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1.
Yokokawa R Miwa J Tarhan MC Fujita H Kasahara M 《Analytical and bioanalytical chemistry》2008,391(8):2735-2743
Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled
molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were
digested by a restriction enzyme, XhoI, for DNA analysis. One end of the λDNA was modified with biotin and the other end was modified with digoxin molecules by
fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin
antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was
attached to a biotinylated microtubule via avidin–biotin biding and the DNA was stretched by a kinesin-based gliding assay.
A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was
supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method
of performing molecular surgery at the single-molecule scale.
Figure DNA molecule manipulation by motor proteins for analysis at the single-molecule level 相似文献
2.
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
3.
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
4.
Microfluidics offers an ideal platform to integrate cell-based assays with electric measurements. The technological advances
in microfluidics, microelectronics, electrochemistry, and electrophysiology have greatly inspired the development of microfluidic/electric
devices that work with a low number of cells or single cells. The applications of these microfluidic systems range from the
detecting of cell culture density to the probing of cellular functions at the single-cell level. In this review, we introduce
the recent advances in the electric analysis of cells on a microfluidic platform, specifically related to the quantification
and monitoring of cells in static solution, on-chip patch-clamp measurement, and examination of flowing cells. We also point
out future directions and challenges in this field.
Figure Different microfluidic devices applied to electrical analysis of cells 相似文献
5.
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
6.
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
7.
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
8.
Emily O’Neill Danielle Harrington John Allison 《Analytical and bioanalytical chemistry》2009,393(8):2029-2038
Monitoring of cell cultures in microbioreactors is a crucial task in cell bioassays and toxicological tests. In this work
a novel tool based on a miniaturized sensor array fabricated using low-temperature cofired ceramics (LTCC) technology is presented.
The developed device is applied to the monitoring of cell-culture media change, detection of the growth of various species,
and in toxicological studies performed with the use of cells. Noninvasive monitoring performed with the LTCC microelectrode
array can be applied for future cell-engineering purposes.
Figure Microelectrode array for monitoring of cell cultures 相似文献
9.
A miniaturized fluorescence detector utilizing a three-dimensional windowless flow cell has been constructed and evaluated. The inlet and outlet liquid channels are collinear and are located in the same plane as the excitation paths, while the optical fiber used to collect the emission light is perpendicular to this plane. The straightforward arrangement of the flow path minimizes band dispersion and eliminates bubble formation or accumulation inside the cell. The use of high-brightness light-emitting diodes (LEDs) as the excitation source and a miniaturized metal package photomultiplier tube (PMT) results in a compact and sensitive fluorescence detector. The detection limit obtained from the system for fluorescein isothiocyanate (FITC) in flow injection mode is 2.6 nmol/L. The analysis of riboflavin and FITC by packed capillary liquid chromatography is demonstrated.
相似文献
10.
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.
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.
Addition of gold nanoparticles to real-time PCR: effect on PCR profile and SYBR Green I fluorescence
Haber AL Griffiths KR Jamting AK Emslie KR 《Analytical and bioanalytical chemistry》2008,392(5):887-896
Real-time quantitative polymerase chain reaction (qPCR) is the industry standard technique for the quantitative analysis of
nucleic acids due to its unmatched sensitivity and specificity. Optimisation and improvements of this fundamental technique
over the past decade have largely consisted of attempts to allow faster and more accurate ramping between critical temperatures
by improving assay reagents and the thermal geometry of the PCR chamber. Small gold nanoparticles (Au-NPs) have been reported
to improve PCR yield under fast cycling conditions. In this study, we investigated the effect of Au-NPs on optimised real-time
qPCR assays by amplifying DNA sequences from genetically modified canola in the presence and absence of 0.9 nM Au-NPs of diameter
12 ± 2nm. Contrary to expectations, we found that Au-NPs altered the PCR amplification profile when using a SYBR Green I detection
system due to fluorescence quenching; furthermore, high-resolution melt (HRM) analysis demonstrated that Au-NPs destabilised
the double-stranded PCR product. The results indicate that effects on the assay detection system must be carefully evaluated
before Au-NPs are included in any qPCR assay.
Figure Raw amplification profiles in the presence and absence of gold nanoparticles 相似文献
15.
Following a recently developed concept of MS binding assays based on the quantification of a native marker by LC–MS a procedure
to study binding of a low-affinity marker in kinetic, saturation, and competition experiments was established. Separation
of bound and unbound marker—the most crucial step of the assay—could be effectively achieved by filtration in a 96-well-format.
MS binding assays according to this procedure allowed the reliable characterization of NO 711 binding to mGAT1 in presence
of physiological NaCl concentrations. Comparing the results obtained in the present study with those from experiments using
1 mol L−1 NaCl in the incubation milieu reveals remarkable differences with respect to the marker’s affinity and kinetics and to the
investigated test compound’s potency.
Principle of MS binding assays After incubation of a target with a native marker, bound and unbound marker are separated by filtration. Subsequently, the
bound native marker is liberated from the target and finally quantified by LC-MS-MS.
Dedicated to Prof. Hans-Dietrich Stachel on the occasion of his 80th birthday 相似文献
16.
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
17.
Son A Dhirapong A Dosev DK Kennedy IM Weiss RH Hristova KR 《Analytical and bioanalytical chemistry》2008,390(7):1829-1835
Rapid and accurate detection of genetic mutations based on nanotechnology would provide substantial advances in detection
of polycystic kidney disease (PKD), a disease whose current methods of detection are cumbersome due to the large size and
duplication of the mutated gene. In this study, a nanotechnology-based DNA assay was developed for detection of SNPs (single
nucleotide polymorphisms) in a feline autosomal dominant PKD (ADPKD) model which can readily be adapted to diagnosis of human
ADPKD type 1. Europium and terbium phosphors were doped into gadolinium crystal hosts with a magnetic core, providing stable
luminescence and the possibility of magnetic manipulations in a solution-based assay. A hybridization-in-solution DNA assay
was optimized for feline PKD gene SNP detection using genomic DNA extracted from feline kidney tissue and blood. This assay
showed a substantial differentiation between PKD and control specimens. The nanotechnology-based DNA assay is attractive from
the viewpoint of rapid availability, simple methodology, and cost reduction for clinical use to detect mutations involved
in human ADPKD and other genetic diseases.
Figure Schematic diagram of PKD (Polycystic Kidney Disease) SNPs detection assay using feline genomic DNA in magnetic/luminescent
nanoparticle-based DNA hybridization 相似文献
18.
Posthuma-Trumpie GA Venema K van Berkel WJ Korf J 《Analytical and bioanalytical chemistry》2007,389(6):2029-2033
This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone
filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa.
A flow-injection system was used to deliver the reactants and the device was linked on-line to an electrochemical detector.
The microreactor was used for on-line preparation of apoglucose oxidase in strong acid and its subsequent reactivation with
flavin adenine dinucleotide. In addition we describe a miniaturized version of the microreactor used to assess several characteristics
of femtomole to attomole amounts of glucose oxidase. A low negative potential over the electrodes was used when ferrocene
was the mediator in combination with horseradish peroxidase, ensuring the absence of oxidation of electro-active compounds
in biological fluids. A low backpressure at very low flow rates is an advantage, which increases the sensitivity. A variety
of further applications of the microreactor are suggested.
Figure Preparation of apoGOx and restoration of enzyme activity using a soluton of FAD 相似文献
19.
Zaher M Ravelet C Baussanne I Ravel A Grosset C Décout JL Peyrin E 《Analytical and bioanalytical chemistry》2009,393(2):655-660
In this paper, we describe the preparation and the evaluation of a porous graphitic carbon (PGC) column coated with a new
dinaphthyl derivative of neamine for chiral ligand-exchange (LE) chromatography. It was shown that the graphitic surface/dinaphthyl
anchor system efficiently (1.15 μmol/m2) and stably (three months of intensive use) adsorbs the neamine template onto the chromatographic support. The resulting
coated PGC stationary phase showed appreciable LE-based enantioselective properties towards several native amino acids.
Chromatographic separation of methionine enantiomers using a dinaphtyl neamine-based ligand-exchange chiral stationary phase 相似文献
20.
The unique properties of mesoporous silica materials (MPs) have attracted substantial interest for use as enzyme-immobilization
matrices. These features include high surface area, chemical, thermal, and mechanical stability, highly uniform pore distribution
and tunable pore size, high adsorption capacity, and an ordered porous network for free diffusion of substrates and reaction
products. Research demonstrated that enzymes encapsulated or entrapped in MPs retain their biocatalytic activity and are more
stable than enzymes in solution. This review discusses recent advances in the study and use of mesoporous silica for enzyme
immobilization and application in biosensor technology. Different types of MPs, their morphological and structural characteristics,
and strategies used for their functionalization with enzymes are discussed. Finally, prospective and potential benefits of
these materials for bioanalytical applications and biosensor technology are also presented.
Figure Enzyme-functionalized mesoporous silica fibers and their integration in a biosensor design. The immobilization process takes
place essentially in the silica micropores. 相似文献