Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.

Simple derivatization method for sensitive determination of fatty acids with fluorescence detection by high-performance liquid chromatography using 9-(2-hydroxyethyl)-carbazole as derivatization reagent

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60 degreesC with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C-1-C-20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C-18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (lambda (ex) 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. (n = 6) for each fatty acid derivative are <4%. The detection limits are at 45-68 fmol levels for C-14-C-20 fatty acids and even lower levels for

Influence of electrode structure on the performance of a direct methanol fuel cell

Direct methanol fuel cells (DMFCs) consisting of multi-layer electrodes provide higher performance than those with the traditional electrode. The new electrode structure includes a hydrophilic thin film and a traditional catalyst layer. A decal transfer method was used to apply the thin film to the Nafion(R) membrane. Results show that the performance of a cell with the hydrophilic thin film is obviously enhanced. A cell with the optimal thin film electrode structure operating at I M CH3OH, 2 atm oxygen and 90degreesC yields a current density of 100 mA/cm(2) at 0.53 V cell voltage. The peak power density is 120 mW/cm(2). The performance stability of a cell in a short-term life operation was also increased when the hydrophilic thin film was employed. (C) 2002 Elsevier Science B.V. All rights reserved.

Fast Pose Estimation with Parameter Sensitive Hashing

Example-based methods are effective for parameter estimation problems when the underlying system is simple or the dimensionality of the input is low. For complex and high-dimensional problems such as pose estimation, the number of required examples and the computational complexity rapidly becme prohibitively high. We introduce a new algorithm that learns a set of hashing functions that efficiently index examples relevant to a particular estimation task. Our algorithm extends a recently developed method for locality-sensitive hashing, which finds approximate neighbors in time sublinear in the number of examples. This method depends critically on the choice of hash functions; we show how to find the set of hash functions that are optimally relevant to a particular estimation problem. Experiments demonstrate that the resulting algorithm, which we call Parameter-Sensitive Hashing, can rapidly and accurately estimate the articulated pose of human figures from a large database of example images.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same speciticity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients. (C) 2004 Elsevier B.V. All rights reserved.

Electrode catalysts behavior during direct ethanol fuel cell life-time test

In the present paper, a 60 h life-time test of a direct ethanol fuel cell (DEFC) at a current density of 20 mA cm(-2) (the beginning 38 h) and 40 mA cm(-2) (the last 22 h) was carried out. After the life-time test, the MEA could not achieve the former performance. X-ray diffraction (XRD), transmission electron microscopy (TEM) and energy dispersive X-ray analysis (EDX) were employed to characterize the anode and cathode catalyst before and after the life-time test. The XRD and TEM results showed that the particle size of the anode catalyst increased from 2.3 to 3.3 nm and the cathode from 3.0 to 4.6 nm. The EDX results of PtSn/C anode catalysts before and after the life-time test indicated that the content of the oxygen and tin, especially the content of the platinum, decreased prominently after the life-time test. The results suggest that the agglomeration of electrocatalysts, the destruction of the anode catalyst together with the fuel/water crossover from anode to cathode concurrently contribute to the performance degradation of the DEFC. (C) 2005 Elsevier B.V. All rights reserved.

Parameter Sensitive Detectors

Object detection can be challenging when the object class exhibits large variations. One commonly-used strategy is to first partition the space of possible object variations and then train separate classifiers for each portion. However, with continuous spaces the partitions tend to be arbitrary since there are no natural boundaries (for example, consider the continuous range of human body poses). In this paper, a new formulation is proposed, where the detectors themselves are associated with continuous parameters, and reside in a parameterized function space. There are two advantages of this strategy. First, a-priori partitioning of the parameter space is not needed; the detectors themselves are in a parameterized space. Second, the underlying parameters for object variations can be learned from training data in an unsupervised manner. In profile face detection experiments, at a fixed false alarm number of 90, our method attains a detection rate of 75% vs. 70% for the method of Viola-Jones. In hand shape detection, at a false positive rate of 0.1%, our method achieves a detection rate of 99.5% vs. 98% for partition based methods. In pedestrian detection, our method reduces the miss detection rate by a factor of three at a false positive rate of 1%, compared with the method of Dalal-Triggs.

Query-Sensitive Embeddings

A common problem in many types of databases is retrieving the most similar matches to a query object. Finding those matches in a large database can be too slow to be practical, especially in domains where objects are compared using computationally expensive similarity (or distance) measures. This paper proposes a novel method for approximate nearest neighbor retrieval in such spaces. Our method is embedding-based, meaning that it constructs a function that maps objects into a real vector space. The mapping preserves a large amount of the proximity structure of the original space, and it can be used to rapidly obtain a short list of likely matches to the query. The main novelty of our method is that it constructs, together with the embedding, a query-sensitive distance measure that should be used when measuring distances in the vector space. The term "query-sensitive" means that the distance measure changes depending on the current query object. We report experiments with an image database of handwritten digits, and a time-series database. In both cases, the proposed method outperforms existing state-of-the-art embedding methods, meaning that it provides significantly better trade-offs between efficiency and retrieval accuracy.

Continuous non-destructive monitoring of cell health using impedance based interdigitated electrode structured sensors

This article examines some preliminary tests which were performed in order to evaluate the best electrode configuration (width and spacing) for cell culture analyses. Biochips packaged with indium tin oxide (ITO) interdigitated electrodes (IDEs) were used to perform impedance measurements on A549 cells cultured on the surface of the biochip. Several tests were carried out using a 10 mM solution of Sodium Chloride (NaCl), cell medium and the cell culture itself to characterize some of the configurations already fabricated in the facilities at Tyndall National Institute.

Involvement of incipient hydrous oxide species in electrocatalysis at some metal electrode surfaces

In this thesis a novel theory of electrocatalysis at metal (especially noble metal)/solution interfaces was developed based on the assumption of metal adatom/incipient hydrous oxide cyclic redox transitions. Adatoms are considered as metastable, low coverage species that oxidise in-situ at potentials of often significantly cathodic to the regular metal/metal oxide transition. Because the adatom coverage is so low the electrochemical or spectroscopic response for oxidation is frequently overlooked; however, the product of such oxidation, referred to here as incipient hydrous oxide seems to be the important mediator in a wide variety of electrocatalytically demanding oxidation processes. Conversely, electrocatalytically demanding reductions apparently occur only at adatom sites at the metal/solution interface - such reactions generally occur only at potentials below, i.e. more cathodic than, the adatom/hydrous oxide transition. It was established that while silver in base oxidises in a regular manner (forming initially OHads species) at potentials above 1.0 V (RHE), there is a minor redox transition at much lower potentials, ca. o.35 v (RHE). The latter process is assumed to an adatom/hydrous oxide transition and the low coverage Ag(l) hydrous oxide (or hydroxide) species was shown to trigger or mediate the oxidation of aldehydes, e. g. HCHO. The results of a study of this system were shown to be in good agreement with a kinetic model based on the above assumptions; the similarity between this type of behaviour and enzyme-catalysed processes - both systems involve interfacial active sites - was pointed out. Similar behaviour was established for gold where both Au(l) and Au(lll) hydrous oxide mediators were shown to be the effective oxidants for different organic species. One of the most active electrocatalytic materials known at the present time is platinum. While the classical view of this high activity is based on the concept of activated chemisorption (and the important role of the latter is not discounted here) a vital role is attributed to the adatom/hydrous oxide transition. It was suggested that the well known intermediate (or anomalous) peak in the hydrogen region of the cyclic voltanmogram for platinum region is in fact due to an adatom/hydrous oxide transition. Using potential stepping procedures to minimise the effect of deactivating (COads) species, it was shown that the onset (anodic sweep) and termination (cathodic sweep) potential for the oxidation of a wide variety of organics coincided with the potential for the intermediate peak. The converse was also shown to apply; sluggish reduction reactions, that involve interaction with metal adatoms, occur at significant rates only in the region below the hydrous oxide/adatom transition.

Fabrication, characterisation and electroanalysis at 1-dimensional nanostructures

Integrated nanowire electrodes that permit direct, sensitive and rapid electrochemical based detection of chemical and biological species are a powerful emerging class of sensor devices. As critical dimensions of the electrodes enter the nanoscale, radial analyte diffusion profiles to the electrode dominate with a corresponding enhancement in mass transport, steady-state sigmoidal voltammograms, low depletion of target molecules and faster analysis. To optimise these sensors it is necessary to fully understand the factors that influence performance limits including: electrode geometry, electrode dimensions, electrode separation distances (within nanowire arrays) and diffusional mass transport. Therefore, in this thesis, theoretical simulations of analyte diffusion occurring at a variety of electrode designs were undertaken using Comsol Multiphysics®. Sensor devices were fabricated and corresponding experiments were performed to challenge simulation results. Two approaches for the fabrication and integration of metal nanowire electrodes are presented: Template Electrodeposition and Electron-Beam Lithography. These approaches allow for the fabrication of nanowires which may be subsequently integrated at silicon chip substrates to form fully functional electrochemical devices. Simulated and experimental results were found to be in excellent agreement validating the simulation model. The electrochemical characteristics exhibited by nanowire electrodes fabricated by electronbeam lithography were directly compared against electrochemical performance of a commercial ultra-microdisc electrode. Steady-state cyclic voltammograms in ferrocenemonocarboxylic acid at single ultra-microdisc electrodes were observed at low to medium scan rates (≤ 500 mV.s-1). At nanowires, steady-state responses were observed at ultra-high scan rates (up to 50,000 mV.s-1), thus allowing for much faster analysis (20 ms). Approaches for elucidating faradaic signal without the requirement for background subtraction were also developed. Furthermore, diffusional process occurring at arrays with increasing inter-electrode distance and increasing number of nanowires were explored. Diffusion profiles existing at nanowire arrays were simulated with Comsol Multiphysics®. A range of scan rates were modelled, and experiments were undertaken at 5,000 mV.s-1 since this allows rapid data capture required for, e.g., biomedical, environmental and pharmaceutical diagnostic applications.