共查询到20条相似文献,搜索用时 15 毫秒
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.
FRET primer real-time PCR chemistry depends on internally labeled primers with FRET dyes linked to their 3′ end. The best
distance between the FRET dyes for obtaining the largest signal and the lowest background is six nucleotides. In this study
the forward primer was labeled with FAM and the reverse primer with Texas red; the labeled primers meet in cycle two of PCR.
At the end of the elongation step FAM is excited to emit fluorescence which will excite Texas red to emit new fluorescence
that correlates directly with the quantity of PCR product accumulated. FRET primer techniques amplify short amplicons with
unique thermal cycling steps, 0 s at 85 °C for denaturation, 7 s for annealing, and 2 s for elongation. The FRET primer technique
was very efficient (92.6, 97.2, and 100%), correlation coefficients were high (1.0, 0.999, and 0.999), and total run time
was very short (20, 45, and 40 min per 40 cycles with LightCycler, iCycler, and RotorGene 3000, respectively). When FRET-labeled
primers were compared with similar but unlabeled primers it was observed that the FRET primer technique had a lower Ct value
and was more efficient than use of unlabeled primers detected by use of SYBR Green I.
Figure Schematic diagram of FRET prime real-time PCR
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
3.
Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase
activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of
these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method
is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the
specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover
rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures,
reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products
and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase
activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific
detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly.
Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
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
5.
Webb A 《Analytical and bioanalytical chemistry》2007,388(3):525-528
Figure Schematic diagram of a typical arrangement used for hyphenating chemical microseparations (e.g. capillary HPLC, CE, or CEC)
with microcoil NMR detection 相似文献
6.
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
7.
Evaporative light scattering detection (ELSD) is widely recognized as a universal tool for liquid and supercritical chromatographies.
In addition, this detection technique is fully compatible with continuous-flow systems. In fact, the combination of continuous
non-chromatographic techniques and ELSD affords the design of simple, reliable systems for extracting qualitative information.
This paper reviews instrumental innovations regarding the miniaturization of evaporative light scattering detectors and their
uses in micro and capillary liquid chromatography; also, it discusses their increasingly important role in the development
of vanguard configurations for sample screening and the determination of total indices without the need for chromatographic
separation. Moreover, it compares them with other types of chromatographic detectors in terms of performance. Finally, the
potential of ELSD for solving real-life analytical problems arising from the need to meet (bio)chemical information needs
is illustrated with various selected applications.
Figure New trends in evaporative light scattering detection: recent chromatographic uses and its role in vanguard/rearguard strategies 相似文献
8.
An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence
detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively.
In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity
and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K
d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H2O2-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator
for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary.
This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective.
Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for
using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or
modification step 相似文献
9.
Human immunoglobulin E (hIgE) is such an important protein, because of its involvement in allergic disease, that it is of
significance to study the interactions between it and its recognizing elements. In this report an analytical strategy based
on surface plasmon resonance (SPR) was developed to probe the pattern of interaction between hIgE and its recognizing molecules,
including aptamers and antibodies. The affinity constants of hIgE for the antibody and the aptamer were compared first; the
aptamer has more affinity than the antibody for human IgE. To study their pattern of interaction, three different binding
approaches, including adding the antibody and the streptavidin-coupled aptamer to the sensing surface, were designed. The
results showed that hIgE captured on the sensing surface could form a multivalent complex with the aptamer. An ELISA-like
assay using the aptamer as both capture and detection probes was then developed. This work highlights an SPR method for characterizing
the interaction between the protein and aptamers that is useful for study of biomolecular interaction patterns and binding
properties.
Figure Schematic diagram of the use of surface plasmon resonance for detection of the pattern of interaction of human IgE with its
DNA aptamer and antibody 相似文献
10.
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
11.
Hideya Kawasaki Tarui Akira Takehiro Watanabe Kazuyoshi Nozaki Tetsu Yonezawa Ryuichi Arakawa 《Analytical and bioanalytical chemistry》2009,395(5):1423-1431
The availability of robust methods for the species-specific detection of meat and bone meal (MBM) in compound feedingstuffs
is an important prerequisite to enforce current and upcoming European legislation on the use of processed animal proteins
in animal nutrition. Among possible methods, those based on DNA turned out to be a reliable tool for this aim, since DNA is
a quite thermostable molecule able to resist severe heat treatments applied in the manufacturing of animal meals. The application
of such methods by control laboratories implies that the method has been validated including an assessment of its robustness.
Successful transferability between laboratories is considered an important robustness criterion of the method. However, corresponding
guidelines regarding the design of such a study relevant to this field are missing. Here, we demonstrate the feasibility of
an alternative concept that was applied to check for the transferability of a qualitative assay for the detection of banned
MBM in feedingstuffs at trace level based on real-time PCR. The concept was based on an experimental nested design applying
analysis of variance (ANOVA) that was conducted independently in two laboratories and which allows for establishing major
factors influencing the result of analysis. Statistical assessment of the results confirmed the importance of the DNA extraction/purification
step utilised, whereas the PCR step turned out to be a minor factor regarding the overall variability of the results. Furthermore,
blind samples comprised of compound feed adulterated with MBM at 0.1 % and blank compound feed were correctly classified as
"positive" or "negative" samples, thus confirming fitness of purpose for the method. This approach can be of interest for
other research groups working in the development of real-time PCR methods and in their use by control laboratories.
Nested design of the study to check for the transferability of the real-time PCR method 相似文献
12.
Fakhrullin RF Vinter VG Zamaleeva AI Matveeva MV Kourbanov RA Temesgen BK Ishmuchametova DG Abramova ZI Konovalova OA Salakhov MK 《Analytical and bioanalytical chemistry》2007,388(2):367-375
We report the development of a novel quartz crystal microbalance immunosensor with the simultaneous measurement of resonance
frequency and motional resistance for the detection of antibodies to double-stranded DNA (dsDNA). The immobilization of poly(l-lysine) and subsequent complexation with DNA resulted in formation of a sensitive dsDNA-containing nanofilm on the surface
of a gold electrode. Atomic force microscopy has been applied for the characterization of a poly(l-lysine)–DNA film. After the blocking with bovine serum albumin, the immunosensor in flow-injection mode was used to detect
the antibodies to dsDNA in purified protein solutions of antibodies to dsDNA and to single-stranded DNA, monoclonal human
immunoglobulin G, DNase I and in blood serum of patients with bronchial asthma and systemic lupus erythematosus. Experimental
results indicate high sensitivity and selectivity of the immunosensor.
In memoriam Prof. Victor G. Vinter 相似文献
13.
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
14.
A fiberoptic evanescent-wave sensor has been developed for the measurement of antinuclear antibodies in sera from patients
and healthy individuals. The sensor was constructed on the basis of modification of the unclad portion of an optical fiber
with self-assembled gold colloids, where the colloidal gold surface was further functionalized with extractable nuclear antigens.
Results show that detection of antinuclear antibodies by this sensor agrees quantitatively with the clinically accepted enzyme-linked
immunosorbent assay (ELISA) method. This sensing platform has the following advantages: label-free and real-time detection
capability, simple to construct and use, highly sensitive, and does not require a secondary antibody. The sensitivity of this
platform is at least an order of magnitude higher than that of the ELISA method and thus may lead to a new direction in recognition
of immune response.
Biomolecular binding of antinuclear antibodies (ANA) with extractable nuclear antigens (ENA)-functionalized gold nanoparticles
results in a change of surface plasmon absorption. When light propagates in an optical fiber by multiple total internal reflection,
such a change in signal can be significantly enhanced. 相似文献
15.
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
16.
A simple method using an unmodified edge plane pyrolytic graphite electrode (EPPGE) is reported for the simultaneous determination
of dopamine (DA), serotonin (ST) and ascorbic acid (AA). The performance of this electrode is superior to other unmodified
carbon-based electrodes and also to many modified electrodes in terms of detection limit, sensitivity and peak separation
for determination of DA, ST and AA. Using this method, detection limits of 90 nM, 60 nM and 200 nM were obtained for DA, ST
and AA respectively. No electrode fouling is observed during a set of experiments and good sensitivity is obtained for the
simultaneous determination of DA, ST and AA. The peaks for the three species are well resolved from each other and the electrode
is successfully utilised for their determination in standard and real samples.
相似文献
17.
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
18.
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
19.
A chemiluminescent (CL) detection method has been developed for DNA hybridization. The assay relies on a sandwich-type DNA
hybridization in which gold nanoparticles modified with alkylthiol-capped oligonucleotide strands are used as probes to monitor
the presence of the specific target DNA. The , which is the dissolving product of the gold nanoparticles anchored on the DNA hybrids, serves as an analyte in the H2O2–luminol– CL reaction for the indirect measurement of the target DNA. The combination of the remarkable sensitivity of the CL analysis
with the large number of released from each DNA hybrid allows a detection limit at levels as low as 0.1 pM of the target DNA. Moreover, with a further
silver amplification step, the detection limit will be pushed down to the femtomolar domain.
相似文献
20.
An innovative scheme for signal amplification using random tetramer-modified gold nanoparticles, termed “nanoamplicons,” has
been developed for hybridization assay without PCR. Large numbers of nanoamplicons could be integrated onto one target, providing
much greater amplification than the larger nanoparticles usually adopted. Using M13mp18 single-strand DNA as a target, this
concept is shown to be a feasible approach to detecting 0.17 amol L−1 DNA without target amplification, based on microgravimetric detection of the adsorption of the probe–target–nanoamplicons
complex via thiol–gold binding. To our knowledge, this method has a sensitivity that is close to that of PCR and superior
to those of nanoparticle-based methods reported previously. Additionally, this novel nanoamplicon could be prepared in the
same way and used for all diagnostic tests; such universality would make the nanoamplicons highly advantageous for the generalization
and standardization of bioassays, and when applying this new technology in clinical laboratories.
Figure A novel signal amplification method for DNA detection with subattomolarsensitivity has been developed using random tetramer-modified
gold nanoparticlesas nanoamplicons, which are easily prepared with high uniformity and can be universally adaptedto any sequences 相似文献