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1.
The microarray of DNA probes with 5' -NH2 and 5' -Tex/3' -NH2 modified terminus on 10 um carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is characterized in the preseni paper. it was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentra-tion of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface. 相似文献
2.
An unnatural amino acid, β-[6′-(N, N-dimethyl)amino-2′-naphthoyl]alanine (Ald) showing polarity-sen sitive fluorescence characteristics, was synthesized. A thorough Ald-scan of dynorphin A (Dyn A), the putative endogenous ligand for κ opioid receptors, was then performed. Replacement of the amino acid residues in positions 5, 8, 10, 12 or 14 of Dyn A(1-13)-NH2 with Ald resulted in compounds that had almost equal κ binding affinity compared with that of the parent compound; on the other hand, substi-tution o... 相似文献
3.
He H Niu CG Zeng GM Ruan M Qin PZ Liu J 《Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy》2011,82(1):493-497
The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA)3(5-NH2-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA)3(5-NH2-phen) on the 5′-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10−10 mol L−1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment. 相似文献
4.
Sung Han Park 《Analytica chimica acta》2006,564(2):133-140
The potential for a new biochip design based on a continuous gradient of density of immobilized single-stranded DNA oligonucleotide probes (ssDNA) is explored. This gradient resolved information platform (GRIP) can provide sequence identification based on the spatial location and extent of hybridization by a target sequence. Surfaces based on indium-tin oxide (ITO) on glass were first functionalized by 3-aminopropyltriethoxysilane (APTES) followed by attachment of glutaraldehyde, prior to immobilization of oligonucleotide probe that was terminated with amine. The use of Cy3 and Cy5 dye-labelled ssDNA probes and targets allowed estimation of density and correlation of the location of binding of labelled targets. Probe molecules of 20 mer lengths were loaded to produce density gradients in the range of 1.0-200 ng/mm2. The biochips could resolve a mixture of fully complementary five base-pair mismatched targets by the location of binding on the surface. Thermal control provided additional selectivity. Thermal cycling and washing provided for regeneration of the surface, and the fluorescence intensities showed no deterioration in at least five cycles of hybridization reactions. 相似文献
5.
Different Strategies of Covalent Attachment of Oligonucleotide Probe onto Glass Beads and the Hybridization Properties 总被引:1,自引:0,他引:1
The glass bead is a new biochip support material for immobilization biomolecules, due to its independence and convenient rearrangement.
In order to optimize the immobilization efficiency of oligonucleotides onto glass beads and obtain the highest hybridization
efficiency, three commonly used coupling strategies have been studied for covalently attaching oligonucleotides onto large
glass beads. Glass beads with 250 μm diameter were amino-silaned with 2% 3-aminopropyltrimethoxysilane (APTMS) and then reacted
separately with glutaraldehyde, succinic anhydride and 1,4-phenylene diisothiocyanate (PDITC) to derive CHO beads, COOH beads
and isothiocyanate-modified beads (NCS-Beads) accordingly. Afterwards, amino-terminal oligonucleotides were covalently attached
onto the surface of beads achieved by three strategies mentioned above. The immobilization efficiency were studied to compare
the three strategies, which turned out 2.55 × 1013 probes/cm2 for CHO-Beads, 3.21 × 1013 probes/cm2 for COOH beads and 6.68 × 1013 probes/cm2 for NCS beads. It meant that the immobilization efficiency based on NCS beads was most acceptable. And the method, developed
by attaching amino-terminal oligonucleotides onto these cyanate active beads, could be regarded as an efficient one for immobilizing
oligonucleotides onto a solid surface. Moreover, in this paper, the hybridization properties of NCS bead-based oligonucleotides
have been studied by employing Cy5-tagged complementary oligonucleotides. It was found that the high probe density NCS beads
led to low hybridization efficiency possibly due to the existence of steric crowding. In addition, the equilibrium binding
constant K
A was determined by employing Langmuir isotherm model, which was 7.0 × 106 M−1 for NCS beads with the density of 6.7 × 1013 probes/cm2. Furthermore, it only took 60 min to reach hybridization equilibrium. These large microspheres (>100 μm) can be employed
in the mesofluidic systems for automated heterogeneous assays. 相似文献
6.
In order to understand the structure of DNAs and their interactions when on microarray surfaces, we performed the first all-atom
molecular dynamics simulation of DNA tethered to a surface. On the surface, the binding of the DNA was enhanced, and its average
equilibrium conformation was the B form. The DNA duplex spontaneously tilted towards its nearest neighbor and settled in a leaning position with a interaxial
distance of 2.2 nm. This close packing of the DNAs, which affects both in situ synthesis and deposition of probes on microarray
surfaces, can thus be explained by salted-induced colloidlike DNA–DNA attractions.
Received: 30 November 2000 / Accepted: 7 February 2001 / Published online: 22 May 2001 相似文献
7.
Reaction of the dinuclear complex [Pd{κ2-N2′,C1-2-(2′-NH2C6H4)C6H4}Cl]2 (1) with ligands (L = 4-picoline, sym-collidine) gave the six-membered palladacycles [Pd{κ2-N2′,C1-2-(2′-NH2C6H4)C6H4}Cl(L)] (2). The complex 1 reacted with AgX (X = CF3SO3, BF4) and bidentate ligands [L–L = phen (phenanthroline), dppe (bis(diphenylphosphino)ethane), bipy(2,2′-bipyridine) and dppp
(bis(diphenylphosphino)propane)] giving the mononuclear orthopalladated complexes [Pd{κ2-N2′,C1-2-(2′-NH2C6H4)C6H4}(L–L)] (3) [L–L = phen, dppe, bipy and dppp]. These compounds were characterized by physico-chemical methods, and the structure of
[Pd{κ2-N2′,C1-2-(2′-NH2C6H4)C6H4}Cl(L)] (L = sym-collidine) was determined by single-crystal X-ray analysis. 相似文献
8.
We use lattice Monte Carlo simulations to study the thermodynamics of hybridization of single-stranded "target" genes in solution with complementary "probe" DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct, with each segment representing a sequence of nucleotides that interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how surface density (number of probes per unit surface area) and concentration of target molecules affect the extent of hybridization. For short probe lengths, as the surface density increases, the probability of binding long stretches of target segments increases at low surface density, reaches a maximum at an intermediate surface density, and then decreases at high surface density. Furthermore, as the surface density increases, the target is less likely to bind completely to one probe; instead, it binds simultaneously to multiple probes. At short probe lengths, as the target concentration increases, the fraction of targets binding completely to the probes (specificity) decreases. At long probe lengths, varying the target concentration does not affect the specificity. At all target concentrations as the probe length increases, the fraction of target molecules bound to the probes by at least one segment (sensitivity) increases while the fraction of target molecules completely bound to the probes (specificity) decreases. This work provides general guidelines to maximizing microarray sensitivity and specificity. Our results suggest that the sensitivity and specificity can be maximized by using probes 130-180 nucleotides long at a surface density in the range of 7 x 10(-5)- 3 x 10(-4) probe molecules per nm(2). 相似文献
9.
Min Ruan Cheng-Gang Niu Pin-Zhu Qin Guang-Ming Zeng Zhao-Hui Yang Hui He Jing Huang 《Analytica chimica acta》2010,664(1):95-99
A two-probe tandem DNA hybridization assay based on time-resolved fluorescence was employed to detect Escherichia coli strain. The amino modified capture probe was covalently immobilized on the common glass slide surface. The Eu(TTA)3(5-NH2-phen) with the characteristics of long lifetime and intense luminescence was labeled with reporter probe. The original extracted DNA samples without the purification and amplification process were directly used in the hybridization assay. The concentration of capture probe, hybridization temperature, hybridization and washing time were optimized. The detection limit is about 1.49 × 103 CFU mL−1E. coli cells, which is comparable to the value of most microbiology methods. The proposed method has the advantages of easy operation, satisfactory sensitivity and specificity, which can provide a promising technique for monitoring the microorganisms. 相似文献
10.
Carboxyl group-functionalized single-walled carbon nanotubes (SWNTs) and 2,6-pyridinedicarboxylic acid (PDC) were electropolymerized
by cyclic voltammetry on a glassy-carbon electrode (GCE) surface to form composite films (SWNTs/PDC). Zirconia was then electrodeposited
on the SWNTs/PDC/GCE from an aqueous electrolyte containing ZrOCl2 and KCl by cycling the potential between −1.1 V and +0.7 V at a scan rate of 20 mV s−1. DNA probes with a phosphate group at the 5′ end were easily immobilized on the zirconia thin films, because of the strong
affinity between zirconia and phosphate groups. The sensors were characterized by cyclic voltammetry (CV) and electrochemical
impedance spectroscopy (EIS). EIS was used for label-free detection of the target DNA by measuring the increase of the electron
transfer resistance (R
et) of the electrode surface after the hybridization of the probe DNA with the target DNA. The PAT gene fragment and polymerase
chain reaction (PCR) amplification of the NOS gene from transgenically modified beans were satisfactorily detected by use
of this DNA electrochemical sensor. The dynamic range of detection of the sensor for the PAT gene fragment was from 1.0 × 10−11 to 1.0 × 10−6 mol L−1 and the detection limit was 1.38 × 10−12 mol L−1. 相似文献
11.
MP2 calculations were used to perform an energy scan of 2-hydroxyadenine (2-OH-A) stacked with four canonical DNA bases. The
structures that were studied correspond to potential energy surface points of B-DNA. Eight stacking complexes were analyzed
in detail: 5′-2-OH-A/A-3′, 5′-2-OH-A/C-3′, 5′-2-OH-A/G-3′, 5′-2-OH-A/T-3′, 5′-A/2-OH-A-3′, 5′-C/2-OH-A-3′, 5′-G/2-OH-A-3′,
and 5′-T/2-OH-A-3′. The stabilization energy, including electron correlation terms, suggests that the 5′-G/2-OH-A-3′ pair
is the most stable among all of the studied complexes. The dependence of the stacking energy on the vertical separation and
on the twist angle between the two stacked bases were studied in great detail. 相似文献
12.
Hong BJ Oh SJ Youn TO Kwon SH Park JW 《Langmuir : the ACS journal of surfaces and colloids》2005,21(10):4257-4261
We have prepared solid substrates modified with a cone-shaped dendron that generates mesospacing (3.2 nm on average) on the surface. This nanoscale-controlled surface provided an ideal DNA microarray in which each probe DNA strand was given ample space for the incoming target DNA, resulting in selectivity as high as that in solution (100: < 1). In addition, high hybridization yield confirms that DNA probes on the mesospaced surface are sterically unhindered for the hybridization. 相似文献
13.
Devarayapalli KC Pack SP Kamisetty NK Nonogawa M Watanabe S Kodaki T Makino K 《Analytical and bioanalytical chemistry》2007,388(4):919-928
DNA chips prepared on a flat glass surface have unavoidable drawbacks when used for quantitative analysis. In an attempt to
overcome this problem, we constructed an HPLC-type system suitable for quantitative analysis that enables base sequence- and
T
m-dependent DNA oligomer separation in a flow system. A small open tubular capillary column (300-mm × 100-μm I.D.) was used.
The DNA oligomers used as probes had an amino group at the 5′-end and were immobilized on the inner silica surface of the
capillary column which had been sequentially treated with 3-aminopropyltriethoxysilane, butyltrimethoxysilane, and disuccinimidylglutarate.
Using the combination of probe-immobilized column placed in a column oven equipped with temperature gradient function, a nano-flow-controllable
pump, a small sample-loading injector, and a capillary-fitted UV detector, we succeeded in separating complementary and non-complementary
DNA oligomers in specific and quantitative modes. We also designed a temperature gradient strategy for efficient separation
of target DNA oligomers in DNA mixture samples. Using a column carrying two different probes with similar T
m values, their complementary target DNA oligomers were also separated and detected. The developed DNA open tubular capillary
column system investigated in the present study could be further improved as an alternative tool to DNA chips to be used for
the quantitative analysis of DNA or mRNA samples.
Kamakshaiah Charyulu Devarayapalli and Seung Pil Pack contributed equally to this paper. 相似文献
14.
A new electrochemiluminescence (ECL) method based on the proximity-dependent surface hybridization assay and Ru(bpy)32+-doped silica nanoparticles (Ru-DSNPs) as labels were proposed for detecting DNA. The hybridization process involves two steps:
firstly, the 3′ thiolated capture probe was self-assembled on the gold electrode. Secondly, the proximity-dependent surface
hybridization assay was carried out. This proximity-dependent surface hybridization assay depended on the simultaneous recognition
of a target DNA by a capture probe and Ru-DSNP-labeled probe and the formation of a duplex complex, which results in the luminophor
approach to the electrode surface. Thus, sensitive ECL signals were obtained. Under optimum conditions, the intensity of the
ECL of Ru-DSNPs was linearly related to the concentration of the target sequence in the range of 2.0 × 10−15 to 2.0 × 10−11 mol/L. The detection limit was 1.0 × 10−15 mol/L (S/N = 3). 相似文献
15.
Dye-encapsulating unilamellar DNA oligonucleotide-tagged liposomes were prepared and characterized for use as signal-enhancing
reagents in a microtiter plate sandwich-hybridization analyses of single-stranded RNA or DNA sequences. The liposomes were
synthesized using the reversed-phase evaporation method and tagged with DNA oligonucleotides by adding cholesteryl-modified
DNA reporter probes to the initial lipid mixture. Liposomes were prepared using probe coverages of 0.0013–0.103 mol% of the
total lipid input, several hydrophobic and poly(ethylene glycol)-based spacers between the cholesteryl anchor and the probe,
and liposome diameters ranging from 200 nm to 335 nm. Their signal enhancement functionality was compared by using them in
microtiter plate sandwich-hybridization assays for the detection of single-stranded DNA sequences. In these assays, an optimal
reporter probe concentration of 0.103 mol%, a liposome diameter of 274 nm, and a phospholipid concentration of 0.3 mM were
found. The length between the cholesteryl anchor and the probe was optimal when a spacer composed of TEG+(CH2O)3 was used. Under optimal conditions, a detection limit of 0.5 nM for a truncated synthetic DNA sequence was found with a coefficient
of variation of 4.4%. A 500-fold lower limit of detection using fluorescence was found using lysed dye-encapsulating liposomes
versus a single fluorescein-labeled probe. Finally, when this method was applied to the detection of atxA RNA extracted from E.coli SG12036-pIu121 and amplified using NASBA, a minimum extracted concentration of RNA of 1.1×10−7 μg/μL was found. 相似文献
16.
C. M. Niemeyer B. Ceyhan S. Gao L. Chi S. Peschel U. Simon 《Colloid and polymer science》2001,279(1):68-72
The organization of metal and semiconductor nanoparticles to form micro- and nanostructured assemblies is currently of tremendous
interest. This communication reports on the utilization of DNA molecules as positioning elements for generating microstructured
surface architecture from gold nanoparticles. Citrate-passivated 40 nm gold colloids were modified by chemisorptive coupling
with a 5′-thiol-derivatized DNA oligomer. The nucleic acid was used as a molecular handle for the specific immobilization
on solid supports, previously functionalized with capture DNA oligomers, complementary to the nanoparticle-bound DNA. As a
consequence of the enormous specificity of nucleic acid hybridization, the DNA-directed immobilization (DDI) allows, to site-specifically
target the hybrid nanoparticles to microlocations which contain the complementary oligomers. The site-selectivity of the surface
adsorption is demonstrated by immobilizing the gold colloids on a DNA microarray on a glass cover slide. Moreover, scanning
force microscopy (SFM) analysis, used to characterize the intermediate steps of the DDI on a gold substrate, provided initial
insights into the specificity and efficiency of this technique. The application of the DDI to fabricate complex colloidal
micro- and nanostructures is anticipated.
Received: 26 July 2000/Accepted: 5 October 2000 相似文献
17.
Aptasensor for ampicillin using gold nanoparticle based dual fluorescence–colorimetric methods 总被引:1,自引:0,他引:1
A gold nanoparticle based dual fluorescence–colorimetric method was developed as an aptasensor to detect ampicillin using
its single-stranded DNA (ssDNA) aptamer, which was discovered by a magnetic bead-based SELEX technique. The selected aptamers,
AMP4 (5′-CACGGCATGGTGGGCGTCGTG-3′), AMP17 (5′-GCGGGCGGTTGTATAGCGG-3′), and AMP18 (5′-TTAGTTGGGGTTCAGTTGG-3′), were confirmed
to have high sensitivity and specificity to ampicillin (K
d, AMP7 = 9.4 nM, AMP17 = 13.4 nM, and AMP18 = 9.8 nM, respectively). The 5′-fluorescein amidite (FAM)-modified aptamer was
used as a dual probe for observing fluorescence differences and color changes simultaneously. The lower limits of detection
for this dual method were a 2 ng/mL by fluorescence and a 10 ng/mL by colorimetry for ampicillin in the milk as well as in
distilled water. Because these detection limits were below the maximum residue limit of ampicillin, this aptasensor was sensitive
enough to detect antibiotics in food products, such as milk and animal tissues. In addition, this dual aptasensor will be
a more accurate method for antibiotics in food products as it concurrently uses two detection methods: fluorescence and colorimetry. 相似文献
18.
Kamila Malecka Iwona Grabowska Jerzy Radecki Anna Stachyra Anna Góra‐Sochacka Agnieszka Sirko Hanna Radecka 《Electroanalysis》2013,25(8):1871-1878
A DNA biosensor for the detection of specific oligonucleotide sequences of Avian Influenza Virus type H5N1 has been proposed. The NH2‐ssDNA probe was deposited onto a gold electrode surface to form an amide bond between the carboxyl group of thioacid and the amino group from ssDNA probe. The signals generated as a result of hybridization were registered in square wave voltammetry and electrochemical impedance spectroscopy in the presence of [Fe(CN)6]3?/4? as a redox marker. The genosensor is capable to determine 20‐mer and 180‐bp (PCR products) oligonucleotides complementary sequences with detection limit in the fM range. The genosensor displays good selectivity and sensitivity. The 20‐mer as well as 180‐bp oligonucleotides without a complementary sequence generate very low signal. 相似文献
19.
Paul A.E. Piunno 《Analytica chimica acta》2005,534(1):53-61
A significant challenge exists in the creation of an environment for immobilized probe oligonucleotides that offer good structural regularity and reproducibility, where nearest neighbour interactions provide for control of selectivity, yet where the degree of hybridization does not alter nearest neighbour interactions. This new work explores whether a “matrix isolation” method will produce the desired environment for the probe molecules. The DNA oligonucleotide probes are polyelectrolytes with charged backbones and significant flexibility. It is possible to isolate the probe molecules by surrounding each, on average, with a sheath of immobilized oligomer that is not based on complementary nucleic acid, yet that is a polyelectrolyte in order to control the surface density and charge within the mixed film. Preliminary work investigates a mixture of dT20 as the probe oligonucleotide, and a 20-mer oligomer primarily containing ethylene glycol phosphate, as a matrix isolation material in a 1:20 mole ratio, respectively. Melt temperature (Tm) measurements indicate that the thermodynamic stability of the probe molecules can be adjusted using the oligomer matrix to achieve lower Tm values by up to 5 °C, with full retention of selectivity for discrimination of single base pair mismatches even under conditions where the probes at a surface are saturated with complementary target. 相似文献
20.
Xiaofeng WangUlrich J Krull 《Analytica chimica acta》2002,470(1):57-70
Single-stranded DNA (ssDNA) oligonucleotide in solution, or that is immobilized onto a surface to create a biosensor, can be used as a selective probe to bind to a complementary single-stranded sequence. Fluorescence enhancement of thiazole orange (TO) occurs when the dye intercalates into double-stranded DNA (dsDNA). TO dye has been covalently attached to probe oligonucleotides (homopolymer and mixed base 10mer and 20mer) through the 5′ terminal phosphate group using polyethylene glycol linker. The tethered TO dye was able to intercalate when dsDNA formed in solution, and also at fused silica surfaces using immobilized ssDNA. The results indicated the potential for development of a self-contained biosensor where the fluorescent label was available as part of the immobilized oligonucleotide probe chemistry. The approach was shown to be able to operate in a reversible manner for multiple cycles of detection of targeted DNA sequences. 相似文献