counts demonstrated that the axotomized ION contained an average (+/- SD) of 13,945 +/- 10,335, 14,112 +/- 3,501, 16,531 +/- 1,904, 9,045 +/- 1,465, 7,018 +/- 4,212, and 8,672 +/- 1,030 axons at the above-listed ages, respectively. These values are well below the 33,059 axons in the normal adult ION (Jacquin et al. [1984] Brain Res. 290:131-135) and the 42,219 axons in the newborn ION (Renehan and Rhoades [1984] Brain Res. 322:369-373). The axotomized ION also contained lower than normal percentages of myelinated axons (26.7% +/- 6.3% on postnatal day 90 vs. 59.7% +/- 6.2% in normal adults). Unmyelinated fibers constituted the vast majority of the remaining fiber types; degenerating fibers never accounted for > 1.6% of all the axons. The number of fascicles making up the axotomized ION overlapped significantly with those found in the normal newborn and adult ION. We conclude that 1) extensive, though variable, axon elimination occurs proximally within one day of the lesion; 2) the 74% reduction in fiber number seen at 90 days is not reliably achieved until postnatal day 7; 3) the higher than normal proportion of unmyelinated axons in the injured ION may underly many of the known effects of neonatal ION injury on the developing whisker-barrel neuraxis; 4) gross changes in ION fasciculation patterns are not prerequisite to injury-induced pattern alterations in the developing trigeminal system.
Assuntos
Animais Recém-Nascidos/anatomia & histologia , Axônios/fisiologia , Órbita/inervação , Ratos/anatomia & histologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Feminino , Masculino , Microscopia Eletrônica , Ratos Sprague-DawleyRESUMO
Infraorbital nerve damage at birth kills neurons and alters anatomical, physiological, and biochemical properties of surviving cells in all portions of the trigeminal brainstem complex, with the exception of laminae I and II of the medullary dorsal horn. The resiliency of laminae I and II may be due to rapid terminal sprouting and reactive synaptogenesis in this region. To test this hypothesis, quantitative electron microscopy revealed the types and numbers of terminals, synapses, and degenerating and growth cone-like profiles in the left laminae I and II at 1, 4, 17, and 90 days after left infraorbital nerve section. Control data were derived from normal newborns and from the right laminae I and II and the left infraorbital nerve of every experimental animal. Deafferented laminae I and II contained a median of 11.7, 8.2, 21.8, and 38.2 synapses/100 microm3 on days 1, 4, 17, and 90, respectively. At corresponding ages, there were 17.1, 19.4, 36.2, and 32 terminals; 14.4, 4.2, 5.1, and 0.3 degenerating profiles; and 4.6, 2.2, 0.1, and 0 growth cone-like profiles/100 microm2. Significant differences from the control right side are: 1) The percentage area occupied by terminals is less on days 1 and 17; 2) terminal density does not increase from day 0 to day 4 as it does on the control side; 3) the density of degenerating profiles is higher on day 17; 4) growth cones are less dense on days 4 and 17; and 5) synapse density is lower on days 1 and 4. Axon number in the infraorbital nerve was highly predictive of terminal and synapse densities in deafferented laminae I and II at all ages. Thus, in laminae I and II, 1) the time course and nature of development are altered by deafferentation at birth; 2) reorganization of terminals and synapses occurs within a day of the lesion; 3) by day 90, there are no remaining lesion effects; and 4) the status of the injured nerve predicts central terminal and synapse densities. These are signs of injury-induced transganglionic degeneration and sprouting. The source of the latter is unknown, although areal fraction data suggest that "replacement" terminals may not be of primary afferent origin.
Assuntos
Bulbo/crescimento & desenvolvimento , Ratos Sprague-Dawley/fisiologia , Núcleos do Trigêmeo/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Denervação , Feminino , Masculino , Bulbo/citologia , Microscopia Eletrônica , Degeneração Neural/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios Aferentes/fisiologia , Neurônios Aferentes/ultraestrutura , Órbita/inervação , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Sinapses/fisiologia , Sinapses/ultraestrutura , Nervo Trigêmeo/cirurgia , Núcleos do Trigêmeo/citologia , Vibrissas/inervaçãoRESUMO
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.
Assuntos
Proteínas de Drosophila , Camundongos Endogâmicos ICR/fisiologia , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Fatores Etários , Animais , Química Encefálica/fisiologia , Tronco Encefálico/química , Tronco Encefálico/citologia , Cerebelo/química , Cerebelo/citologia , Feminino , Expressão Gênica/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Hipotálamo/química , Hipotálamo/citologia , Hibridização In Situ , Mesencéfalo/química , Mesencéfalo/citologia , Camundongos , Neurturina , Bulbo Olfatório/química , Bulbo Olfatório/citologia , Prosencéfalo/química , Prosencéfalo/citologia , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/análise , Medula Espinal/química , Medula Espinal/citologia , Tálamo/química , Tálamo/citologiaRESUMO
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.
Assuntos
Técnicas Biossensoriais , Equipamentos Descartáveis , Processamento de Imagem Assistida por Computador , Imunoensaio , Óptica e Fotônica , Método Duplo-Cego , ImunofluorescênciaRESUMO
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.
Assuntos
Bactérias/isolamento & purificação , Toxinas Bacterianas/análise , Técnicas Biossensoriais , Monitoramento Ambiental , Ricina/análise , Reações CruzadasRESUMO
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.
Assuntos
Técnicas Biossensoriais , Substâncias Perigosas/análise , Fluorescência , Sensibilidade e EspecificidadeRESUMO
A fiber-optic biosensor capable of remote continuous monitoring has recently been designed. To permit sensing at locations separate from the optoelectronic instrumentation, long optical fibers are utilized. An evanescent wave immuno-probe is prepared by removing the cladding near the distal end of the fiber and covalently attaching antibodies to the core. Probes with a radius unaltered from that of the original core inefficiently returned the signal produced upon binding the fluorescent-labelled antigen. To elucidate the limiting factors in signal acquisition, a series of fibers with increasingly reduced probe core radius was examined. The results were consistent with the V-number mismatch, the difference in mode carrying capacity between the clad and unclad fiber, being a critical factor in limiting signal coupling from the fiber probe. However, it was also delineated that conditions which conserve excitation power, such that power in the evanescent wave is optimized, must also be met to obtain a maximal signal. The threshold sensitivity for the optimal step-etched fiber probe was improved by over 20-fold in an immunoassay, although, it was demonstrated that signal acquisition decreased along the probe length, suggesting that a sensor region of uniform radius is not ideal.
Assuntos
Técnicas Biossensoriais , Tecnologia de Fibra Óptica/instrumentação , Imunoensaio de Fluorescência por Polarização/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Desenho de Equipamento , Estudos de Avaliação como Assunto , Monitorização Fisiológica/instrumentação , Fibras Ópticas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A biosensor was developed using antibodies, fluorescence and the evanescent wave to detect antigen binding at the surface of an optical fiber. Cladding was removed from the core along the distal end of a step-index optical fiber, and recognition antibodies were immobilized on the declad core to form the probe sensing region. Immersing the declad probe in aqueous solution creates a V-number mismatch between the immersed probe and the clad fiber. Probes created with reduced sensing region radius exhibited improved response by decreasing the V-number mismatch. Tapering the radius of this region has further improved probe response. Ray tracing analysis of the tapered probe demonstrated that the evanescent wave penetration depth increases along the length of the taper. Experiments correlating position of refraction along the taper with launch angle at the proximal end were realized in the ray tracing model. An evanescent wave immunoassay was performed with a series of the tapered fiber probes, each tapered from the fiber core radius (100 microns) to different end radii. An end radius of 29 microns was found to produce maximal signal from the tapered probe. Factors leading to the determination of the optimized probe are discussed.
Assuntos
Técnicas Biossensoriais , Tecnologia de Fibra Óptica/instrumentação , Imunoensaio de Fluorescência por Polarização/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Desenho de Equipamento , Estudos de Avaliação como Assunto , Fibras Ópticas , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Técnicas Biossensoriais/instrumentação , Escherichia coli O157/isolamento & purificação , Citometria de Fluxo/instrumentação , Separação Imunomagnética/instrumentação , Robótica/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de SistemasRESUMO
Because of their light weight and general wave-front-transforming ability, holograms appear potentially useful as beam correctors and collimators for diode-laser arrays in intersatellite optical data links. However, to survive in space a hologram must withstand damage from electrons and protons trapped in the Van Allen belts. We have found that holograms made with Polaroid DMP128 photopolymer on Suprasil-2 can withstand 63-MeV protons up to a total dose of 2 Mrad (Si) and withstand (60)Co gamma rays up to a total dose of 2 Mrad (Si) without loss of diffraction efficiency. It appears that these holograms are sufficiently radiation hard for space application.
RESUMO
We have found that holograms made in a dichromated gelatin emulsion sandwiched between Suprasil-2 plates are resistant to damage caused by 4.8 x 10(13)-Cm(-2) [1-Mev(Si) equivalent] fission neutrons. These holograms are, therefore, suitable for use in certain radiation environments.
RESUMO
Fiber optic biosensors have proven their ability to detect antigens rapidly in a variety of environmental and clinical samples. These biosensors are based on the technique of covalently linking antibodies to the core of an optical fiber and detecting antigen binding via measurement of fluorescence induced in the evanescent wave. One problem associated with these biosensors is the fiber-to-fiber variability in measured signal. We have addressed this problem by labeling a portion of the immobilized capture antibody with the fluorescent cyanine dye Cy5.5 (emission lambda max = 696 nm). The antigen was then labeled with fluorescent Cy5 (emission lambda max = 668 nm). Both fluorophores were excited by 635-nm light, and their emission was collected using both a fiber optic spectrometer and a biosensor optimized to collect fluorescence at two wave-lengths. The fluorescence from the Cy5.5-labeled capture antibody served as a calibration signal for each fiber and corrected for differences in optics, fiber defects, and varying amounts of capture antibody present on the fiber. Our data show that normalizing the signal measured from Cy5-labeled antigen binding to the Cy5.5 signal provides a standardization process for greatly reducing signal variance among individual fibers.