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.     

Spectroscopic Electrochemical Studies of Interaction Between Fuchsin Basic DNA

Visible spectroscopic and electrochemical methods were used to study the interactions between DNA and fuchsin basic(FB). FB has an irreversible electro-oxidation peak in 5 mmol/L Tris-HCl buffer solution at pH = 7.4 on a glassy carbon electrode(GCE). After adding certain concentration of dsDNA, the oxidation peak current of FB decreases, but the peak potential hardly changs. The visible absorption spectroscopic study shows that the binding mode of FB to dsDNA is intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is smaller than 0. 2, and a new substance, which produces a new absorption peak, is obtained via a covalent binding between dsDNA and FB apart from intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is larger than 0. 2. The visible absorption spectra varies no longer when the ratio of the concentration of dsDNA to FB is larger than 1.5. A mean binding ratio of dsDNA to FB was determined to be 1.4: 1,suggesting that two complexes FB-dsDNA and FB-2dsDNA be formed. The interaction between FB and ssDNA was only electrostatic binding. The more powerful interaction of FB with dsDNA than with ssDNA may be applied for the recognition of dsDNA and ssDNA, and in DNA biosensor as hybridization indicator.     

烟酰胺与DNA相互作用的电化学研究

利用示差脉冲伏安法研究了烟酰胺(NA)与小牛胸腺DNA在pH 8.0条件下相互作用的电化学行为.双链DNA(dsDNA)或单链DNA(ssDNA)的存在导致NA的峰电流明显降低且峰电位负移,表明NA与DNA发生相互作用,生成了复合物,且其作用模式主要是静电模式,但NA与dsDNA的相互作用强于与ssDNA的相互作用,可用于识别dsDNA和ssDNA.通过dsDNA加入前后峰电流的变化,计算得出NA与dsDNA结合常数β=4.946×10(11),结合位点数m=3.此外,NA的峰电流Ip与DNA质量浓度在1～14mg/L的范围内呈线性关系,线性回归方程为Ip(10-5A)=-0.03451cDNA(mg/L)+1.7408,相关系数R为0.9998.该法具有良好的回收率和选择性,可用于样品中DNA的测定.     

Towards the optimization of an optical DNA sensor: control of selectivity coefficients and relative surface affinities

Short single-stranded DNA (ssDNA) oligonucleotides can be grown on the surface of fused silica by automated nucleic acid synthesis. The immobilized ssDNA can be deposited at a desired average density. The density of ssDNA provides a controlled parameter that in combination with temperature, ionic strength and pH, can be used to define the selectivity of hybridization. Furthermore, the density of ssDNA can be used to control the affinity of complementary DNA so that it associates with the nucleic acids on the surface rather than areas that are not coated with ssDNA. The characteristic melt temperature observed for immobilized double-stranded DNA (dsDNA) 20mer shifts by up to 10 °C when a single base pair mismatch is present in the center of a target oligonucleotide. Optimization of quantitative analysis of such single base pair mismatches requires use of select experimental conditions to maximize the formation of the fully matched target duplex while minimizing the formation of the mismatched duplex. Results based on fiber optic biosensors that are used to study binding of fluorescein-labeled complementary DNA demonstrate that it is possible to achieve a selectivity coefficient of fully matched to single base pair mismatch of approximately 85-1, while maintaining >55% of the maximum possible signal that can be obtained from the fully matched target duplex.     

Hybridization with nanostructures of single-stranded DNA

Nanostructures of single-stranded DNA (ssDNA) were produced within alkanethiol self-assembled monolayers using nanografting, an atomic force microscopy (AFM) based lithography technique. Next, variations of the fabrication parameters, such as the concentration of ssDNA or lines per frame, allowed for the regulation of the density of ssDNA molecules within the nanostructures. The label-free hybridization of nanostructures, monitored using high-resolution AFM imaging, has proven to be highly selective and sensitive; as few as 50 molecules can be detected. The efficiency of the hybridization reaction at the nanometer scale highly depends on the ssDNA packing density within the nanostructures. This investigation provides a fundamental step toward sensitive DNA detection and construction of complex DNA architectures on surfaces.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

Optical properties of an immobilized DNA monolayer from 255 to 700 nm

The real (n) and imaginary (k) refractive indices of an immobilized monolayer of 27 nucleotide (nt) single stranded DNA (ssDNA) and the corresponding double stranded DNA (dsDNA) are measured in the 255-700 nm range. Multiple techniques are used to obtain consistent estimation. The coverage is approximately 6.5% with an average interchain distance of tethered ssDNA molecules of approximately 11.8 nm, which is significantly larger than the "footprint" of the chain on the surface. The measured increase in n by approximately 5% between the ssDNA and the dsDNA is 20% smaller than the expected change due to doubling of the molecular weight. The change in k is not significant, indicating that the electron delocalization effect expected in dsDNA due to base pair stacking is not important at optical frequencies.     

水溶性碳纳米粒子的制备及其在DNA和单核苷酸多态性分析中的应用

利用电化学氧化的方法制备了水溶性好、粒径为7～12nm的碳纳米粒子,该碳纳米粒子通过π-π相互作用吸附荧光标记的单链DNA探针,并能有效地猝灭其荧光．当单链DNA探针与匹配的DNA目标分子杂交形成双链DNA时,猝灭的荧光被恢复,由此可以检测1-200nmol／L的DNA目标分子。此外,在碳纳米粒子存在时,由荧光标记的DNA探针和DNA目标分子形成的双链DNA的熔解温度可以简便地被测定,当双链DNA有错配碱基时,其熔解温度降低,由此可方便、快速地分析单核苷酸多态性．     

Salt‐induced formation of DNA double helices from single stranded DNA investigated by analytical ultracentrifugation

The effect of monovalent and divalent cations on the ssDNA to dsDNA transformation was systematically examined. For salts containing monovalent cations (LiCl, NaC1, KCl, CsCl), the conversion of ssDNA to dsDNA increased with ionic strength up to a value of I = 0.01 M and then plateaued, confirming that all four of the monovalent cations behaved similarly and promoted the formation of dsDNA. The monovalent cation type influenced the equilibrium constant for the conversion of ssDNA to dsDNA, indicating a degree of ion‐specificity in dsDNA formation. In the case of salts containing divalent cations (e.g., MgCl₂), the conversion of ssDNA to dsDNA also increased with increasing ionic strength, though the plateau region was reached at a much lower ionic strength (I = 5.0 × 10⁻⁴ M), which can be attributed to the higher electrostatic screening efficiency of Mg²⁺ cations and thus their superior ability to link DNA chains. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 501–508     

A simple visual method for DNA detection based on the formation of gold nanoparticles

A simple visual method for DNA detection during the formation of gold nanoparticles (AuNPs) was developed based on different electrostatic properties of single strand DNA (ssDNA) and double strand DNA (dsDNA). Since the ssDNA is easy to bind to AuNPs due to its exposed bases which could prevent salt-induced aggregation of AuNPs. The dsDNA always present negative charge because its negatively charged phosphate backbone is exposed. In this case, the dsDNA could disturb the adsorption between dsDNA and AuNPs and result in non-aggregation of AuNPs. After hybridization, chloroauric acid and ascorbic acid were added to the mixture solution, and the solution changed to red immediately and turned to purple in 10 min in the present of target DNA. TEM results confirmed that the change of color stemed from aggregation of AuNPs. In order to obtain accurate results by naked eye, the DNA detection assay should be conducted under pH 7.0.     

A simple visual method for DNA detection based on the formation of gold nanoparticles

A simple visual method for DNA detection during the formation of gold nanoparticles (AuNPs) was developed based on different electrostatic properties of single strand DNA (ssDNA) and double strand DNA (dsDNA). It could identify target DNA in 10 min.     

灿烂甲酚蓝在DNA修饰金电极上的电化学行为

利用自组装技术将巯基乙醇固定在金电极表面形成巯基乙醇自组装膜修饰金电极, 用乙基-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为偶联试剂, 分别将鲱鱼精单链DNA(ssDNA)和双链DNA(dsDNA)固定于金电极表面形成ssDNA和dsDNA 修饰电极. 考察了灿烂甲酚蓝(BCB)在不同DNA 修饰电极上的电化学行为,结果表明, BCB 在ssDNA 和dsDNA 修饰电极上的吸附常数分别为1.67×10^4和3.22×10^4 L·mol-1, BCB 与ssDNA 主要以静电作用结合, 而与dsDNA作用存在静电和嵌插两种模式. dsDNA 对BCB 具有更高的亲和力, 使BCB 可以作为一种有效的电化学杂交指示剂.     

Investigation of Metal Ion Effect on Specific DNA Sequences and DNA Hybridization

In this article, we investigated the sequence specific interaction of single (ssDNA) and double stranded (dsDNA) with silver ions (Ag⁺) with electrochemical methods. We, for the first time, examined the effect of base sequences, base content and physiochemical properties of different DNA sequences on interaction with Ag⁺ in detail. We used different base contents to show how the composition of nucleic acid influences the electrochemical signals. We first immobilized ssDNA probes on bare graphite electrodes. Then, we showed the sequence effect on oxidation signals of AgDNA complex by sensing Ag⁺ to the probe coated surfaces to interact with different ssDNA sequences. Furthermore, we investigated the effect of Ag⁺ on dsDNA. We measured the oxidation signals obtained from Ag⁺‐ssDNA and Ag⁺‐dsDNA complex at approximately 0.2 V and 1.0 V (vs Ag/AgCl), respectively with Differential Pulse Voltammetry (DPV). We showed that the oxidation signals of the AgDNA complex obtained from dsDNA‐modified electrodes is higher than the electrodes modified with ssDNA. More importantly, we showed that Ag⁺‐ssDNA and Ag⁺ ion‐dsDNA exhibit different electrochemical behaviors.     

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.     

Electrochemical DNA Biosensors Applicable to the Study of Interactions Between DNA and DNA Intercalators

Electrochemical DNA biosensors, based either on carbon paste electrode (CPE) or hanging mercury drop electrode (HMDE) were prepared. These biosensors were used in the study of interaction between double stranded DNA (dsDNA) and single stranded DNA (ssDNA) and acridine orange, a well known DNA intercalator. The different electrochemical behaviors were compared in the article.     

Voltammetric Studies on the Recognition of a Copper Complex to Single‐ and Double‐Stranded DNA and Its Application in Gene Biosensor

A mixed‐ligands copper complex [Cu(phendione)(DAP)]SO₄ (phendione=1,10‐phenanthroline‐5,6‐dione, DAP=2,3‐diaminophenazine) was synthesized. Cyclic voltammetry showed that the complex underwent an obvious decrease of redox peak currents and positive shift of formal potential after interaction with double‐stranded DNA (dsDNA), suggesting that the copper complex behaved as a typical metallointercalator for dsDNA, The recognition properties of the copper complex to single‐stranded DNA (ssDNA) and dsDNA were assessed using surface‐based electrochemical methods and the results suggested that the complex had obviously different redox signals at ssDNA and dsDNA modified electrodes. The copper complex was further used as an electroactive indicator for the detection of cauliflower mosaic virus (CaMV) 35S promoter gene.     

Considerations for the quantitative transduction of hybridization of immobilized DNA

Immobilized single-stranded DNA (ssDNA) can be used as a selective ‘reagent’ to bind complementary DNA or RNA for applications such as the detection of pathogenic organisms, gene therapy agents and genetic mutations. The density of ssDNA on a surface will determine the charge density due to ionizable phosphate groups. Such a negatively charged interface will attract positive counter-ions from solution, which may result in a local ionic strength, pH and dielectric constant on the surface that is substantially different from that in bulk electrolyte solution. It is the local conditions which influence the thermodynamics of hybridization, and this can studied by the melt temperature (T_m) of double-stranded DNA (dsDNA). Experimental work and theoretical models have been used to examine whether hybridization reactions on a surface can cause dynamic changes in local charge density, and therefore, changes in selectivity and drift in calibration for quantitative analysis. Organosilane chemistry has been used to covalently immobilize hexaethylene glycol linkers and to control the subsequent density of dT₂₀ that was prepared by automated synthesis. Fiber-optic biosensors based on fused silica that was coated with DNA were used in a total internal reflection fluorescence instrument to determine T_m from the dissociation of duplexes of fluorescein-labeled dA₂₀ : dT₂₀. The experimental results suggest that the thermodynamic stability of duplexes that are immobilized on a surface is dependent on the density of immobilized DNA and on the extent of hybridization of DNA. The experimental results show that the thermodynamic stability of immobilized dsDNA is significantly different than that of dsDNA in bulk solution, and include observations of the variation of enthalpy at different ionic strengths, asymmetry in the melt curves, and the possibility of a reduced dielectric constant within a DNA layer relative to that in bulk solution.     

Selective interactions of a few acridinium derivatives with single strand DNA: study of photophysical and DNA binding interactions

Novel acridinium derivatives 1-3, wherein steric factors have been varied systematically through substitution at the ninth position of the acridinium ring, were synthesized and their interactions with single strand and double strand DNA have been investigated through photophysical, biophysical, and microscopic techniques. The acridinium derivative 1 exhibited quantitative fluorescence yields (phi f approximately =1) and high lifetime of 35 ns, while significantly lower fluorescence yields of 0.11 and 0.02 and lifetimes of 3.5 and 1.2 ns were observed for 2 and 3, respectively. The derivatives 1 and 2 having 2-methylphenyl and 2,4-dimethylphenyl substituents at the ninth position of the acridinium ring showed selective interactions with single strand DNA (ssDNA) with association constants of KssDNA = 6.3-6.6 x 10(4) M(-1), while negligible interactions were observed with double strand DNA (dsDNA). In contrast, the derivative 3 with 2,6-dimethylphenyl substitution showed negligible interactions with both ssDNA and dsDNA. Studies with a series of 19-mer oligonucleotides indicate that these derivatives exhibit significant selectivity for the sequences rich in guanosine (ca. 3-fold) as compared to the cytosine-rich sequences. These derivatives with high water solubility and the ability to distinguish between ssDNA and dsDNA through changes in fluorescence emission can be used as fluorescent probes for understanding the role of ssDNA in various biological processes and to study various DNA-ligand interactions.     

Monitoring human telomere DNA hybridization and G-quadruplex formation using gold nanorods

A facile and multi-response strategy for studying the transformations of human telomere DNA from single strand (ss) to double strand (ds) and G-quadruplex has been established by using positively charged gold nanorod (AuNR) as an optical label. The conformation change information of the telomere DNA was transferred into multiple optical signals, including changes in fluorescence emission, near infrared (NIR) absorption, plasma resonance light scattering (PRLS) and dynamic light scattering (DLS) response. The formations of dsDNA and G-quadruplex DNA induced fluorescence quenching of dye on DNA, and were accompanied by the intensity decrease and blue shift of the longitudinal absorption peak of AuNRs. Meanwhile, PRLS and DLS results revealed slightly increased AuNR aggregation due to increased charge density of dsDNA and G-quadruplex DNA as compared to ssDNA. Control experiment suggests that the AuNR-based assay is highly sequence specific; and the high sensitivity allows the study of human telomere DNA at a concentration as low as 58 nM.     

以乙二胺为手臂分子制备的DNA修饰电极及其伏安性能

Carboxyl was formed on the surface of glassy carbon electrode(GCE) by electrochemical oxidation. Ethylenediamine(En) was used as the arm molecule to link carboxyl with dsDNA using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) as the activators to prepare dsDNA modified electrode(dsDNA/En/GCE). It was shown that dsDNA couM be covalently immobilized on the surface of GCE. ssDNA modified electrode(ssDNA/En/GCE) was obtained via the thermal denaturation of dsDNA/En/GCE. The dsDNA/En/GCE and ssDNA/En/GCE were characterized by voltammetry with methylene blue(MB) as the indicator. The results indicated that the currents of the redox peaks of MB at ssDNA/En/GCE were larger than those at dsDNA/En/GCE, and the currents of the redox peaks at En/GCE were the smallest. The peak-currents of MB at the DNA modified electrode had good reproducibility after multi-denaturation and hybridization cycles.