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Signal Amplification (signal + amplification)
Selected AbstractsThe Determination of Methylmercury in Real Samples Using Organically Capped Mesoporous Inorganic Materials Capable of Signal Amplification,ANGEWANDTE CHEMIE, Issue 45 2009Estela Climent Die Affinität von Methylquecksilber zu Leukosquarain-Gruppen (weiß im Bild) bewirkt das Öffnen damit bedeckter Poren eines mesoporösen Hybridmaterials und das Freisetzen eingeschlossener Safraninfarbstoffe (orange), was den selektiven optischen Nachweis von Methylquecksilber in komplexen biologischen Proben ermöglicht. [source] Post-transplant recurrent hepatitis C: immunohistochemical detection of hepatitis C virus core antigen and possible pathogenic implicationsLIVER INTERNATIONAL, Issue 6 2008Mário G. Pessôa Abstract Introduction: The mechanisms by which severe cholestatic hepatitis develops after liver transplantation are not fully understood. Reports on immunohistochemical distribution of hepatitis C virus (HCV) antigens are still scarce, but recently, HCV immunostaining was suggested for early diagnosis of cholestatic forms of recurrent hepatitis C in liver grafts. After purification, Rb246 pab anticore (aa1-68) yielded specific, granular cytoplasmic staining in hepatocytes. Signal amplification through the Envision-Alkaline Phosphatase System avoided endogenous biotin and peroxidase. Aims/Methods: Rb246 was applied to liver samples of explants of 12 transplant recipients, six with the most severe form of post-transplantation recurrence, severe cholestatic hepatitis (group 1) and six with mild recurrence (group 2). We also assessed immuno-reactivity at two time-points post-transplantation (median 4 and 22 months) in both groups. HCV-core Ag was semiquantified from 0 to 3+ in each time point. Serum HCV-RNA was also measured on the different time points by branched DNA. Results: In the early post-transplant time point, one patient had a mild staining (1+), two patients had a moderate staining (2+) and the other three had no staining in group 1, compared with five patients with no staining (0) and one patient with mild staining (1+) in group 2. Late post-transplant liver samples were available in nine patients, and two out of four samples in group 1 showed a mild staining, compared with no staining patients in five patients in group 2. Strikingly, on the explant samples, HCV immunostaining was strongly positive in group 1, and mildly positive in group 2. Two out of five samples showed 3+ staining, and three samples showed 2+ staining in group 1; two out of five samples showed no staining, two samples showed 1+ staining and one sample showed 2+ staining in group 2. Serum HCV-RNA was significantly higher in group 1, on both time-points post-transplantation. HCV-core Ag was not directly associated with serum HCV-RNA on the different time points. Conclusion: These preliminary results suggest that strong HCV immunostaining in the explant is predictive of more severe disease recurrence. [source] DNA Aptamers that Bind to PQQGDH as an Electrochemical Labeling ToolELECTROANALYSIS, Issue 11 2009Yuko Osawa Abstract We screened DNA aptamers that bind to pyrroquinoline quinone glucose dehydrogenase (PQQGDH) for the development of an electrochemical labeling tool. PQQGDH is an excellent enzyme for the signal amplification of biosensors. We focused on DNA aptamers as labeling agents and tried to select those DNA aptamers that bind to PQQGDH without affecting its enzymatic activity. After 7 rounds of screening, one aptamer was obtained: ,PGa4'. It bound to PQQGDH with specificity and showed no effect on the glucose dehydrogenase (GDH) activity. Moreover, beads labeled with PQQGDH via PGa4 generated an electrical current upon glucose addition. Therefore, we believe that the PGa4 aptamer against PQQGDH may become a powerful labeling tool for electrochemical biosensors. [source] Amperometric Detection of Catecholamine Neurotransmitters Using Electrocatalytic Substrate Recycling at a Laccase ElectrodeELECTROANALYSIS, Issue 2 2005Yvonne Ferry Abstract An enzyme electrode based on the coimmobilization of an osmium redox polymer and laccase on glassy carbon electrodes has been applied to ultra sensitive amperometric detection of the catecholamine neurotransmitters dopamine, epinephrine and norepinephrine, resulting in nanomolar detection limits, as low as 4,nM for dopamine. The sensitivity of the electrode is due to signal amplification via oxidation of the catecholamine by the immobilized laccase, which is regenerated by concomitant reduction of oxygen to water, coupled to the electrocatalytic re-reduction of the oxidized catecholamine by the osmium redox complex: electrocatalytic substrate recycling. In addition because the sensor can be operated in reductive mode at ,0.2,V (vs. Ag/AgCl), noise and interferences are diminished. Combined with its high sensitivity this enzyme electrode also exhibited excellent selectivity allowing the detection of catecholamines in the presence of ascorbic acid. However, differentiation between the current responses achieved for the three catecholamines is not possible. The effective mode of constant recycling, resulting in amplification of the current response, of the laccase enzyme electrode sensor combined with the inherent advantages of using electrochemical techniques holds great promise for the future of catecholamine detection and monitoring. [source] Application of Exchangeable Biochemical Reactors with Oxidase-Catalase-Co-immobilizates and Immobilized Microorganisms in a Microfluidic Chip-CalorimeterENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 5 2008M. Leifheit Abstract Several methods for the quantitative detection of different compounds, e.g., L -amino acids, sugars or alcohols in liquid media were developed by application of an automatic measuring unit including a fluid chip-calorimeter FCC-21. For this purpose, enzymes were immobilized covalently on the inner and outer surface of CPG (controlled porous glass)-spherules with an outer diameter of 100,,m and filled into a micro flow-through reaction chamber (VR = 20,,L). The design of the measuring cell allows for easy insertion into the calorimeter device of a stored series of comfortably pre-fabricated measuring cells. These cells can be filled with different enzyme immobilizates. Different oxidases were used and co-immobilized with catalase for the improvement of the detection sensitivity. A signal amplification could be achieved up to a factor of 3.5 with this configuration. ,- D -glucose, ethanol and L -lysine could be detected in a range of 0.25,1.75,mM using glucose oxidase, alcohol oxidase and lysine oxidase. The group of oxidases in combination with the enzymatic catalysis of the intermediate H2O2 allows the quantitative detection of a large number of analytes. A good measurement and storage stability could be achieved for several weeks by this immobilization method. In addition to enzyme-based detection reactions, it was shown that living microorganisms can be immobilized in the reaction chamber. Thus, the system can be used as a whole-cell biosensor. The quantitative detection of phenol in the range of 10,100,,M could be performed using the actinomycete Rhodococcus sp. immobilized on glass beads by means of embedding into polymers. [source] Cationic Polyelectrolyte Amplified Bead Array for DNA Detection with Zeptomole Sensitivity and Single Nucleotide Polymorphism SelectivityADVANCED FUNCTIONAL MATERIALS, Issue 16 2010Chun Wang Abstract A highly sensitive strand specific DNA assay, which consists of a peptide nucleic acid (PNA) probe, a cationic conjugated polymer (PFVP), and self-assembled polystyrene beads in microwell arrays on silicon chip, is reported. PFVP, as an efficient signal amplifier and signal reporter, has been specially designed and synthesized to be compatible with commercial confocal microscopes for sensing on solid substrates. The assay operates on the net increase in negative charge at the PNA surface that occurs upon single-stranded DNA hybridization, which subsequently allows complex formation with the positively charged PFVP to favor energy transfer between the polymer and Cy5-labeled target. With maximized surface contact provided by bead arrays and signal amplification provided by PFVP, this assay allows detection of ,300 copies of Cy5-labeled DNA using a commercial confocal microscope. In addition, the same strategy is also extended for label-free DNA detection with a detection sensitivity of 150 attomole. Excellent discrimination against single nucleotide polymorphism (SNP) is also demonstrated for both Cy5-labeled and label-free target detection. This study indicates that cationic conjugated polymers have great potential to be incorporated into the widely used microarray technology for simplified process with improved detection sensitivity. [source] Chemical Nanosensors Based on Composite Molecularly Imprinted Polymer Particles and Surface-Enhanced Raman ScatteringADVANCED MATERIALS, Issue 21 2010Marc Bompart Chemical nanosensors with a submicrometer core,shell composite design, based on a polymer core, a molecularly imprinted polymer (MIP) shell for specific analyte recognition, and an interlayer of gold nanoparticles for signal amplification, are described. SERS measurements on single nanosensors yield detection limits of 10,7,M for the , -blocker propranolol, several orders of magnitude lower than on plain MIP spheres. [source] Protein Nanoparticle Templates: Constrained Synthesis and Organization of Catalytically Active Metal Nanoparticles by Self-Assembled Protein Templates (Adv. Mater.ADVANCED MATERIALS, Issue 34 200934/2009) Novel geometrical architectures of hybrid nanoparticle/protein complexes can be generated by chemically synthesizing monodisperse metal nanoparticles in situ in the presence of a stable, stress-related protein, report Silke Behrens, Oded Shoseyov, and co-workers on p. 3515. The particles are catalytically active and can reduce 4-nitrophenol, and are potentially useful as future biofunctional nanoparticle labels for catalytic signal amplification in optical assays. [source] Constrained Synthesis and Organization of Catalytically Active Metal Nanoparticles by Self-Assembled Protein TemplatesADVANCED MATERIALS, Issue 34 2009Silke Behrens Novel geometrical architectures of hybrid nanoparticle,protein complexes are generated by chemically synthesizing monodisperse metal nanoparticles in situ in the presence of a stable, stress-related protein. The catalytic activity of the protein,particle hybrids is examined for the reduction of 4-nitrophenol, providing future biofunctional nanoparticle labels for catalytic signal amplification in optical assays. [source] Low-Noise Fully Differential Amplifiers Using JFET-CMOS Integration Technology for Smart SensorsIEEJ TRANSACTIONS ON ELECTRICAL AND ELECTRONIC ENGINEERING, Issue 3 2008Hidekuni Takao Member Abstract In this paper, CMOS-based low-noise amplifiers with JFET-CMOS technology for high-resolution sensor interface circuits are presented. A differential difference amplifier (DDA) configuration is employed to realize differential signal amplification with very high input impedance, which is required for the front-end circuit in many sensor applications. Low-noise JFET devices are used as input pair of the input differential stages or source-grounded output load devices, which are dominant in the total noise floor of DDA circuits. A fully differential amplifier circuit with pure CMOS DDA and three types of JFET-CMOS DDAs were fabricated and their noise performances were compared. The results show that the total noise floor of the JFET-CMOS amplifier was much lower compared to that of the pure CMOS configuration. The noise-reduction effect of JFET replacement depends on the circuit configuration. The noise reduction effect by JFET device was maximum of about , 18 dB at 2.5 Hz. JFET-CMOS technology is very effective in improving the signal-to-noise ratio (SNR) of a sensor interface circuit with CMOS-based sensing systems. © 2008 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc. [source] IDENTIFICATION OF BACTERIA ASSOCIATED WITH DINOFLAGELLATES (DINOPHYCEAE) ALEXANDRIUM SPP.JOURNAL OF PHYCOLOGY, Issue 2 2002FLUORESCENT IN SITU HYBRIDIZATION AND CONFOCAL MICROSCOPY, USING TYRAMIDE SIGNAL AMPLIFICATION In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification,fluorescent in situ hybridization (TSA-FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA-FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton. [source] An overview on S-band erbium-doped fiber amplifiersLASER PHYSICS LETTERS, Issue 1 2007S. W. Harun Abstract An erbium-doped fiber amplifier (EDFA) for S-band signal amplification is designed by using a depressed cladding erbium-doped fiber (DC-EDF). The fiber's characteristics are described in terms of the effects of the fiber spooling diameter on the amplifier's performance. In this experiment, the spooling diameter required for optimum amplifier gain was around 5,7 cm. By using a typical two-stage configuration (with a 27 m long DC-EDF and a 260 mW pump laser power), the maximum small signal gain obtained was about 32 dB. Yet, by employing a double pass amplifier configuration with a shorter DC-EDF length and a lower pump laser power (15 m and 135 mW, respectively), a similar maximum small signal gain of approximately 30 dB was achieved. This improvement in gain characteristics however, incurred an increased noise figure penalty of about 1 dB in comparison to single-pass amplifier configurations. In order to reduce the amplifier's noise figure while maintaining its high gain, a partial double-pass S-band EDFA configuration was introduced. This configuration provides a high 26.9 dB gain and an improved noise figure comparable to a single pass configuration. Gain clamping in S-band EDFAs are also demonstrated by utilizing a fiber Bragg grating to form an oscillating laser at around 1530 nm. This technique enables good gain clamping with a gain variation of less than 1 dB. (© 2007 by Astro, Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA) [source] How to use virological tools for optimal management of chronic hepatitis CLIVER INTERNATIONAL, Issue 2009Stéphane Chevaliez Abstract Chronic hepatitis C is a global health problem that may cause cirrhosis and progression to hepatocellular carcinoma. Currently available antiviral treatments are moderately effective. Several virological assays are available to help diagnose and manage patients infected with the hepatitis C virus (HCV). These include the anti-HCV antibody assays, measurement of HCV RNA viral load and HCV genotyping. HCV RNA can be assayed by two types of molecular biology-based techniques: target amplification as in polymerase chain reaction methods and signal amplification such as the branched DNA assay. Monitoring of viral kinetics during the early phases of antiviral treatment is crucial in making treatment decisions such as early stopping rules and also in optimizing the length of treatment. The HCV genotype can be determined by several methods. Whatever the method, pretreatment determination allows treatment length and ribavirin dose to be optimized and also offers prognostic information on treatment outcomes as certain genotypes respond more favourably to treatment. Thus, virological assays are indispensable in the diagnosis and management of individuals infected with the HCV. [source] Cationic and Anionic Conjugated Polyelectrolytes: Aggregation-Mediated Fluorescence Energy Transfer to Dye-Labeled DNAMACROMOLECULAR RAPID COMMUNICATIONS, Issue 16 2008Youngeup Jin Abstract An electrostatic complex of water-soluble conjugated polyelectrolytes (CPs) between anionic poly(9,9-bis(4,-sulfonatobutyl)fluorene- co-alt -1,4-phenylene) disodium salt (a-PFP) and cationic poly(9,9-bis((6,- N,N,N,-trimethylammonium)hexyl)fluorene- co -2,1,3-bezothiadiazole) dibromide (85:15) (c-PFB15) was tested as a fluorescence resonance energy transfer (FRET) donor to Texas Red (TR)-labeled single-stranded DNA (ssDNA-TR) via two-step FRET processes. Electrostatic complexation of a-PFP and c-PFB15 in water leads to aggregation of polymer chains, a concomitant reduction of intersegment distances, and energy transfer to the benzothiadiazole (BT) segments. The following complexation with ssDNA-TR leads to energy transfer from BT to TR via two-step FRET processes. This detection schematic shows an FRET-induced signal amplification, which can be achieved by adjusting the charge ratio in the cationic/anionic CP complex and controlling the number density of the binding CPs around the acceptor, resulting in enhanced antenna effects and sensitivity in CP-based FRET DNA detection assays. [source] Non-radioactive in situ detection of mRNA in ES cell-derived cardiomyocytes and in the developing heartMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2002Arnoud C. Fijnvandraat Abstract Non-radioactive in situ hybridisation is an excellent method to visualise mRNA molecules within their topographical context. Recently we have reported a new non-radioactive in situ hybridisation procedure on tissue sections that is essentially based on the whole mount in situ hybridisation procedure. This method is superior in spatial resolution and sensitivity compared to the radioactive in situ hybridisation procedure. Generally, low levels of gene expression, such as found with the developmental onset of gene expression and in differentiating embryonic stem cells, are difficult to detect by in situ hybridisation. Here an application of the protocol is presented which is based on tyramide signal amplification, which enables the detection of very low abundant mRNAs. The significance of this method is two-fold: (1) the molecular phenotype of embryonic stem cell-derived cardiomyocytes can be examined at the cellular level with high sensitivity, and (2) the number of cells that express the gene of interest can be assessed. Microsc. Res. Tech. 58:387,394, 2002. © 2002 Wiley-Liss, Inc. [source] Human papillomavirus as a risk factor in oral carcinogenesis: a study using in situ hybridization with signal amplificationMOLECULAR ORAL MICROBIOLOGY, Issue 4 2008R. Acay Introduction:, It is still controversial whether human papillomavirus (HPV) can be considered a risk factor in oral carcinogenesis. The aim of this study was to detect HPV DNA in 50 cases diagnosed as oral leukoplakias, with different degrees of epithelial dysplasia, and as oral squamous cell carcinomas, using in situ hybridization with signal amplification (CSA-ISH). Methods:, HPV DNA was assessed in paraffin sections using CSA-ISH with a wide-spectrum biotinylated DNA probe. In HPV-positive cases, genotyping with specific probes to HPV types 6/11, 16/18 and 31/33 was performed. Results:, The overall prevalence of HPV infection was 24%, markedly higher than that found in the control group. Results showed a discrete proportional relationship in the indices found in leukoplakia with no dysplasia, leukoplakia with dysplasia, and squamous cell carcinoma, but this was not statistically significant. When separating the group of leukoplakia by degrees of dysplasia, this relation of proportion was not observed. In genotyping, HPV types 16/18 were the most prevalent, and types 6/11 were only found in groups of mild or no dysplasia. Conclusion:, The results suggest that HPV is not likely to play a role in the progression of malignant transformation in oral lesions. Nevertheless, the increased prevalence of HPV infection compared to normal oral mucosa and the fact that high-risk HPV types were the most frequently identified do not allow the exclusion of HPV as a risk factor in oral carcinogenesis. [source] Alternans in QRS Amplitude During Ventricular TachycardiaPACING AND CLINICAL ELECTROPHYSIOLOGY, Issue 2 2002PHILIPPE MAURY MAURY, P., et al.: Alternans in QRS Amplitude During Ventricular Tachycardia. Although the value of T wave alternans as an index of electrical instability has been extensively investigated, little is known about QRS alternans during VT. Intracardiac electrograms of 111 episodes of spontaneous monomorphic regular VT retrieved from implantable defibrillators in 25 patients were retrospectively selected. Three beat series, representing the total amplitudes and amplitudes from baseline to summit and from baseline to lower point of 16 or 32 successive QRS complexes before deliverance of electrical therapy were generated for each episode. Spectral analysis was then performed using the fast Fourrier transform. VT was considered as alternans if the magnitude of the spectral power at the 0.5-cycle/beat frequency was greater than the mean ± 3 SD of the noise in at least one of the three spectral curves. QRS alternans was present in 23 (20%) of 111 episodes and in 9 (36%) of 25 patients. Alternans was not related to the VT cycle length, QRS duration, QRS amplitude, signal amplification, nor to clinical variables. Alternans was more frequently detected in unipolar configuration and when a higher number of complexes was included in analysis. Failure of antitachycardia pacing was more frequent in case of alternans VT (50% vs 75% success in non-alternans VT, P = 0.05). Spontaneous termination before deliverance of therapy occurred in 16 nonalternans VT but never in alternans episodes (P = 0.02). Alternans in QRS amplitude is a relatively common finding during VT and could be associated with failure of antitachycardia pacing and lack of spontaneous termination. Lower efficacy of electrical therapies in case of QRS alternans must be confirmed in a way to improve the effectiveness of antitachycardia pacing. [source] Characterization of neuropeptide Y2 receptor protein expression in the mouse brain.THE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 3 2006Abstract Neuropeptide Y (NPY), a 36-amino-acid peptide, mediates biological effects by activating Y1, Y2, Y5, and y6 receptors. NPY neurons innervate many brain regions, including the hypothalamus, where NPY is involved in regulation of a broad range of homeostatic functions. We examined, by immunohistochemistry with tyramide signal amplification, the expression of the NPY Y2 receptor (Y2R) in the mouse brain with a newly developed rabbit polyclonal antibody. Y2R immunoreactivity was specific with its absence in Y2R knockout (KO) mice and in adjacent sections following preadsorption with the immunogenic peptide (10,5 M). Y2R-positive processes were located in many brain regions, including the olfactory bulb, some cortical areas, septum, basal forebrain, nucleus accumbens, amygdala, hippocampus, hypothalamus, substantia nigra compacta, locus coeruleus, and solitary tract nucleus. However, colchicine treatment was needed to detect Y2R-like immunoreactivity in cell bodies in many, but not all, areas. The densest distributions of cell bodies were located in the septum basal forebrain, including the bed nucleus, and amygdala, with lower density in the anterior olfactory nucleus, nucleus accumbens, caudal striatum, CA1, CA2, and CA3 hippocampal fields, preoptic nuclei lateral hypothalamus, and A13 DA cells. The widespread distribution of Y2R-positive cell bodies and fibers suggests that NPY signaling through the Y2R is common in the mouse brain. Localization of the Y2R suggests that it is mostly presynaptic, a view supported by its frequent absence in cell bodies in the normal mouse and its dramatic increase in cell bodies of colchicine-treated mice. J. Comp. Neurol. 499:357,390, 2006. © 2006 Wiley-Liss, Inc. [source] C-fiber (Remak) bundles contain both isolectin B4-binding and calcitonin gene-related peptide-positive axonsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 4 2005Beth Brianna Murinson Abstract Unmyelinated nerve fibers (Remak bundles) in the rodent sciatic nerve typically contain multiple axons. This study asked whether C-fiber bundles contain axons arising from more than one type of neuron. Most small neurons of the lumbar dorsal root ganglion (DRG) are either glial cell line-derived neurotrophic factor dependent or nerve growth factor dependent, binding either isolectin B4 (IB4) or antibodies to calcitonin gene-related peptide (CGRP), respectively. Injection of IB4-conjugated horseradish peroxidase into a lumbar DRG resulted in intense labeling of IB4 axons, with very low background. Visualized by confocal fluorescence, IB4-binding and CGRP-positive nerve fibers orginating from different DRG neurons came together and remained closely parallel over long distances, suggesting that these two types of axon occupy the same Remak bundle. With double-labeling immunogold electron microscopy (EM), we confirmed that IB4 and CGRP axons were distinct and were found together in single Remak bundles. Previous studies indicate that some DRG neurons express both CGRP and IB4 binding. To ensure that our immunogold results were not a consequence of coexpression, we studied large populations of unmyelinated axons by using quantitative single-label EM. Tetramethylbenzidine, a chromogen with strong intrinsic signal amplification of IB4-horseradish peroxidase, labeled as many as 52% of unmyelinated axons in the dorsal root. Concomitantly, 97% of the Remak bundles with more than one axon contained at least one IB4-labeled axon. Probabilistic modeling using binomial distribution functions rejected the hypothesis that IB4 axons segregate into IB4-specific bundles (P < 0.00001). We conclude that most Remak bundle Schwann cells simultaneously support diverse axon types with different growth factor dependences. J. Comp. Neurol. 484:392,402, 2005. © 2005 Wiley-Liss, Inc. [source] Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplificationTHE PLANT JOURNAL, Issue 6 2001Ludmila I. Khrustaleva Summary The sensitivity of fluorescence in situ hybridization (FISH) for mapping plant chromosomes of single-copy DNA sequences is limited. We have adapted for plant cytogenetics a new signal-amplification method termed tyramide-FISH (Tyr-FISH). Until present this technique has only been applied to human chromosomes. The method is based on enzymatic deposition of fluorochrome-conjugated tyramide. With Tyr-FISH it was possible to detect target T-DNA sequences on plant metaphase chromosomes as small as 710 bp without using a cooled CCD camera. Short detection time and high sensitivity, in combination with a low background, make the Tyr-FISH method very suitable for routine application in plant cytogenetic research. With Tyr-FISH we analysed the position of T-DNA inserts in transgenic shallots. We found that the inserts were preferentially located in the distal region of metaphase chromosomes. Sequential fluorescence in situ hybridization with a 375 bp satellite sequence suggested that a specific T-DNA insert was located within the satellite sequence hybridization region on a metaphase chromosome. Analysis of less-condensed prophase and interphase chromosomes revealed that the T-DNA was integrated outside the satellite DNA-hybridization region in a more proximal euchromatin region. [source] Application of cellulose-based self-assembled tri-enzyme system in a pseudo-reagent-less biosensor for biogenic catecholamine detectionBIOTECHNOLOGY JOURNAL, Issue 5 2007Mikhail L. Rabinovich Professor Abstract Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine. [source] | ||||||||||||||||||||||