Characterization of single- and double-stranded DNA on gold surfaces

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.     

Methylene blue as an electrochemical indicator for DF508 cystic fibrosis mutation detection

Cystic fibrosis is one of the most common life-shortening, childhood-onset inherited diseases. Among the 1,000 known cystic fibrosis-related mutations, DF508 is the most common, with a frequency varying between 50% and 70% according to geographical areas and population typology. In this work, we report the use of methylene blue as an electrochemical reporting agent in the discrimination of synthetic PCR analogue of the DF508 cystic fibrosis mutation (Mut) from the wild type (Wt). At optimum experimental condition, a discrimination factor between mutant and wild type of approximately 1.5-fold was found. The proposed assay was quantitative and linear in the range of 10–100 nM, exhibiting a limit of detection of 2.64 nM. Electrochemical studies at variable ionic strength conditions allowed further elucidation of the mechanism of the methylene blue (MB)–DNA interaction. To the best of our knowledge, this is the first report of detection of hybridisation solely via guanine-specific MB–DNA interaction simultaneously in MB solution, independent of electrostatic interaction as demonstrated in the ionic strength study. The introduction of formamide in the hybridization buffer, to improve discrimination, was also investigated. Finally, mutant wild type discrimination was demonstrated, at 10 nM concentration, with the use of a multi-sensor setup.     

Methylene blue and neutral red electropolymerisation on AuQCM and on modified AuQCM electrodes: an electrochemical and gravimetric study

The phenazine monomers neutral red (NR) and methylene blue (MB) have been electropolymerised on different quartz crystal microbalance (QCM) substrates: MB at AuQCM and nanostructured ultrathin sputtered carbon AuQCM (AuQCM/C), and NR on AuQCM and on layer-by-layer films of hyaluronic acid with myoglobin deposited on AuQCM (AuQCM-{HA/Mb}(6)). The surface of the electrode substrates was characterised by atomic force microscopy (AFM), and the frequency changes during potential cycling electropolymerisation of the monomer were monitored by the QCM. The study investigates how the monomer chemical structure together with the electrode morphology and surface structure can influence the electropolymerisation process and the electrochemical properties of the phenazine-modified electrodes. Differences between MB and NR polymerisation, as well as between the different substrates were found. The electrochemical properties of the PNR-modified electrodes were analysed by cyclic voltammetry and electrochemical impedance spectroscopy and compared with the unmodified AuQCM. The results are valuable for future applications of modified AuQCM as substrates for electroactive polymer film deposition and applications in redox-mediated electrochemical sensors and biosensors.     

A theoretical and experimental study of two thiazole orange derivatives with single- and double-stranded oligonucleotides, polydeoxyribonucleotides and DNA

The effect of interaction with DNA and oligonucleotides on the photophysical properties of two thiazole orange (TO) derivatives, with different side chains (-(CH2)3-N+(CH3)3 and -(CH2)6-I)) linked to the nitrogen of the quinoline ring of the thiazole orange, is presented here. The first one called TO-PRO1 is a commercially available dye, whereas the second one called TO-MET has been specially synthesized for further covalent binding to oligonucleotides with the aim of being used for specific in situ detection of biomolecular interactions. Both photophysical measurements and molecular calculations have been done to assess their possible mode of interaction with DNA. When dissolved in buffered aqueous solutions both derivatives exhibit very low fluorescence quantum yields of 8 x 10(-5) and 2 x 10(-4), respectively. However, upon binding to double-stranded DNA, large spectroscopic changes result and the quantum yield of fluorescence is enhanced by four orders of magnitude, reaching values up to phi F = 0.2 and 0.3, respectively, as a result of an intercalation mechanism between DNA base pairs. A modulation of the quantum yield is observed as a function of the base sequence. The two derivatives also bind with single-stranded oligonucleotides, but the fluorescence quantum yield is not so great as that when bound to double-stranded samples. Typical fluorescence quantum yields of 7 x 10(-3) to 3 x 10(-2) are observed when the dyes interact with short oligonucleotides, whereas the fluorescence quantum yield remains below 10(-2) when interacting with single-stranded oligonucleotides. This slight but significant quantum-yield increase is interpreted as a folding of the single strand around the dye, which reduces the internal rotation of the two heterocycles around the central methine bridge that links the two moieties of the dye. From these properties, it is proposed to link monomer covalently to oligonucleotides for the subsequent detection of target sequences within cells.     

Detection protein biomarker with gold nanoparticles functionalized hollow mesoporous Prussian blue nanoparticles as electrochemical probes

Gold nanoparticles functionalized hollow mesoporous Prussian blue nanoparticles(Au@HMPB NPs)were synthesized and its peroxidase-like activity was explored for electrochemical probe.The Au@HMPB NPs can reduce H2O2 low detection potential of-0.1 V with high sensitivity.After physically adsorption of antibodies onto the gold nanoparticle surface,the functionalized nanoparticles were turned into immuno-probe.The soluble a-chain of interleukin-2(IL-2)receptor(sCD25)was chosen as a model protein biomarker to test the performance of the probe.sCD25 in the samples were captured and enriched by capture anti-CD25 antibody functionalized magnetic nanospheres.Detection antibody functionalized Au@HMPB can then be linked onto the nanospheres and generate electrochemical current towards H2O2 reduction.The electrochemical responses to 1 mmol/L H2O2 was increased with the increasing concentration of CD25.     

DNA nanosensors based on the use of single gold nanowire electrodes and Methylene Blue as an intercalator

The authors describe an electrochemical DNA nanosensor based on the use of single gold nanowire electrodes (AuNWEs). The probe DNA is immobilized on the AuNWE via Au-S bonds that are formed between thiol-terminated DNA and the gold surface. Single AuNWEs were prepared by an improved laser-assisted pulling method and hydrofluoric acid etching. The nanoelectrodes were characterized by cyclic voltammetry and COMSOL simulation. Square wave voltammetry was used to monitor the DNA hybridization event between probe DNA and target DNA by using Methylene Blue (MB) as an intercalator of dsDNA. Under optimal conditions, the peak current for MB (best measured at a potential of ?0.2 V vs. Ag/AgCl) increases linearly with the logarithm of the analyte concentration in the 1.0 f. to 10 nM range, with a 0.48 fmM detection limit at an S/N ratio of 3. The assay is highly selective, reproducible and stable. Considering the small overall dimensions and high sensitivity, this nanoelectrode potentially can be applied to in-vivo sensing of DNA inside living cells

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Graphical abstract Schematic presentation of an electrochemical DNA nanosensor using single gold nanowire electrodes and based on the interaction of thiol-terminated DNA and gold surface. It was used to detect complementary DNA with high selectivity and sensitivity.

     

Comparative electrochemical and impedance studies of self-assembled rigid-rod molecular wires and alkanethiols on gold substrates

A study of the charge transfer and self-assembly characteristics of two new rigid-rod molecular wires 1 and 2 assembled on polycrystalline gold electrodes was carried out using electrochemical impedance spectroscopy and cyclic voltammetry. This class of wires have precisely controlled (ca. 1.5-2.5 nm) lengths of π-conjugation, with extended HOMO and LUMO wavefunctions. While rotations can occur around the C-C single bonds, the molecules cannot isomerise or fold due to their rigid backbone structures. The behaviour of these wires was compared with SAMs of heptanethiol (HPT) and dodecanethiol (DDT). It was found that SAMs of 1, which bears flexible hexyloxy sidechains, had randomly distributed pinholes which show microelectrode behaviour even when diluted with DDT. SAMs of 2, which do not have any sidechains, were well-organised at open-circuit potentials enabling evaluation of electron transfer kinetics assuming an average film thickness. However, impedance studies show that deviations from open circuit potentials resulted in an exponential decrease in charge transfer resistance, whereas capacitance remained constant, possibly attributable to conformational changes of the SAM. The syntheses and characterisation of the molecules is described.     

Electrochemical method for monitoring the progress of polymerase chain reactions using Methylene blue as an indicator

We report on the proof-of-principle of an amperometric method to monitor the progress of a polymerase chain reaction (PCR) in real-time. It is based on the finding that the intercalating redox probe Methylene Blue (MB) becomes less easily electrochemically detectable once intercalated into double-stranded DNA (ds-DNA) compared to its free form. This was studied by cyclic voltammetry before and after addition of salmon sperm ds-DNA. Under optimized conditions, the products of the PCR of mitochondrial DNA fragments were quantitatively detected at different stages of amplification cycles. This strategy is potentially cheaper and easier to integrate into a hand-held miniaturized device than fluorescence-based real-time PCR.

DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label

Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

An additive-free electrochemical route to rapid synthesis of large-area copper nano-octahedra on gold film substrates

We report the first fabrication of large-area Cu nano-octahedra with well-defined shape, good monodispersity and uniform distribution on a gold film substrate (GFS) by a very simple, rapid and cost-effective low-potential electrodeposition (LPED) technique. Different from general surfactant-assisted electrochemical approaches, LPED is an additive-free strategy that exploits the subtle surface energy differences among various crystallographic planes of metals themselves. The size of the octahedra depends on deposition time and is adjustable over a wide range. The shape evolution is found to be remarkably different from that of octahedral particles formed in solution. A formation mechanism is proposed for the unique evolution of Cu octahedra as resulting from the anisotropic growth under appropriate applied potentials.     

Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution.

The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNA-dyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye-DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA strains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4',6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye-DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye-DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and dsDNA DNA-cyanine dye complexes were small. Broadening of dye-ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye-dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former.     

尿酸在聚亚甲基蓝修饰金电极上的电化学及电化学动力学性质

尿酸(Uric acid,UA)是人体内嘌呤核苷酸分解代谢过程中的最终产物.体液中尿酸的含量变化可以充分反映出人体新陈代谢、免疫等机能状况,同时也可间接反映出与嘌呤代谢的有关疾病[1].     

Creation of mammalian single- and double-stranded DNA surfaces: a real-time QCM-D study

The quartz crystal microbalance with dissipation monitoring (QCM-D) is an excellent method for studying the creation of DNA-based surfaces and films. Previous studies have used QCM-D to focus on the construction of DNA surfaces composed of short synthetic DNA oligomers or plasmid DNA. Here, we have used QCM-D to monitor the creation of genomic single- and double-stranded calf thymus DNA surfaces on a polycation adsorbed to a SiO2 support. We have successfully monitored the hybridization between the ssDNA surfaces and their complementary strands in solution and have also shown that DNA multilayer formation can be observed using denatured calf thymus DNA. We have furthermore established that the ssDNA and dsDNA surfaces show different binding characteristics to ethidium bromide, a common dsDNA intercalator, demonstrating the potential use of such surfaces to identify possible DNA ligands.     

Core size effects on the reactivity of organic substrates as monolayers on gold nanoparticles

Monolayer-protected nanoparticles (MPNs) with average core sizes of 1.7- (small), 2.2- (medium) and 4.5-nm (large) diameter have been prepared and functionalized with a variety of aryl ketone substrates, namely, 11-mercaptoundecaphenone (1), 1-(4-hexyl-phenyl)-11-mercaptoundecanone (2), 1-[4-(11-mercaptoundecyl)phenyl]hexanone (3), or 1-[4-(11-mercaptoundecyl)phenyl]undecanone (4). Upon irradiation in benzene solution, the aryl ketone-modified MPNs undergo the Norrish type II photoreaction and yield alkene- or acetophenone-modified MPNs exclusively, with no evidence for the generation of cyclobutanol. The extent of the photoreaction for the entire series of aryl ketones is dependent on the size of the MPN core. For 11-mercaptoundecaphenone, the reaction proceeds nearly to completion on the smallest MPN cores (99 +/- 1%) but occurs to a much lesser extent on medium (85 +/- 5%) and large cores (66 +/- 6%). The differences in the extents of reaction are rationalized by the decreased reactivity of substrates on terrace regions, which become increasingly larger with the core size. In lending support to this hypothesis, the edge and vertex sites of medium-sized MPNs were selectively populated with an aryl ketone probe and shown to react quantitatively, whereas selective population of the terrace sites on the same-sized MPNs results in a much lower extent of reaction. Together, these results indicate differences in reactivity of monolayer substrates on terrace versus edge/vertex sites of MPNs. The differences in reactivity with site will play a role in the design of modified MPNs for applications.     

Adsorption properties of the penicillin derivative DTPA on gold substrates.

Despite the large number of articles and patents dealing with penicillin and other beta-lactam antibiotics, there have been no reports about the self-assembly of such substances as monolayers on gold surfaces. The main reason stems from the high reactivity of the beta-lactam ring, which hinders the development of molecules possessing this entity together with a metal-anchoring function. Herein, we present the synthesis of a novel molecule, 6-[(R,S)-5-(1,2-dithiolan-3-yl)pentanoyl-amino]-penicillanic acid, which combines the beta-lactam ring and a metal-anchoring group. Using spectroscopic tools, we demonstrate the chemisorption of this compound on gold as self-assembled monolayers without any alteration of the penicillin pharmacophore and document its reactivity towards a penicillin-binding protein, BlaR-CTD. Our work is a preliminary step towards the development of new biosensors and well-ordered protein arrays, both based on the high affinity of penicillin for penicillin-binding proteins.     

Immobilization of amine-modified oligonucleotides on aldehyde-terminated alkanethiol monolayers on gold

Chemistry is described for the fabrication of DNA arrays on gold surfaces. Alkanethiols modified with terminal aldehyde groups are used to prepare a self-assembled monolayer (SAM). The aldehyde groups of the monolayer may be reacted with amine-modified oligonucleotides or other amine-bearing biomolecules to form a Schiff base, which may then be reduced to a stable secondary amine by treatment with sodium cyanoborohydride. The surface modifications and reactions are characterized by polarization modulation Fourier transform infrared reflection absorption spectroscopy (PM-FTIRRAS), and the accessibility, binding specificity, and stability of the DNA-modified surfaces are demonstrated in hybridization experiments.     

Potentiodynamic electrochemical impedance spectroscopy. Copper underpotential deposition on gold

Copper underpotential deposition on polycrystalline gold was examined by potentiodynamic electrochemical impedance spectroscopy (PDEIS). The time evolution of the ac response was acquired as 3D plots of frequency response versus applied potential, and these data were later processed with an equivalent circuit analyser built into the virtual spectrometer. The dependences of the equivalent circuit parameters on potential revealed different responses of nitrate and sulphate. Nitrate adsorption pseudocapacitance, and the inverse of the adsorption pseudoresistance, both exhibited peaks in a narrow range of Cu monolayer growth, while sulphate affected a wider potential range and gave two desorption peaks in the anodic scan. Intrinsic irreversibility of the constituent processes was revealed by cyclic redox transformations of the Cu monolayer.