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Analytical and Bioanalytical Chemistry (v.377, #6)
Protease sensors for bioimaging by Martin Funovics; Ralph Weissleder; Ching-Hsuan Tung (pp. 956-963).
Optical imaging of specific molecular targets and pathways in vivo has recently become possible through continued developments in imaging equipment, reconstruction algorithms, and more importantly the availability of imaging reporter molecules. These reporter molecules encompass photoproteins expressed in vivo and exogenously administered probes detectable by fluorescence and/or bioluminescence imaging. One particularly enticing aspect of optical imaging is the ability to design activatible probes with inherent amplification. This review summarizes our experience in developing novel near-infrared fluorescent (NIRF) imaging agents that report on protease activities. These agents are designed to be biocompatible, highly activatible, and able to produce bright NIRF following protease cleavage.
Keywords: Optical Imaging; Neoplasms, detection; Fluorescence; Contrast agents, activatible
Optical imaging: bacteria, viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals by Yong A. Yu; Tatyana Timiryasova; Qian Zhang; Richard Beltz; Aladar A. Szalay (pp. 964-972).
Early detection of tumors and their metastases is crucial for the prognosis of cancer treatment. Traditionally, tumor detection is achieved by various methods, including magnetic resonance imaging and computerized tomography. With the recent cloning, cellular expression, and real-time imaging of light-emitting proteins, such as Renilla luciferase (Ruc), bacterial luciferase (Lux), firefly luciferase (Luc), green fluorescent protein (GFP), or Ruc-GFP fusion protein, significant efforts have been focused on using these marker proteins for tumor detection. It has also been demonstrated that certain bacteria, viruses, and mammalian cells (BVMC), when administered systemically, are able to gain entry and replicate selectively in tumors. In addition, many tissue/tumor specific promoters have been cloned which allow transgene expression specifically in tumor tissues. Therefore, when light-emitting protein encoded BVMC are injected systemically into rodents, tumor-specific marker gene expression is achieved and is detected in real time based on light emission. Consequently, the locations of primary tumors and previously unknown metastases in animals are revealed in vivo. In the future it will likely be feasible to use engineered light-emitting BVMC as probes for tumor detection and as gene-delivery vehicles in vivo for cancer therapy.
Keywords: Optical imaging ; Tumors ; Metastases ; Luciferase ; Green fluorescent protein ; Bacteria ; Vaccinia virus ; Mammalian cells
Characterization of CNS disorders and evaluation of therapy using structural and functional MRI by Markus Rudin; Thomas Mueggler; Peter R. Allegrini; Diana Baumann; Martin Rausch (pp. 973-981).
Modern drug development requires technologies that allow rapid translation from the preclinical to the clinical stage. It is obvious that non-invasive imaging modalities such as magnetic resonance imaging (MRI) will play a central role in this regard. This article reviews the use of structural and functional MRI readouts for characterization of central nervous system (CNS) disorders and evaluation of the efficacy of potential CNS drugs. Examples comprise dementia of Alzheimer's type, cerebral ischemia, and neuroinflammation covering both clinical and preclinical aspects. In these examples MRI has been used to obtain relevant structural information on brain atrophy, on the location and extent of ischemic brain areas, and on the number and distribution of demyelinated plaques. These structural data are complemented by readouts assessing the functional consequences associated with the pathomorphological changes. In the last decade, MRI has evolved into a standard tool for the development of CNS drugs. With regard to target-specific/molecular imaging applications MRI is limited by its inherently low sensitivity; complementary imaging modalities utilizing optical and radionuclear reporter systems will thus be required.
Keywords: Magnetic resonance imaging (MRI); Functional MRI; Cerebral ischemia; Alzheimer's dementia; Multiple sclerosis; Experimental allergic encephalitis; Drug development
Three-dimensional ultrasound imaging and its use in quantifying organ and pathology volumes by Aaron Fenster; Dónal B. Downey (pp. 982-989).
Although ultrasonography is an important cost-effective imaging modality, technical improvements are needed before its full potential is realized for accurate and reproducible monitoring of disease progression or regression. Two-dimensional viewing of three-dimensional anatomy, using conventional ultrasonography, limits our ability to quantify and visualize pathology and is partly responsible for the reported variability in diagnosis and monitoring of disease progression. Efforts of investigators have focused on overcoming these deficiencies by developing 3D ultrasound imaging techniques that are capable of acquiring B-mode images using existing conventional ultrasound systems, reconstructing the information into 3D images, and then allowing interactive viewing of the 3D images on inexpensive desktop computers. In addition, the availability of 3D ultrasound images has allowed the development of manual and semi-automated techniques to quantify normal and abnormal anatomical volumes. In this paper we review our semi-automated 3D segmentation approaches for segmenting the surface of the carotid arteries and plaques, and segmenting the prostate. These techniques demonstrate that efficient segmentation techniques can be used with 3D ultrasound images to quantify anatomical organ volumes and morphology.
Keywords: 3D Ultrasonography; 3D Imaging; Volume measurements
Quantitative magnetic resonance (QMR) method for bone and whole-body-composition analysis by Gersh Z. Taicher; Frank C. Tinsley; Arcady Reiderman; Mark L. Heiman (pp. 990-1002).
Objective: to evaluate the applicability, precision, and accuracy of the new EchoMRI quantitative magnetic resonance (QMR) method for in-vitro bovine bone analysis and in-vivo whole-body-composition analysis of conscious live mice. Research methods and procedures: bovine tibia bone samples were measured by QMR and dual-energy X-ray adsorptiometry (DEXA). Repeated measures of whole-body composition were made using live and dead mice with different levels of fat by QMR and DEXA and by classic chemical analysis of the mouse carcass. Results: bone-mineral density (BMD) and bone-mineral content (BMC) measured in bovine tibia by QMR and DEXA were highly correlated. Precision of fat and lean measurement in mice was found to be better for QMR than for DEXA. The coefficient of variation (CV) for fat was 0.34–0.71% for QMR compared with 3.06–12.60% for DEXA. Discussion: QMR offers more specific parameters of bone structure than does DEXA. QMR and DEXA did not differ in the total amount of fat detected in live mice but QMR had improved precision. QMR was superior to DEXA in measuring fat in very small mice. Conclusions: in bone tissue there is a strong correlation between hydrogen NMR signal and bone-mineral density as measured by X-ray. QMR provides a very precise, accurate, fast, and easy to use method for determining fat and lean mass of mice without the need for anesthesia. Its ability to detect differences and monitor changes in body composition in mice with great precision should be of great value in characterizing phenotypes and studying drugs affecting obesity.
Keywords: QMR; Bone; Osteoporosis; Body composition; Obesity; New technology
A new approach for assessing early osteoarthritis in the rat by M. J. Roberts; S. B. Adams Jr; N. A. Patel; D. L. Stamper; M. S. Westmore; S. D. Martin; J. G. Fujimoto; M. E. Brezinski (pp. 1003-1006).
Several animal models have been developed to investigate osteoarthritis and potential disease-modifying therapeutics. However, early disease data from these models are limited by the resolution of current imaging modalities. In this in-vitro study, an optical coherence tomography (OCT) system with an axial resolution of 15 µm was used to track sequential changes in osteoarthritic rat knees. Osteoarthritis was induced via transection of the medial collateral ligament and an artificial full thickness meniscal tear. Imaging occurred at one, two, and three weeks after surgery. OCT successfully detected early signs of osteoarthritic change, including alteration of the cartilage surface and disruption of the bone–cartilage interface. This study demonstrates that OCT, along with the induction of mechanical injury, provides an excellent model for monitoring the sequential changes of osteoarthritis.
Keywords: Optical coherence tomography; Osteoarthritis; Articular cartilage; Imaging; MRI
Analysis of serotonin release from single neuron soma using capillary electrophoresis and laser-induced fluorescence with a pulsed deep-UV NeCu laser by Hai Miao; Stanislav S. Rubakhin; Jonathan V. Sweedler (pp. 1007-1013).
The use of capillary electrophoresis (CE) with laser-induced fluorescence excited by ultraviolet (UV) lasers in the range 200–300 nm has been restricted by the available wavelengths and expense of UV lasers. The integration of a NeCu deep UV laser operating at 248.6 nm with a single channel CE system with post-column sheath flow detection allows detection limits for serotonin and tryptophan of 3.9×10-8 M and 4.5×10-8 M respectively. Single cell analysis of serotonergic metacerebral cells from the sea slug Aplysia californica yields a value of 800±85 fmol of serotonin in each cell soma. For the first time, serotonin is directly detected in electrically stimulated release from single metacerebral cell soma, with approximately 4% of the serotonin contained in the soma released from a semi-intact preparation with a 2 min electrical stimulation.
Keywords: NeCu laser; Capillary electrophoresis; Single cell analysis; Release; Serotonin
Analysis of silica hydride and surface hydrosilation reactions by solid-state NMR in the preparation of p-chlorobenzamide bonded silica phase by B. Lynch; G. H. Müller; L. O. Healy; J. D. Glennon; M. Pursch; K. Albert (pp. 1014-1019).
29Si and 13C CP-MAS NMR spectroscopy was used to follow the conversion of native silica to a p-chlorobenzamide bonded silica material. The benzamide bonded phase was prepared via a hydrosilation reaction of a hydride silica intermediate with p-chloro-N-allylbenzamide. Solid-state NMR was used to show the disappearance of reactive surface hydride species (MH) and to identify newly formed bonded chemical species on the silica surface. DRIFT spectroscopy, elemental analysis, and specific surface-area determinations (BET) of the prepared phases are also reported.
Keywords: Solid-state NMR spectroscopy; Liquid chromatography; Preparation of bonded silica phases; Hydrosilation on hydride silica
Determination of ultra-trace amounts of Fe in AgNO3 solutions by means of isotope dilution analysis applying an inductively coupled plasma mass spectrometer equipped with a dynamic reaction cell by Lieve Balcaen; Ingrid Geuens; Luc Moens; Frank Vanhaecke (pp. 1020-1025).
The development of an ICP-MS method for the determination of ultra-trace amounts of Fe in AgNO3 solutions using isotope dilution for calibration is described. AgNO3 solutions are used as raw materials in the production of traditional photographic materials, and it is known that contamination with metal traces can influence the quality of the films thus produced. After adding an appropriate amount of an 54Fe-enriched spike and permitting isotopic equilibration to take place, Ag was selectively removed from the solutions by precipitation as AgBr. Although to some extent, co-precipitation of Fe is possible under the given circumstances, an incomplete recovery of the analyte element did not affect the accuracy of the results, owing to the use of isotope dilution for calibration. NH3 was used as a reaction gas in a quadrupole-based ICP-MS instrument, equipped with a dynamic reaction cell (DRC), providing interference-free measurement of the 54Fe/56Fe ratio. The limit of detection (LOD) obtained using this procedure was approximately 0.01 µg g-1. This is an excellent value in comparison with the detection limit obtained with the more traditional approach: sample dilution and external calibration with a Fe standard solution (LOD ~1 µg g-1). To validate the method, recovery experiments were carried out. In all instances, a quantitative recovery was established. Finally, the method was applied to the analysis of AgNO3 solutions. A large variation in Fe concentration was observed. Depending on the Fe content in the samples, relative standard deviations typically ranged between 1 and 14%.
Keywords: ICP-MS; Dynamic reaction cell; Isotope dilution; Trace elements; Photographic materials
Preconcentration of selenium compounds on a porous graphitic carbon column in view of HPLC-ICP-AES speciation analysis by K. Abbas-Ghaleb; N. Gilon; G. Crétier; J. M. Mermet (pp. 1026-1031).
The retention of organic selenium compounds on a porous graphitic carbon stationary phase was investigated. Different acids were studied as mobile phases to elute selenocystamine, selenoethionine, selenomethionine and selenocystine. Detection was achieved using inductively coupled plasma-atomic emission spectrometry to provide selenium-specific and sensitive detection. The separation of the four species was carried out using methanoic acid. An important on-column preconcentration was obtained when solutes were injected in nitric acid or trifluoroacetic acid (TFA) media. The large injection volume employed (2,500 µL) allowed us to reach low relative detection limits (2–6 µg/L). The method, employing TFA as injection solvent and methanoic acid as the eluent was found to be robust with respect to different matrices spiked with selenocompounds.
Keywords: Porous graphitic carbon; Selenium; Speciation; Preconcentration
Spectroscopic characterization of natural corals by B. Kaczorowska; A. Hacura; T. Kupka; R. Wrzalik; E. Talik; G. Pasterny; A. Matuszewska (pp. 1032-1037).
The FTIR, micro-Raman, NMR, and XPS spectra of 25 different natural corals have been compared. Reflectance and transmission absorbance IR and Raman data have been used as rapid and efficient means of classification of natural corals containing aragonite (non-precious white species), calcite (red, pink, precious white species), and organic material (black protein polymer). The combination of reflectance IR and infrared microscopy could serve as a rapid, non-destructive method for distinguishing natural corals from artificial, fake jewels.
Keywords: Coral; FTIR; Raman; XPS; NMR; Molecular structure
Rapid pesticide analysis, in post-harvest plants used as animal feed, by low-pressure gas chromatography–tandem mass spectrometry by A. Garrido-Frenich; F. J. Arrebola; M. J. González-Rodríguez; J. L. Martínez Vidal; N. Mora Díez (pp. 1038-1046).
A wide range of pesticides used to control pests in vegetables have been determined in agricultural plant waste from beans, watermelons, and melons grown in greenhouses located in a predominantly agricultural area in Southeast Spain (Almería). Analysis of the pesticides was carried out by low-pressure gas chromatography (LP-GC) with mass spectrometry in tandem (MS–MS) mode, after extraction of the lyophilized samples with dichloromethane. The influence of the sample matrix on the analysis was avoided by use of matrix-matched standards. Linearity, detection limit (LOD), quantitation limit (LOQ), recovery, and precision for each pesticide were calculated. The most frequently encountered pesticides were endosulfan (>73% of the analyzed samples) and buprofezin (>55% of the samples), followed by cypermethrin, pirimifos-methyl, bifentrin, and chlorpyrifos (>30% of the samples). The pesticide found at the highest concentration level was endosulfan (223.33 mg kg−1) in a watermelon sample.
Keywords: Low-pressure gas chromatography; Mass spectrometry; GC–MS; Post-harvest plants; Animal feed; Pesticides
Electrospray ionization mass spectrometry of terrestrial humic substances and their size fractions by A. Piccolo; M. Spiteller (pp. 1047-1059).
Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the average molecular mass of terrestrial humic substances, such as humic (HA) and fulvic (FA) acids from a soil, and humic acid from a lignite (NDL). Their ESI mass spectra, by direct infusion, gave average molecular masses comparable to those previously obtained for aquatic humic materials. The soil HA and FA were further separated in size-fractions by preparative high performance size exclusion chromatography (HPSEC) and analyzed with ESI-MS by both direct infusion and a further on-line analytical HPSEC. Unexpectedly, their average molecular mass was only slightly less than for the bulk sample and, despite different nominal molecular size, did not substantially vary among size-fractions. The values increased significantly (up to around 1200 Da) after on-line analytical HPSEC for the HA bulk sample, at both pH 8 and 4, and for the HA size-fractions when pH was reduced from 8 to 4. It was noticed that HA size-fractions at pH 8 were separated by on-line HPSEC in further peaks showing average masses which progressively increased with elution volume. Furthermore, when the HA and NDL bulk samples were sequentially ultracentrifuged at increasing rotational speed, their supernatants showed mass values which were larger than bulk samples and increased with rotational speed. These variations in mass values indicate that the electrospray ionization is dependent on the composition of the humic molecular mixtures and increases when their heterogeneity is progressively reduced. It is suggested that the dominance of hydrophobic compounds in humic supramolecular associations may inhibit the electrospray ionization of hydrophilic components. Our results show that ESI-MS is reasonably applicable to humic substances only after an extensive reduction of their chemical complexity.
Keywords: Electrospray ionization mass spectrometry; Humic substances; Soil; Supramolecular associations; Molecular mixtures; Molecular sizes; Hydrophobic compounds
Quantifying selectivity: a statistical approach for chromatography by Rudolf Kapeller (pp. 1060-1070).
A parameter for quantitative description of selectivity is defined. A model for quantification of selectivity based on a statistical approach is drawn up. Formulas for quantification of selectivity are derived, especially for chromatography. These are based on theoretical and—as far as needed—empirical functions (made evident by experimental data or numerical simulations) or at least worst-case assumptions. An example (GC–MS determination of atrazine in drinking water) shows how quantification of selectivity can be achieved in practice. The approach is intended to be used for validation purposes.
Keywords: Selectivity; Specificity; Quantification; Statistical approach; Chromatography
Flow-injection spectrophotometric determination of cyanate in bioremediation processes by use of immobilised inducible cyanase by V. M. Luque-Almagro; R. Blasco; J. M. Fernández-Romero; M. D. Luque de Castro (pp. 1071-1078).
A new flow injection (FI) method for photometric monitoring of cyanate in bioremediation processes using immobilised native cyanase is described. The method is based on the catalytic reaction between cyanate and bicarbonate to produce ammonia and carbon dioxide in the presence of an inducible native cyanase, immobilised in a reactor packed with glass beads. Two degrees of purification of the biocatalyst were used—heated cell-free extract and purified extract of cyanase from Pseudomonas pseudoalcaligenes CECT 5344. The ammonia produced by the enzymatic reaction is finally monitored photometrically at 700 nm using a modification of the conventional Berthelot method. The method furnishes different calibration curves depending on the degree of purification of the cyanase, with linear ranges between 1.23 and 616.50 μmol L−1 (r 2=0.9979, n=7) and between 1.07 and 308.25 µmol L−1 (r 2= 0.9992, n=7) for the heated cell-free extract and the purified cyanase extract, respectively. No statistically significant differences between the samples were found in the precision study evaluated at two cyanate concentration levels using one-way analysis of variance. A sampling frequency of 15 h−1 was achieved. The method was used to monitor cyanate consumption in a cyanate bioremediation tank inoculated with Pseudomonas pseudoalcaligenes CECT 5344 strain. The correlation between cyanate degradation and ammonia production was tested using a conventional method. Finally, the method was applied to different samples collected from the bioremediation tank using the standard addition method; recoveries between 85.9 and 97.4% were obtained.
Keywords: Cyanate; Cyanase; Purification; Flow injection; Immobilised enzyme
2,3-Dihydroxypyridine-loaded cellulose: a new macromolecular chelator for metal enrichment prior to their determination by atomic absorption spectrometry by Vibha Gurnani; Ajai K. Singh; B. Venkataramani (pp. 1079-1086).
New macromolecular chelators have been synthesized, by loading 2,3-dihydroxypyridine (DHP) on cellulose via linkers -NH-CH2-CH2-NH-SO2-C6H4-N=N- and -SO2-C6H4-N=N-, and characterized by elemental analysis, TGA, IR, and CPMAS 13C NMR spectra. The cellulose with DHP anchored by the shorter linker had better sorption capacity (between 69.7 and 431.1 μmol g−1) for Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Pb(II), and Fe(III)) than the other (51.9–378.1 μmol g−1); the former was therefore studied in detail as a solid extractant for these metal ions. The optimum pH ranges for quantitative sorption (recovery 97.6–99.8%) on this matrix were: 7.0–9.0, 6.0–9.0, 3.0–8.0, 6.0–8.0, 6.0–9.0, 6.0–7.0, and 2.0–6.0 respectively. Desorption was quantitative with 0.5 mol L−1 HCl and 0.5 mol L−1 HNO3 (for Pb). Simultaneous sorption (at pH 7.0) of all metal ions other than Fe(III) was possible if their total concentration did not exceed the sorption capacity (lowest value). The recovery of seven metal ions from their mixture at pH 6.0 was nearly quantitative when the concentration level of each metal ion was 0.2 μg mL−1. The optimum flow rate of metal ion solutions for quantitative sorption of metal onto a column packed with DHP-modified cellulose was 2–7 mL min−1, whereas for desorption the optimum flow rate for the acid solution was 2–4 mL min−1. The time needed to reach 50% of the total loading capacity (t 1/2) was <5 min for all the metal ions except Ni and Pb. The limit of detection (blank+3s) was from 0.70 to 4.75 μg L−1 and the limit of quantification (blank+10s) was between 0.79 and 4.86 μg L−1. The tolerance limits for NaCl, NaBr, NaI, NaNO3, Na2SO4, Na3PO4, humic acid, EDTA, Ca(II), and Mg(II) for sorption of all metal ions are reported. The column packed with DHP-anchored cellulose can be reused at least 20 times for enrichment of metal ions in water sample. It has been used to enrich all the metal ions in pharmaceutical and water samples before their determination by flame AAS. RSD for these determinations was between 1.1 and 6.9%.
Keywords: 2,3-Dihydroxypyridine; Cellulose; Atomic absorption spectrometry; Metal ion; Macromolecular chelator; Enrichment