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1.
A variety of techniques exist that provide chemical information in the form of a spatially resolved image: electron microprobe
analysis, nuclear microprobe analysis, synchrotron radiation microprobe analysis, secondary ion mass spectrometry, and confocal
fluorescence microscopy. Linear (LINAC) and circular (synchrotrons) particle accelerators have been constructed worldwide
to provide to the scientific community unprecedented analytical performances. Now, these facilities match at least one of
the three analytical features required for the biological field: (1) a sufficient spatial resolution for single cell (< 1 μm)
or tissue (<1 mm) analyses, (2) a temporal resolution to follow molecular dynamics, and (3) a sensitivity in the micromolar
to nanomolar range, thus allowing true investigations on biological dynamics. Third-generation synchrotrons now offer the
opportunity of bioanalytical measurements at nanometer resolutions with incredible sensitivity. Linear accelerators are more
specialized in their physical features but may exceed synchrotron performances. All these techniques have become irreplaceable
tools for developing knowledge in biology. This review highlights the pros and cons of the most popular techniques that have
been implemented on accelerator-based sources to address analytical issues on biological specimens.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
2.
The standard methods currently used to read out microarrays are fluorescent and chemiluminesent imaging techniques. These
methods require labeling of a component with a marker and, usually, only the concentration of the marker molecule is detected.
A label-free imaging method that also enables quantitative spectroscopic analysis of the composition and component interaction
would be of great advantage. In this article it is shown for the first time that IR mapping ellipsometry enables label-free
imaging of a biochip before and after incubation with peptide solution. The measurements prove that IR ellipsometry is a sensitive
tool for laterally resolved identification of the different materials and determination of the composition of a biochip. The
lateral resolution required was achieved by using radiation from an infrared synchrotron beamline.
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3.
Limits of detection have been studied for several elements in aluminium and steel alloys, at atmospheric pressure in air,
by use of the single and collinear double-pulse configurations of laser-induced breakdown spectroscopy. For this purpose,
calibration plots were constructed for Mg, Al, Si, Ti, Cr, Mn, Fe, Ni, and Cu using a set of certified aluminium alloy samples
and a set of certified steel samples. The investigation included optimization of the experimental conditions to furnish the
best signal-to-noise ratio. Inter-pulse delay, gate width, and acquisition delay were studied. The detection limits for the
elements of interest were calculated under the optimum conditions for the double-pulse configuration and compared with those
obtained under the optimum conditions for single-pulse configuration. Significantly improved detection limits were achieved,
for all the elements investigated, and in both aluminium and steel, by use of the double-pulse configuration. The experimental
findings are discussed in terms of the measured plasma conditions (particle and electron density, and temperature). 相似文献
4.
Photoacoustic spectroscopy is a new analytical tool that provides a simple nondestructive technique for obtaining information about the electronic absorption sprctrum of samples such as powders, semisolids, gels, and liquids. It can also be applied to samples which cannot be examined by conventional optical methods. Numerous applications of this technique in the field of inorganic and organic semiconductors, biology, and catalysis have been described. Among the advantages of photoacoustic spectroscopy, the signal is almost insensitive to light scattering by the sample and information can be obtained about nonradiative deactivation processes. Signal saturation, which can modify the intensity of individual absorption bands in special cases, is a drawback of the method. 相似文献
5.
The molecular aggregation of oxazine 1 (Ox1) and oxazine 4 (Ox4) in reduced charge montmorillonite (RCM) colloids was investigated
by absorption and fluorescence spectroscopies. The aggregation was significantly influenced by the structure of dye cations.
Presence of four hydrophobic ethyl groups attached to the ammonium substituents in Ox1 cation prevented formation of closely
packed sandwich-type assemblies ( H-aggregates). Significant effect of the layer charge was observed for Ox4/RCMs dispersions. Large amounts of the Ox4 H-aggregates were formed in the systems with RCMs of the highest layer charge and reflected in quenched fluorescence. The presence
of J-aggregates was proven by absorption spectra for the systems with Ox4 and low-charge RCMs. The flocculation of the lowest
charge RCM colloids led to an extensive reduction of the luminescence. The trends and effects of the dye molecular structure
and RCM properties are compared with the results previously published for other types of dyes. 相似文献
6.
The laccase-catalysed transformation of indigo carmine (IC) with and without a redox active mediator was studied using online
UV–visible spectroscopy. Deconvolution of the mixture spectra obtained during the reaction was performed on a model-free basis
using multivariate curve resolution (MCR). Thereby, the time courses of educts, products, and reaction intermediates involved
in the transformation were reconstructed without prior mechanistic assumptions. Furthermore, the spectral signature of a reactive
intermediate which could not have been detected by a classical hard-modelling approach was extracted from the chemometric
analysis. The findings suggest that the combined use of UV–visible spectroscopy and MCR may lead to unexpectedly deep mechanistic
evidence otherwise buried in the experimental data. Thus, although rather an unspecific method, UV–visible spectroscopy can
prove useful in the monitoring of chemical reactions when combined with MCR. This offers a wide range of chemists a cheap
and readily available, highly sensitive tool for chemical reaction online monitoring. 相似文献
7.
We report the first combined use of analytical spectroscopy, guest–host chemistry, and multivariate regression analysis for
determination of enantiometric composition of multicomponent samples of chiral analytes. Sample solutions containing multicomponent
analytes of ephedrine, tryptophan, propranolol, and proline of varying enantiomeric composition with beta-cyclodextrin (BCD)
or methyl-beta-cyclodextrin (Me-BCD) as chiral host molecules were investigated using ultraviolet (UV)–visible spectroscopy.
The interactions of enantiomers of chiral analytes with chiral hosts resulted in the formation of transient diastereomeric
inclusion complexes with varying spectral properties. Multivariate analysis using partial-least-square (PLS) regression was
used to correlate subtle changes in the UV–visible spectra of the guest–host complexes with the enantiomeric composition of
the calibration samples. These PLS regressions were carefully optimized and then used to predict the enantiomeric composition
of multicomponent chiral analytes of validation samples. The results of these validation studies demonstrate the predictive
ability of the regression models for determination of future enantiomeric composition of samples. The accuracy of the models
to correctly predict the enantiomeric composition of samples, evaluated by use of the root mean square percent relative error
(RMS%RE) was analyte and chiral host dependent. In general, better prediction of enantiomeric composition of samples and low
RMS%RE values were obtained when Me-BCD was used as the chiral host. The analyses procedure reported here is simple, rapid,
and inexpensive. In addition, this approach does not require prior separation of chiral analytes, thus reducing analysis time
and eliminating the need for expensive chiral columns. 相似文献
8.
Abstract UV–visible spectral observations indicate that the J-aggregation of protonated meso-tetra(4-sulfonatophenyl)porphyrin ([H 2TSPP] 2+) under acidic conditions is completely inhibited by the π–π counteraction between 1-butyl-pyridinium tetrafluoroborate ([bpy]BF 4) and [H 2TSPP] 2+. The studies also suggest that the intermolecular π–π force is of relative importance for the J-aggregates of [H 2TSPP] 2+ and the intermolecular electrostatic force for the H-aggregates.
Graphical Abstract
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9.
Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes
such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence
detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements
reported average properties for the organelles of cell populations. Similar measurements for organelles released from single
cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an
approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either
the fluorescent mitochondrion-specific 10- N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced
into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment,
an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram,
the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities
are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which
labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users 相似文献
10.
Guanine nucleotide binding proteins, such as Ras proteins, play a pivotal role in maintaining the regular life cycle of cells.
The involvement of Ras mutants in the progress of cancer has attracted many efforts to find detection methods for Ras activity.
In this study we present a luminescent microwell plate assay for monitoring GTPase activity of Ras proteins. The luminescence
intensity of the Tb–norfloxacin complex is influenced by nucleoside phosphates as well as by inorganic phosphates. Real-time
kinetics of the GTPase activity of wild-type Ras and Ras mutants can be monitored online. The effect of a GTPase activating
protein as well as of a downstream effector (Ras-binding domain of human Raf-1) on the GTPase activity of different Ras mutants
is examined. In contrast to other methods, this assay does not require the use of radioactively labeled substrates or chromatographic
separation steps. Moreover, the application of fluorescently labeled GTP substrates which often interfere with enzymatic activity
can be avoided. This in vitro assay can serve as a model system for the screening of regulators affecting the GTPase activity
of Ras proteins.
Figure The emission of the lanthanide complex Tb(III)-norfloxacin is influenced by nucleoside phosphates as well as by inorganic
phosphates. Ras proteins display a specific GTPase activity which converts protein-bound GTP to GDP and phosphate, the latter
being released. The Ras activity can be monitored by a significant decrease in luminescence intensity of Tb(III)-norfloxacin
owing to the strong quenching effect induced by the enzymatically hydrolyzed phosphate anions. This luminescent assay enables
the monitoring of real-time kinetics of the GTPase activity of Ras proteins and Ras mutants and a fast screening of their
regulators. 相似文献
11.
The evaluation of the effects of drugs or chemicals on the functions of the immune system is an increasingly important task.
Due to the accessibility of primary cells and cell lines, in vitro cellular functional tests are frequently being performed
with cells representing the innate immune system, in particular those with phagocytotic activities, such as neutrophils and
macrophages. Suitable functional parameters are the efficiency of phagocytosis, the efficiency with which viable pathogens
are killed, the production of reactive oxygen and nitrogen species (ROS and RNS) and that of cytokines. Corresponding analytical
procedures are available, but standardization is required, as varying the procedure may influence the outcomes of the assays. 相似文献
12.
Tip-enhanced Raman spectroscopy (TERS), which utilizes the strong localized optical field generated at the apex of a metallic
tip when illuminated, has been shown to successfully probe the vibrational spectrum of today’s and tomorrow’s state-of-the-art
silicon and next-generation semiconductor devices, such as quantum dots. Collecting and analyzing the vibrational spectrum
not only aids in material identification but also provides insight into strain distributions in semiconductors. Here, the
potential of TERS for nanoscale characterization of strain in silicon devices is reviewed. Emphasis will be placed on the
key challenges of obtaining spectroscopic images of strain in actual strained silicon devices.
Figure Figure Concept of Tip Enhanced Raman Spectroscopy (TERS), which utilizes the strong localized optical field generated at the
apex of a metallic tip when illuminated. TERS has been demonstrated to successfully probe the vibrational spectrum of today’s
and tomorrow’s state-of-the-art silicon and next generation semiconductor devices 相似文献
13.
To date, lasers are widely accepted tools in analytical spectroscopy, involved in various stand-alone and hyphenated techniques. Furthermore, significant progress can be noted in this field. In this paper, first of all some laser characteristics are discussed. Subsequently, five selected topics are outlined to illustrate recent achievements and future developments: 1. Laser-induced fluorescence for detection in capillary electrophoresis, including the use of ultraviolet, continuous-wave lasers in combination with wavelength-resolved emission detection; the use of diode laser-induced fluorescence in the red region of the electromagnetic spectrum and the use of Ti:sapphire lasers for multiphoton-excited fluorescence detection. 2. Degenerate four-wave mixing for detection in liquid microseparation systems (based on the coherence of laser light). 3. Fluorescence line-narrowing spectroscopy for identification purposes, a cryogenic high-resolution molecular fluorescence technique with a high potential in environmental analysis. 4. Recent developments in Raman spectroscopy (including ultraviolet-Resonance Raman and hyphenation of liquid chromatography and Raman spectroscopy). 5. Use of lasers for sample introduction in inorganic analysis based on controlled material ablation.
Author Keywords: Laser spectroscopy; Capillary electrophoresis; Fluorescence; Degenerate four-wave mixing; Fluorescence line-narrowing; Raman spectroscopy; Inorganic analysis 相似文献
14.
A method is presented for the electroanalytical characterization of interactions of dsDNA with a drug, under conditions that
both agents are dissolved in the phosphate buffer solution and both are electroactive. Normal pulse, square wave, differential
pulse, and cyclic voltammetries were employed in the measurements of the drug and dsDNA oxidation signals at carbon electrodes.
UV–Vis spectroscopy was used as a non-electrochemical method to support the electroanalytical data. An anticancer drug, C-1311
(5-diethylaminoethyl-amino-8-hydroxyimidazoacridinone), has been selected for the examination. Normal pulse voltammetry was
particularly useful in showing that under the conditions employed neither dsDNA nor the drug were adsorbed at the electrode
surface. Necessary conditions for the appearance of the well-defined dsDNA voltammetric signal (guanine peak) are: rigorous
chemical and biological purity in the cell and appropriate purity of DNA. An analysis of the obtained results confirmed that
there were two modes of interaction between C-1311 and dsDNA: by intercalation and electrostatically. In the presence of excess
NaCl the electrostatic interactions deteriorate. The binding constants ( K
1 and K
2, respectively) and the number ( n) of nucleic base pairs (bp) and the number ( m) of phosphate groups (pg) interacting with one molecule of drug have been determined. For strong interactions (intercalation)
the values of the binding constant, K
1, and the binding-site size, n, equal 3.7 × 10 4 M −1 and 2.1, respectively. For the weak electrostatic interactions the K
2 and m parameters equal 0.28 × 10 4 M −1 and 4.7. The intercalation process is rather slow and its rate (the conditions of pseudo-first-order reaction) was estimated
to equal 7 × 10 −4 s −1. The possibility of independent determination of both interacting agents was very useful in the study.
Figure Intercalation of C-1311 into a dsDNA fragment 相似文献
15.
A polypseudorotaxane was prepared using 4-( N, N-dimethylamino) benzoyl modified β-cyclodextrin (DMAB-β-CD) and biotin-terminated poly(propylene glycol) (biotin-terminated
PPG) in water for 7 days. When the biotin-terminated PPG was added to a saturated solution of DMAB-β-CD at room temperature,
the polypseudorotaxane was not obtained. However, with increasing temperature to 60 °C, precipitation was observed. The 1H-NMR spectrum indicated that the obtained precipitate was the polypseudorotaxane. The final yield was 54%, which was higher
than that previously reported for polypseudorotaxane formation using chemically modified β-CD. The polypseudorotaxane can
be useful to make a polyrotaxane via avidin–biotin molecular recognition using the terminal biotin group. 相似文献
16.
Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial
growth by binding to carboxy-terminal d-Ala- d-Ala intermediates in the peptidoglycan of the cell wall of Gram-positive bacteria. In this paper we report the derivatization
of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding
properties of the microspheres to two d-Ala- d-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled d-Ala- d-Ala- d-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 × 10 −6 and 3.0 × 10 −6 mol L −1, respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial
detection was determined to be 30 colon-forming units; cfu) of Gram-positive bacteria has been demonstrated. A simple microfluidic
experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide–antibiotic
interaction. 相似文献
17.
The sandwich-type [Na(UO 2) 2(H 2O) 4(BiW 9O 33) 2] 13− uranium ( VI) has been synthesized by reacting the trivacant species of B-α-[BiW 9O 33] 9− with
and investigated by IR and UV–Vis spectroscopy, and elemental analysis. The X-ray single crystal analysis was carried out
on Na 13[Na(UO 2) 2(H 2O) 4(BiW 9O 33) 2] · 33H 2O ( I) which crystallizes in the orthorhombic system, space group Pna2 1 with a = 33.8454(19) ?, b = 21.1484(12) ?, c = 13.2403(7) ?, α = 90°, β = 90°, γ = 90°, and Z = 4. The polyanion consists
of two lacunary B-α-[BiW 9O 33] 9− groups which sandwich two uranyl cations and one sodium cation. The uranium atoms adopt distorted pentagonal–bipyramidal coordination,
achieved by two equatorial bonds to each BiW 9O 33 unit and one external water ligand. The coordination of each uranium atom is evident by the shift of ν as(W–O b–W) and ν as(Bi–O) stretching vibrational bonds.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination
and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of
the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially
available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody
as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method
based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic
range was 4–15 ng mL −1 with a limit of detection (LOD) of 2 ng mL −1 ( R=0.9247), whereas for the electrochemical protocol the dynamic range was 2–50 ng mL −1 and the LOD was1 ng mL −1. 相似文献
19.
Intrinsically disordered proteins (IDPs) that undergo structural transition upon binding their target molecules are becoming increasingly known. IDPs, because of their binding specificity and induced folding properties, can serve as biological recognition elements for sensing applications. In this paper, BRCA1, an IDP, was utilized as the biological recognition element to detect tumor suppressor protein p53 through the BRCA1/p53 binding interaction to serve as a proof-of-concept for the use of IDPs as recognition elements. The binding resulted in a disordered-to-ordered BRCA1 conformational change, as seen in our circular dichroism (CD) measurements. This conformational change in BRCA1 (residues 219-498) was utilized in the detection of p53 (residues 311-393) via both intrinsic and extrinsic fluorescent probes. Intrinsic tryptophan residues within the BRCA1 sequence detected p53 (311-393) with a detection limit of 0.559 nM (0.112 pmol). Two environmentally sensitive fluorophores, tetramethylrhodamine-5-maleimide (TMR) and 6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid, succinimidyl ester (dansyl-X, SE) were conjugated to BRCA1 (219-498). Dansyl-X, SE-conjugated BRCA1 (219-498) detected p53 (311-393) with a detection limit of 1.50 nM (0.300 pmol). The sensitivities for TMR and dansyl-X, SE-conjugated BRCA1 for the detection of p53 were nearly threefold and twofold higher, respectively, than the sensitivity reported using intrinsic BRCA1 tryptophan fluorescence. CD measurements did not reveal a disruption of p53/dye-conjugated BRCA1 binding, thus validating the applicability of environmentally sensitive fluorophores as transduction moieties to detect molecules which bind to IDPs and induce a structural change. 相似文献
20.
The quenching process of the laser-induced luminescence of Co(II)-doped zirconia samples prepared by the sol–gel method is
reported. Zirconia monoliths doped with Co(II) at different concentrations, were laser irradiated producing fluorescence;
its intensity, measured by the slope at the low energy side of the Raman spectra, is reduced with the irradiation time. The
rate the fluorescence decays can be modeled as a double exponential function of the irradiation time; the characteristic times
involved in this quenching process are in the range of seconds. The suppression of the luminescence has been associated to
the local heating produced when the laser beam is focused in a small area (≈2 microns in diameter) on the sample. This heating
process reduces physical (such as grain boundaries and surface states) and chemical (oxygen vacancies) defects; however, some
residual fluorescence still remains after long periods of illumination.
R. Rodríguez is on sabbatical leave at Cinvestav, Unidad Queretaro. 相似文献
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