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1.
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
2.
Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater
for high throughput applications, one area of immense research potential is single-molecule bioassays where miniaturized devices
are developed to perform rapid and effective biological reactions and analyses. With the success of various emerging technologies
for engineering miniaturized structures down to the nanoscale level, supported by specialized equipment for detection, many
investigations in the field of life science that were once thought impossible can now be actively explored. In this review,
the significance of downscaling to the single-molecule level is firstly presented in selected examples, with the focus placed
on restriction enzyme assays. To determine the effectiveness of single-molecule restriction enzyme reactions, simple and direct
analytical methods based on DNA stretching have often been reliably employed. DNA stretching can be realized based on a number
of working principles related to the physical forces exerted on the DNA samples. We then discuss two examples of a nanochannel
system and a microchamber system where single-molecule restriction enzyme digestion and DNA stretching have been integrated,
which possess prospective capabilities of developing into highly sensitive and high-throughput restriction enzyme assays.
Finally, we take a brief look at the general trends in technological development in this field by comparing the advantages
and disadvantages of performing assays at bulk, microscale and single-molecule levels.
Figure Minaturization of Restriction Enzyme Assays and DNA Stretching 相似文献
3.
Baek TJ Park PY Han KN Kwon HT Seong GH 《Analytical and bioanalytical chemistry》2008,390(5):1373-1378
We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA
analysis. The PDA chip comprises an 8 × 6 array of photodiodes each with a diameter of 600 μm. Each photodiode element acts
both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray
platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction
(PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the
chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles
bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated
from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which
allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative
signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating
HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development
time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a
broader range of target DNA concentration by controlling the silver development time.
Figure An optical image of the PDA chip and target DNA detection through silver enhancement
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
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
5.
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
6.
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
7.
We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG)
on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from
poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface
was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated
anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved
by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities
towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi
responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the
target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement
of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the
avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The
combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement
of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant
molecules.
Figure Microchannel SPR imaging for protein–protein interactions 相似文献
8.
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
9.
Nicolas F. Y. Durand Elli Saveriades Philippe Renaud 《Analytical and bioanalytical chemistry》2009,394(2):421-425
In this work, we present theoretical and experimental studies of nanofluidic channels as a potential biosensor for measuring
rapid protein complex formation. Using the specific properties offered by nanofluidics, such as the decrease of effective
diffusion of biomolecules in confined spaces, we are able to monitor the binding affinity of two proteins. We propose a theoretical
model describing the concentration profile of proteins in a nanoslit and show that a complex composed by two bound biomolecules
induces a wider diffusion profile than a single protein when driven through a nanochannel. To validate this model experimentally,
we measured the increase of the fluorescent diffusion profile when specific biotinylated dextran was added to fluorescent
streptavidin. We report here a direct and relatively simple technique to measure the affinity between proteins.
Figure We present theoretical and experimental studies of nanofluidic channels as potential biosensors for rapidly measuring protein
complex formation. Our system is based on steady-state diffusion effects which are observed inside a nanoslit. 相似文献
10.
Applications of microelectromechanical systems (MEMS) technology are widespread in both industrial and research fields providing
miniaturized smart tools. In this review, we focus on MEMS applications aiming at manipulations and characterization of biomaterials
at the single molecule level. Four topics are discussed in detail to show the advantages and impact of MEMS tools for biomolecular
manipulations. They include the microthermodevice for rapid temperature alternation in real-time microscopic observation,
a microchannel with microelectrodes for isolating and immobilizing a DNA molecule, and microtweezers to manipulate a bundle
of DNA molecules directly for analyzing its conductivity. The feasibilities of each device have been shown by conducting specific
biological experiments. Therefore, the development of MEMS devices for single molecule analysis holds promise to overcome
the disadvantages of the conventional technique for biological experiments and acts as a powerful strategy in molecular biology.
Figure Towards single bio molecular handling and characterization by MEMS 相似文献
11.
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
12.
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
13.
The use of electrochemical impedance spectroscopy for biosensing 总被引:1,自引:0,他引:1
This review introduces the basic concepts and terms associated with impedance and techniques of measuring impedance. The focus
of this review is on the application of this transduction method for sensing purposes. Examples of its use in combination
with enzymes, antibodies, DNA and with cells will be described. Important fields of application include immune and nucleic
acid analysis. Special attention is devoted to the various electrode design and amplification schemes developed for sensitivity
enhancement. Electrolyte insulator semiconductor (EIS) structures will be treated separately.
Figure An alternating current which is forced to pass an interface is sensitive to surface changes and will detect impedance changes
due to biomolecule immobilisation or formation of a recognition complex. This can be used for the construction of biosensor
electrodes 相似文献
14.
Melphalan is a bifunctional alkylating agent that covalently binds to the nucleophilic sites present in DNA. In this study
we investigated oligonucleotides prepared enzymatically from DNA modified with melphalan. Calf thymus DNA was incubated in-vitro
with melphalan and the resulting modifications were enzymatically cleaved by means of benzonase and nuclease S1. Efficient
sample preconcentration was achieved by solid-phase extraction, in which phenyl phase cartridges resulted in better recovery
of the modified species than C18. The applied enzymatic digestion time resulted in production of trinucleotide adducts which were efficiently separated and
detected by use of reversed-phase HPLC coupled to an ion-trap mass spectrometer with electrospray ionization. It was assumed
that melphalan could act as both a monofunctional and bifunctional alkylating agent. Mono-alkylated adducts were much more
abundant, however, and the alkylation site was located on the nucleobases. On the other hand, we unequivocally identified
cross-link formation in DNA, even though at low abundance and only a few adduct types were detected.
Figure Different Alkylation reactions of Melphalan with DNA 相似文献
15.
Jackson AT Slade SE Thalassinos K Scrivens JH 《Analytical and bioanalytical chemistry》2008,392(4):643-650
The end-group functionalisation of a series of poly(propylene glycol)s has been characterised by means of electrospray ionisation–tandem
mass spectrometry (ESI-MS/MS). A series of peaks with mass-to-charge ratios that are close to that of the precursor ion were
used to generate information on the end-group functionalities of the poly(propylene glycol)s. Fragment ions resulting from
losses of both of the end groups were noted from some of the samples. An example is presented of how software can be used
to significantly reduce the length of time involved in data interpretation (which is typically the most time-consuming part
of the analysis).
Figure Screenshot from Polymerator software of annotated ESI-MS/MS spectrum from the lithiated heptamer of poly(propylene glycol)
di-acrylate 相似文献
16.
The use of polymers in microchip fabrication affords new opportunities for the development of powerful, miniaturized separation
techniques. One method in particular, the use of phase-changing sacrificial layers, allows for simplified designs and many
additional features to the now standard fabrication of microchips. With the possibility of adding a third dimension to the
design of separation devices, various means of enhancing analysis now become possible. The application of phase-changing sacrificial
layers in microchip analysis systems is discussed, both in terms of current uses and future possibilities.
Figure Phase-changing sacrificial materials enable multilayer microfluidic device layouts 相似文献
17.
Kowalczyk A Nowicka AM Karbarz M Stojek Z 《Analytical and bioanalytical chemistry》2008,392(3):463-469
A piece of dry N-isopropylacrylamide polymer was soaked in phosphate buffer to obtain a hydrogel which was then employed in the examination
of interactions between an anticancer drug C-1311 (5-diethylaminoethyl-amino-8-hydroxyimidazoacridinone) and dsDNA. dsDNA
was introduced into the polymer at the polymerization stage. The drug was added to the buffer. Using the volume phase transition
of the gel at 40 °C, the unbound drug could be determined in the solution released during the transition, which made the calculations
more reliable. The interaction parameters were calculated using the McGhee and von Hippel model. It appeared that in the gel
medium, the interaction between the drug and dsDNA is spatially limited, since the number of binding units of the polymer
chain occupied by one drug molecule was found to be one, while it was two in the regular buffer solution.
Figure
The two authors Agata Kowalczyk and Anna M. Nowicka contributed equally to this work. 相似文献
18.
A method based on use of functionalized gold nanoparticles on polyethylenimine film has been developed for colorimetric detection of immunoglobulin G (IgG). The immunogold nanoparticles were immobilized on quartz slides by recognition between antibody and antigen, with the antigen chemically adsorbed on the polyethylenimine film. By measurement of the UV–visible spectra of the immobilized immunogold, detection of h-IgG was achieved. The detection limit for h-IgG by use of this method can be as low as 0.01 μg mL−1. This method is quite promising for numerous applications in immunoassay.
Figure 相似文献
19.
Kang DY Kim MJ Kim ST Oh KS Yuk SH Lee S 《Analytical and bioanalytical chemistry》2008,390(8):2183-2188
Asymmetrical flow field-flow fractionation (AsFlFFF) was used to determine the size distribution of drug-loaded core/shell
nanoparticles which have a lipid core of lecithin and a polymeric shell of a Pluronic. AsFlFFF provided separation of the
drug-loaded core/shell nanoparticles from smaller coreless polymeric micelles, thus allowing accurate size analysis of the
drug-loaded nanoparticles without interference by the coreless micelles. It was found from AsFlFFF that the drug-loaded nanoparticles
have broad size distributions ranging from 100 to 600 nm in diameter. It was also found that, after the nanoparticles had
been stored for 70 days, they disappeared as a result of self-degradation. Being a separation technique, AsFlFFF seems to
be more useful than transmission electron microscopy or dynamic light scattering for size analysis of core/shell nanoparticles,
which have broad and bimodal size distributions.
Figure Separation by AsFlFFF 相似文献
20.
Popot MA Woolfitt AR Garcia P Tabet JC 《Analytical and bioanalytical chemistry》2008,390(7):1843-1852
The insulin-like-growth factor (IGF-I) peptide is considered to be the main indirect marker for growth hormone administration
(GH) in a horse. Further to a previous investigation on measurement of IGF-I in plasma samples by mass spectrometry, this
study focuses on quantitative and qualitative analysis of intact IGF-I in horse plasma. First, protein-transposing software
has been developed for IGF-I to facilitate its quantification by HPLC–electrospray–ion-trap mass spectrometry. Second, product-ion
scan experiments on IGF-I have been conducted on standard samples, non-fortified equine plasma samples, fortified plasma samples,
and equine GH post-administration samples. This “top-down” approach method enables characterisation of fragment ions corresponding
to the carboxy terminal end, which can be useful for the confirmation of the presence of IGF-I in plasma samples.
Figure Structure of IGF-I and amino acid sequences of IGF-I and R3 IGF-I. Deconvolution mass spectra of the IGF-I and R3 IGF-I mixture 相似文献