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1.
Fast and sensitive DNA analysis using changes in the FRET signals of molecular beacons in a PDMS microfluidic channel 总被引:3,自引:0,他引:3
Jung J Chen L Lee S Kim S Seong GH Choo J Lee EK Oh CH Lee S 《Analytical and bioanalytical chemistry》2007,387(8):2609-2615
A new DNA hybridization analytical method using a microfluidic channel and a molecular beacon-based probe (MB-probe) is described.
A stem-loop DNA oligonucleotide labeled with two fluorophores at the 5′ and 3′ termini (a donor dye, TET, and an acceptor
dye, TAMRA, respectively) was used to carry out a fast and sensitive DNA analysis. The MB-probe utilized the specificity and
selectivity of the DNA hairpin-type probe DNA to detect a specific target DNA of interest. The quenching of the fluorescence
resonance energy transfer (FRET) signal between the two fluorophores, caused by the sequence-specific hybridization of the
MB-probe and the target DNA, was used to detect a DNA hybridization reaction in a poly(dimethylsiloxane) (PDMS) microfluidic
channel. The azoospermia gene, DYS 209, was used as the target DNA to demonstrate the applicability of the method. A simple
syringe pumping system was used for quick and accurate analysis. The laminar flow along the channel could be easily controlled
by the 3-D channel structure and flow speed. By injecting the MB-probe and target DNA solutions into a zigzag-shaped PDMS
microfluidic channel, it was possible to detect their sequence-specific hybridization. Surface-enhanced Raman spectroscopy
(SERS) was also used to provide complementary evidence of the DNA hybridization. Our data show that this technique is a promising
real-time detection method for label-free DNA targets in the solution phase.
Figure FRET-based DNA hybridization detection using a molecular beacon in a zigzag-shaped PDMS microfluidic channel 相似文献
2.
Bonanni A Esplandiu MJ Pividori MI Alegret S del Valle M 《Analytical and bioanalytical chemistry》2006,385(7):1195-1201
Impedance spectroscopy is proposed as the transduction principle for detecting the hybridization of DNA complementary strands.
In our experiments, different DNA oligonucleotides were used as model gene substances. The gene probe is first immobilized
on a graphite-epoxy composite working electrode based genosensor. Detection principle is based on changes of impedance spectra
of a redox marker, the ferro/ferricyanide couple, after hybridization with target DNA. Resistance offered to the electrochemical
reaction serves as the working signal, allowing for an unlabelled gene assay.
相似文献
3.
McCarthy EL Egeler TJ Bickerstaff LE Pereira da Cunha M Millard PJ 《Analytical and bioanalytical chemistry》2006,386(7-8):1975-1984
Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia
virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and
rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained
from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary
tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers,
which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock
probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal
cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial,
and protozoan pathogens within localized regions of microcapillary tubes.
相似文献
4.
Nagl S Stich MI Schäferling M Wolfbeis OS 《Analytical and bioanalytical chemistry》2009,393(4):1199-1207
Chemical sensing, imaging and microscopy based on the use of fluorescent probes has so far been limited almost exclusively
to the detection of a single parameter at a time. We present a scheme that can overcome this limitation by enabling optical
sensing of two parameter simultaneously and even at identical excitation and emission wavelengths of two probes provided (a)
their decay times are different enough to enable two time windows to be recorded, and (b) the emission of the shorter-lived
probe decays to below the detectable limit while that of the other still can be measured. We refer to this new scheme as the
dual lifetime determination (DLD) method and show that it can be widely varied by appropriate choice of probes and experimental
settings. DLD is demonstrated to work by sensing oxygen and temperature independently from each other by making use of two
probes, one for oxygen (a platinum porphyrin dissolved in polystyrene), and one for temperature [a europium complex dissolved
in poly(vinyl methylketone)]. DLD was applied to monitor the consumption of oxygen in the glucose oxidase-catalyzed oxidation
of glucose at varying temperatures. The scheme is expected to have further applications in cellular assays and biophysical
imaging.
Figure Principle behind the dual lifetime determination (DLD) method 相似文献
5.
Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO 总被引:1,自引:0,他引:1
There have recently been advances in the application of aptamers, a new class of nucleic acids that bind specifically with
target proteins, as protein recognition probes for biomedical study. The development of a signaling aptamer with the capability
of simple and rapid real-time detection of disease-related proteins has attracted increasing interest. We have recently reported
a new protein-detection strategy using a signaling aptamer based on a DNA molecular light-switching complex, [Ru(phen)2(dppz)]2+. In this work we have used the commercially available DNA-intercalating dye, TOTO, to replace [Ru(phen)2(dppz)]2+ for detection of oncoprotein platelet-derived growth factor BB (PDGF-BB), a potential cancer marker. Taking advantage of
the high affinity of the aptamer to PDGF-BB and the sensitive fluorescence change of the aptamer–TOTO signaling complex on
protein binding, PDGF-BB was detected in physiological buffer with high selectivity and sensitivity. The detection limit was
0.1 nmol L−1, which was better than that of other reported aptamer-based methods for PDGF-BB, including that using [Ru(phen)2(dppz)]2+. The method is very simple with no need for covalent labeling of the aptamer or probe synthesis. It facilitates wide application
of the signaling mechanism to the analysis and study of cancer markers and other proteins.
相似文献
6.
This technical note reports on a new procedure to on-column-label organelles sampled from a tissue cross section into a fused
silica capillary. These organelles are then analyzed by capillary electrophoresis with postcolumn laser-induced fluorescence
detection. In this procedure, the fluorescent label does not come in contact with the tissue, which facilitates visualization
of the sampled tissue cross section. In addition, on-column labeling allows for better control of the reaction time and fluorescent
label concentrations. As a proof-of-principle, we show results of mitochondria from rat gastrocnemius muscle cross sections
that were on-column-labeled with 10-N-nonyl acridine orange (NAO), a mitochondrion-specific probe, and compare them with results for NAO in-tissue labeling of
the same tissue. The new organelle labeling procedure reported here may easily be extended to the analysis of individual organelles
in other biological samples and may become a valuable tool in studies investigating the role of mitochondria in muscle aging
and exercise physiology.
相似文献
7.
Michelle M. Martinez Randall D. Reif Dimitri Pappas 《Analytical and bioanalytical chemistry》2010,396(3):1177-1185
Early detection of apoptotic cells via caspase activity is demonstrated with fast response time. Fluorescence correlation
spectroscopy (FCS) is used to identify the presence of a cleaved fluorogenic probe based on the fluorescence of rhodamine
110 in Jurkat cells. FCS curves are shown to be markedly different for autofluorescent (non-apoptotic) cells, whereas cells
with cleaved probe showed diffusion and molecular brightness characteristic of rhodamine 110. Using FCS measurements, cells
were identified as apoptotic on the basis of the presence of autocorrelated fluorescence, average molecular brightness (η), and molecular dwell time (τ
D). Apoptotic cells identified in this manner were detected as early as 45 min after induction. Unlike other methods with similar
identification times, such as western blotting and electron microscopy, cells remain viable for further analysis. This multi-parameter
approach is rapid, flexible, and does not require transfection of the cells prior to analysis, enabling apoptosis to be identified
early in a wide variety of cell types.
相似文献
8.
On-line NMR detection of microgram quantities of heparin-derived oligosaccharides and their structure elucidation by microcoil NMR 总被引:1,自引:0,他引:1
The isolation and purification of sufficient quantities of heparin-derived oligosaccharides for characterization by NMR is
a tedious and time-consuming process. In addition, the structural complexity and microheterogeneity of heparin makes its characterization
a challenging task. The improved mass-sensitivity of microcoil NMR probe technology makes this technique well suited for characterization
of mass-limited heparin-derived oligosaccharides. Although microcoil probes have poorer concentration sensitivity than conventional
NMR probes, this limitation can be overcome by coupling capillary isotachophoresis (cITP) with on-line microcoil NMR detection
(cITP-NMR). Strategies to improve the sensitivity of on-line NMR detection through changes in probe design and in the cITP-NMR
experimental protocol are discussed. These improvements in sensitivity allow acquisition of cITP-NMR survey spectra facilitating
tentative identification of unknown oligosaccharides. Complete structure elucidation for microgram quantities of the purified
material can be carried out through acquisition of 2D NMR spectra using a CapNMR microcoil probe.
Survey NMR spectrum obtained by cITP-NMR using a second-generation probe (the microcoil of which is shown) facilitates tentative
identification of unknown oligosaccharides (e.g., the heparin-derived tetrasaccharide illustrated) 相似文献
9.
Traditional methods for protein kinase (PK) assay are mainly based on use of 32P-labeled adenosine triphosphate (ATP); applications of such methods are, however, hampered by radioactive waste and short
half-life of 32P-labeled ATP. Therefore non-radioactive methods, such as fluorescence detection techniques are good alternative. In this
review, we describe the principles of four fluorescence techniques (fluorescence intensity endpoint measurement, fluorescence
resonance energy transfer (FRET), fluorescence polarization (FP), and fluorescence lifetime imaging) and provide an overview
of applications of these fluorescence detection techniques in protein kinase assay, underlining their relative advantages
and limitations. Research trends in this field are also highlighted.
Figure Schematic representation of kinase assay based on direct fluorescence polarization measurements. The fluorescent peptide,
on phosphorylation by kinase, binds to a phosphospecific antibody, which leads to a high FP value 相似文献
10.
Thin nanoporous alumina obtained by anodization of aluminum films offers promising advantages for application in fluorescence-based biological sensors including convenient preparation, increased density of binding sites, and improved collection efficiency of fluorescence. These advantages are illustrated in the detection of streptavidin using biotin covalently bound to the surface of alumina nanopores. Fluorescence intensity enhancement as high as 7 times is observed in nanopores in comparison to flat glass surface.
相似文献
11.
Lillian Roth Jutta Zagon Anke Ehlers Lothar W. Kroh Hermann Broll 《Analytical and bioanalytical chemistry》2009,394(2):529-537
A new approach for the detection of DNA target molecules is described, using capture probes and subsequent signal enhancement
by a uniform polymerase chain reaction (PCR). Peptide nucleic acid probes were immobilized in real-time PCR-compatible microtiter
plates. After hybridization of biotinylated DNA targets, detection was performed by real-time immuno-PCR, a method formerly
used for protein detection. We demonstrate the feasibility of this strategy for the qualitative detection of DNA oligonucleotides
with a detection limit (LOD) of 6 attomol. Furthermore, the method was applied to PCR-amplified samples from genetically modified
maize DNA (Mon810). A 483-bp DNA fragment was detected in mixture with 99.9% of noncomplementary DNA with a sensitivity down
to the level of attomole.
Figure 相似文献
12.
Hana Vaisocherová Jan Snášel Tomáš Špringer Hana Šípová Ivan Rosenberg Josef Štěpánek Jiří Homola 《Analytical and bioanalytical chemistry》2009,393(4):1165-1172
Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective
AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time
label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring
of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by
using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length
of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface
density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence
of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical
tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the
binding of ds long terminal repeats–IN complexes with the host DNA.
HIV-1 integrase strand transfer activity was monitored in real time using a multichannel surface plasmon resonance biosensor.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
13.
We screened a series of RNA and DNA aptamers for their ability to serve in the dye displacement assays in which analytes compete
with TO dye. We conclude that, while the performance of the TO dye displacement approach is not always predictable, it is
still a simple and sensitive assay to detect binding between RNA aptamers and small molecules. In particular, we describe
efficient assays for tobramycin and theophylline, with up to 90% displacement of TO observed, and we describe the first aptameric
assay for cAMP.
Figure An RNA or DNA aptamer against a molecule (circle) binds TO dye, resulting in a fluorescent complex. Presence of free molecule in solution results in the displacement of TO
from the complex and a reduction in fluorescence
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
14.
Bai LP Cai Z Zhao ZZ Nakatani K Jiang ZH 《Analytical and bioanalytical chemistry》2008,392(4):709-716
Spectrofluorometric titration, electrospray ionization time-of-flight mass spectrometric and UV melting methods were employed
to study the binding of chelerythrine and sanguinarine to bulged DNA. The results showed that both alkaloids bind specifically
to single pyrimidine (C, T) bulge sites. The ability of sanguinarine to bind to both regular and bulged hairpins was found
to be stronger than that of chelerythrine, but the binding selectivity of chelerythrine toward single-base bulges was much
larger than that of sanguinarine.
Figure Association constants for chelerythrine and sanguinarine toward regular and single-base bulged hairpins obtained from fluorometric
analysis 相似文献
15.
Pérez Pavón JL García Pinto C Guerrero Peña A Moreno Cordero B 《Analytical and bioanalytical chemistry》2008,391(2):599-607
In the present work we report the results obtained with a methodology based on direct coupling of a headspace generator to
a mass spectrometer for the identification of different types of petroleum crudes in polluted soils. With no prior treatment,
the samples are subjected to the headspace generation process and the volatiles generated are introduced directly into the
mass spectrometer, thereby obtaining a fingerprint of volatiles in the sample analysed. The mass spectrum corresponding to
the mass/charge ratios (m/z) contains the information related to the composition of the headspace and is used as the analytical signal for the characterization
of the samples. The signals obtained for the different samples were treated by chemometric techniques to obtain the desired
information. The main advantage of the proposed methodology is that no prior chromatographic separation and no sample manipulation
are required. The method is rapid, simple and, in view of the results, highly promising for the implementation of a new approach
for oil spill identification in soils.
Figure PCA score plots illustrate clear discrimination of types of crude oil in polluted soil samples (e.g. results are shown for
vertisol) 相似文献
16.
Cordes DB Miller A Gamsey S Singaram B 《Analytical and bioanalytical chemistry》2007,387(8):2767-2773
The simultaneous use of several fluorescent reporter dyes in a multicomponent boronic acid-based glucose sensing system is
reported. In one application, two dyes with widely different emission wavelengths are used to report changes in glucose concentration.
A third glucose-insensitive dye was then added to act as a reference dye and provide for a ratiometric correction to the two
reporter dye signals. The inclusion of such a reference dye reduces errors arising from sources such as fluctuations in lamp
intensity and sample dilution.
The simultaneous use of multiple fluorescent reporter dyes 相似文献
17.
New fast methods for the determination of pharmacokinetic behaviour of potential drug candidates are receiving increasing
interest. We present a new homogeneous method for the determination of drug binding and drug competition for human serum albumin
and α1-acid glycoprotein that is amenable to high-throughput-screening. It is based on selective fluorescent probes and the measurement
of fluorescence polarization. This leads to decreased interference with fluorescent drugs as compared with previously published
methods based on similar probes and the measurement of fluorescence intensity. The binding of highly fluorescent drugs that
still interfere with the probes can be measured by simply titrating the drugs in a two-component system with the serum protein.
The assay may also be used to discover strongly binding protein ligands that are interesting for drug-targeting strategies.
Additionally, binding data could be obtained from larger libraries of compounds for in silico predictive pharmacokinetics.
Figure Fluorescence polarization displacement titration of dansylsarcosine (3D-structure as insert) bound to human serum albumin
(HSA) by naproxene
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
Takahashi K Kishine K Matsuyama S Saito T Kato H Kinugasa S 《Analytical and bioanalytical chemistry》2008,391(6):2079-2087
Poly(ethylene glycol) (PEG) is a useful water-soluble polymer that has attracted considerable interest in medical and biological
science applications as well as in polymer physics. Through the use of a well-calibrated evaporative light-scattering detector
coupled with high performance supercritical fluid chromatography, we are able to determine exactly not only the average mass
but also all of the molecular mass fractions of PEG samples needed for certified reference materials issued by the National
Metrology Institute of Japan. In addition, experimental uncertainty was determined in accordance with the Guide to the expression of uncertainty in measurement (GUM). This reference material can be used to calibrate measuring instruments, to control measurement precision, and to confirm
the validity of measurement methods when determining molecular mass distributions and average molecular masses. Especially,
it is suitable for calibration against both masses and intensities for matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry.
Figure Comparison between the molecular mass fractions of PEG 1000 before calibration (si) (○) and after calibration (wi) (⧫). The error bar shows the expanded uncertainty of k = 2 of each mass fraction 相似文献
19.
Lee JO So HM Jeon EK Chang H Won K Kim YH 《Analytical and bioanalytical chemistry》2008,390(4):1023-1032
Recent advances in nanotechnology have enabled the development of nanoscale sensors that outperform conventional biosensors.
This review summarizes the nanoscale biosensors that use aptamers as molecular recognition elements. The advantages of aptamers
over antibodies as sensors are highlighted. These advantages are especially apparent with electrical sensors such as electrochemical
sensors or those using field-effect transistors.
Figure Feeling proteins with aptamer-functionalized carbon nanotubes 相似文献
20.
Gurcel C Vercoutter-Edouart AS Fonbonne C Mortuaire M Salvador A Michalski JC Lemoine J 《Analytical and bioanalytical chemistry》2008,390(8):2089-2097
The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells. This dynamic glycosylation plays
a fundamental role in the activity of many nuclear and cytoplasmic proteins and is associated with pathologies like type II
diabetes, Alzheimer’s disease or some cancers. However the exact link between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Here we report a strategy based
on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues (substituted with an azido or alkyne group) and the corresponding biotinylated probe
to specifically detect, enrich and identify O-GlcNAc-modified proteins. This bio-orthogonal conjugation confirms that only azido analogue of GlcNAc is metabolized by the
cell. Thanks to the biotin probe, affinity purification on streptavidin beads allowed us to identify 32 O-GlcNAc-azido-tagged proteins by LC-MS/MS analysis in an MCF-7 cellular model, 14 of which were previously unreported. This
work illustrates the use of the click-chemistry-based strategy combined with a proteomic approach to get further insight into
the pattern of O-GlcNAc-modified proteins and the biological significance of this post-translational modification.
Figure Detection of biotinylated O-GlcNAz proteins in MCF-7 cells
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Caroline Gurcel and Anne-Sophie Vercoutter-Edouart contributed equally to this work. 相似文献