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1.
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.
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2.
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
3.
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
4.
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
5.
Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids 总被引:1,自引:0,他引:1
Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination
with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms
(SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids
and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive
indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface.
When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached
to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal
from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very
effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic
digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring
the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the
target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism
(GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect
SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and
pharmaceutical applications.
Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant
structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively
choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
6.
In this paper, an electrochemical investigation of (−)-epigallocatechin gallate (EGCG) and its interaction with DNA is presented.
Via an electrochemical approach assisted by ultraviolet–visible (UV–Vis) spectroscopy, we propose that EGCG can intercalate
into DNA strands forming a nonelectroactive complex, which results in the decrease of the anodic peak current of EGCG. Meanwhile,
an electrochemical study with the DNA–Cu(II)–EGCG system shows that damage to DNA can be recognized electrochemically via
the increase in the anodic peak current resulting from the oxidation of guanine and adenine bases. The damage can also be
recognized spectrophotometrically via an increase in the 260 nm absorption band. In addition, it was found that EGCG is able
to discriminate dsDNA from ssDNA, making a potential electrochemical indicator for the detection of DNA hybridization events.
A rapid and convenient method of detecting EGCG was also developed in this work.
Figure Interaction of EGCG with DNA and damage to DNA in the presence of Cu(II)
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
7.
Nanostructured electrochemical DNA biosensors for detection of the effect of berberine on DNA from cancer cells 总被引:2,自引:0,他引:2
Ovádeková R Jantová S Letasiová S Stepánek I Labuda J 《Analytical and bioanalytical chemistry》2006,386(7-8):2055-2062
Multi walled carbon nanotubes (MWNT) in dimethylformamide (DMF) or aqueous sodium dodecyl sulfate (SDS) solution, colloidal
gold nanoparticles (GNP) in phosphate buffer solution (PBS), and a GNP–MWNT mixture in aqueous SDS solution have been investigated
for chemical modification of a screen-printed carbon electrode used as the signal transducer of a dsDNA-based biosensor. Differential
pulse voltammetry of the DNA redox marker and the guanine moiety anodic oxidation and cyclic voltammetry with K3[Fe(CN)6] as indicator revealed substantial enhancement of the response of the biosensor, particularly when MWNT in SDS solution was
used. The biosensor was used in testing of berberine, an isoquinoline plant alkaloid with significant antimicrobial and anticancer
activity. Berberine had a very strong, concentration-dependent, effect on the structural stability of DNA from the human cancer
cells (U937 cells) whereas non-cancer cells were changed only when berberine concentrations were relatively high 75 and 50 μg
mL−1.
Figure Schematic illustration of preparation of the nanostructured films: (a) layer-to-layer coverage (DNA/nanomaterial/SPE); (b) mixed coverage (DNA-nanomaterial/SPE) 相似文献
8.
Cruz Enriquez A Rivero Espejel IA Andrés García E Díaz-García ME 《Analytical and bioanalytical chemistry》2008,391(3):807-815
The interaction of 11-mercaptoundecanoic acid capped gold nanoparticles (MUA-GNPs) with europium ions and aminoacids has been
studied by UV-Vis spectrophotometry, fluorescence, confocal fluorescence microscopy, resonance light scattering and TEM. Results
demonstrated that hyper-Rayleigh scattering emission occurs upon the addition of lysine to the MUA-GNPs–Eu(III) system, thus
providing an inherently sensitive method for lysine determination. The effects of geometrical factors of the gold nanoparticles
(aspect ratio, particle size, cluster formation) and the surrounding medium (pH) on this behavior are discussed. The cooperative
binding interactions of Eu3+ and lysine with gold nanoparticles permitted the discrimination of lysine from other amino acids. The probable mechanism
for the spectral changes and the enhanced resonance light scattering observed is outlined.
Figure Gold nanoparticle resonance light scattering plasmon enhancement through cooperative binding with europium and lysine 相似文献
9.
Concentrations of cationic surfactants in aqueous solutions have been estimated on the basis of changes in the color of gold
nanoparticles, used as reporter probes. We have shown that the colors of gold nanoparticles with anionic protective groups
on their surfaces shift from red to indigo/purple and then back to red in a range of cationic surfactant solutions in which
concentrations vary from very low to above the theoretical CMCs. The color changes occur near the theoretical CMCs, presumably
because the presence of surfactant micelles in the solution prevents the gold nanoparticles from aggregating. We have used
gold nanoparticles as reporter probes to determine the concentrations of cationic surfactants in products such as hair conditioners,
which often contain large amounts of alkyltrimethylammonium halides. Although this approach can only provide an estimate,
it can be performed simply by addition of a given amount of gold nanoparticles to a series of diluted solutions, without the
need for instruments or labor-intensive procedures.
Figure Photographs of a series of diluted hair conditioner solutions with added gold nanoparticles
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10.
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
11.
An X-ray fluorescence method (XRF) is presented that allowed low detection limits (at the 0.1–23 ng mL−1 level) to be obtained for Cr, Mn, Fe, Ni, Zn, Sr, Pb, Bi and Br in water. The samples were prepared using a thin layer method. Trace elements were determined via the calibration curve and standard addition. Absorption effects and inhomogenities in prepared samples were checked for using the emission–transmission method and internal standards, respectively. The results from the XRF method were compared with the results from the inductively coupled plasma atomic emission spectrometry method.
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12.
Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number 总被引:3,自引:0,他引:3
Somanath Bhat Jan Herrmann Paul Armishaw Philippe Corbisier Kerry R. Emslie 《Analytical and bioanalytical chemistry》2009,394(2):457-467
Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. PCR solution is distributed
throughout numerous partitions, and following amplification, target DNA copy number is estimated based on the proportion of
partitions containing amplified DNA. Here, we identify approaches for obtaining reliable digital PCR data. Single molecule
amplification efficiency was significantly improved following fragmentation of total DNA and bias in copy number estimates
reduced by analysis of short intact target DNA fragments. Random and independent distribution of target DNA molecules throughout
partitions, which is critical to accurate digital PCR measurement, was demonstrated by spatial distribution analysis. The
estimated relative uncertainty for target DNA concentration was under 6% when analyzing five digital panels comprising 765
partitions each, provided the panels contained an average of 212 to 3,365 template molecules. Partition volume was a major
component of this uncertainty estimate. These findings can be applied to other digital PCR studies to improve confidence in
such measurements.
Figure Digital PCR amplification plot (left) and panel read out (right) of HindIII-digested pIRMM69. pIRMM69 contains one HindIII restriction enzyme site outside the hmg and transgene fragments used as targets in PCR. Red boxes with white shade denote positive hits containing one or more target
DNA molecules, and white boxes with grey shade refer to no target being amplified. 相似文献
13.
Berchmans S Vergheese TM Kavitha AL Veerakumar M Yegnaraman V 《Analytical and bioanalytical chemistry》2008,390(3):939-946
The present work describes, for the first time, in situ electrochemical preparation of dendrimer-encapsulated Cu nanoparticles
using a self-assembled monolayer of fourth-generation amine-terminated polyamidoamine (PAMAM) dendrimer as the template. Atomic
force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) studies of the modified surface confirmed the presence of
Cu nanoparticles entrapped in dendrimer film. Au electrode modified with a monolayer of the dendrimer enables preconcentration
and subsequent voltammetric detection of Cu2+ at picomolar concentrations. Further, Cu nanoparticles in the dendrimer monolayer could be electrochemically derivatised
to Cu hexacyanoferrate, which exhibits specific crystal planes, unlike the random distribution of crystal planes in bulk-formed
Cu hexacyanoferrate, which is another catalytically active material for sensor applications.
Figure Electrochemical preparation of copper–dendrimer nanocomposite 相似文献
14.
In this paper, direct whole blood PCR amplifications on a static chip thermostat without sample purifications are demonstrated;
in these amplifications, problems such as cross-interferences and contaminations could be avoided. The amplification conditions,
such as the compositions of reagents and thermal programs, were investigated systematically by a GeneAmp PCR system with a
native p53 gene segment (about543 bp) of human genome and an exterior lambda DNA segment (about 500 bp) as targets. Direct
amplifications of p53 and K-ras (about 157 bp) gene segments from 0.5 μL blood samples were successfully demonstrated by a
static PCR chip with an indium tin oxide glass substrate. The chip thermostat has a typical size of 25 mm × 25 mm, and a polyethylene
tube was used as the PCR vial on the glass surface of the chip. Fuzzy proportional integration–differentiation algorithms
were adopted in temperature controls of the chip with an aid of a micro-Pt100 sensor. In the direct PCR with the thermostat
chip, the whole process only involves automatic thermal programs. This work demonstrated that a chip PCR for field test without
desktop facilities is possible either for a point of care test or for forensic analysis.
Figure Photo of the glass static thermostat chip with 2 PCR reaction vials
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
15.
An overview is presented on the application of surface-enhanced infrared absorption (SEIRA) spectroscopy to biochemical problems.
Use of SEIRA results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders
of magnitude and has the potential to enable new studies, from fundamental aspects to applied sciences. This report surveys
studies of DNA and nucleic acid adsorption to gold surfaces, development of immunoassays, electron transfer between metal
electrodes and proteins, and protein–protein interactions. Because signal enhancement in SEIRA uses surface properties of
the nano-structured metal, the biomaterial must be tethered to the metal without hampering its functionality. Because many
biochemical reactions proceed vectorially, their functionality depends on proper orientation of the biomaterial. Thus, surface-modification
techniques are addressed that enable control of the proper orientation of proteins on the metal surface.
Figure Surface enhanced infrared absorption spectroscopy (SEIRAS) on the studies of tethered protein monolayer (cytochrome c oxidase
and cytochrome c) on gold substrate (left), and its potential induced surface enhanced infrared difference absorption (SEIDA)
spectrum 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.
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17.
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
18.
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
19.
Self-assembled monolayers (SAMS) of chemisorbed thioglycollate on a gold electrode surface have been used as a base interface
for the electrostatic adsorption of ferrocenium ion. Electrochemical impedance spectra (EIS) and cyclic voltammetry (CV) were
used to evaluate the electrochemical properties of the supramolecular film. The bare gold electrode failed to distinguish
the oxidation peaks of ascorbic acid (AA) and uric acid (UA) in phosphate buffer solution (PBS, pH 7.0), while the ferricinium–thioglycollate
modified electrode could separate them efficiently. In differiential pulse voltammetric measurements, the prepared gold electrode
could separate AA and UA signals, allowing the simultaneous determination of AA and UA. Under optimal conditions and within
the linear range of 1.0 × 10−6 to 5.0 × 10−4 M, the detection limits of AA and UA achieved were 2.0 × 10−7 and 1.0 × 10−7 M, respectively. The applicability of the prepared electrode was demonstrated by measuring AA and UA in human urine without
any pretreatment.
Figure Fabrication process for the modified electrode 相似文献
20.
Casas V Llompart M Garcia-Jares C Cela R Dagnac T 《Analytical and bioanalytical chemistry》2007,387(5):1841-1849
The aqueous instability of pyrethroids and other compounds usually found in commercial pesticide formulations has been demonstrated
in this work. Several types of sample treatment have been studied to avoid analyte losses during sample manipulation and storage.
Analysis was performed by SPME–GC–MS. Addition of sodium thiosulfate to tap water prevented pyrethroid degradation as a result
of oxidation by free chlorine. The amount added was optimized to minimize the effect of the salt on the analytical results.
Analysis of samples that had been stored at 4 °C for several days revealed loss of some of the pyrethroids in the first period
of storage. The effect of freezing the samples was studied and it was confirmed that samples could be stabilized for at least
one week by freezing. Finally, addition of a miscible organic solvent, for example acetone, led to improvement of the analytical
precision. The quality of the SPME–GC–MS method was studied. Linearity (R > 0.993), repeatability (RSD < 15%), and sensitivity
(detection limits between 0.9 and 35 pg mL−1) were good. When the procedure was applied to real samples including run off and waste water some of the target compounds
were identified and quantified.
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