Detection of mercury and phenylmercury ions using DNA-based fluorescent probe

A DNA probe labeled with a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) quencher and a carboxyfluorescein (FAM) donor at its 5'- and 3'-termini can be used for the detection of Hg(2+) ions and phenylmercury ions (PhHg(+)). This DNA probe possesses a random coil structure that changes into a hairpin-like structure upon binding Hg(2+) and PhHg(+) ions. As a result, the fluorescence of the FAM unit decreased through quenching between the donor and the quencher. In the presence of ethylenediaminetetraacetic acid (EDTA), the DNA probe allowed the selective detection of PhHg(+) ions at concentrations as low as 70.0 nM, mainly as a result of T-Hg(2+)-T coordination and π-π stacking between the Ph unit and DNA bases. A linear correlation existed between the fluorescence intensity and the concentration of PhHg(+) ions over the range from 0.10 to 1.0 μM (R(2) = 0.99). After acid hydrolysis and neutralization of the samples, all of the mercury species are converted to Hg(2+) ions, allowing us to use the DNA-based probe to determine the concentrations of total mercury species at the nM level. The practicality of this probe has been validated by the analyses of pond water and fish samples, showing its advantages of sensitivity, selectivity, and simplicity.     

Hg(II) ion specifically binds with T:T mismatched base pair in duplex DNA

Metal-mediated base pair formation, resulting from the interaction between metal ions and artificial bases in oligonucleotides, has been developed for its potential application in nanotechnology. We have recently found that the T:T mismatched base pair binds with Hg(II) ions to generate a novel metal-mediated base pair in duplex DNA. The thermal stability of the duplex with the T-Hg-T base pair was comparable to that of the corresponding T:A or A:T. The novel T-Hg-T base pair involving the natural base thymine is more convenient than the metal-mediated base pairs involving artificial bases due to the lack of time-consuming synthesis. Here, we examine the specificity and thermodynamic properties of the binding between Hg(II) ions and the T:T mismatched base pair. Only the melting temperature of the duplex with T:T and not of the perfectly matched or other mismatched base pairs was found to specifically increase in the presence of Hg(II) ions. Hg(II) specifically bound with the T:T mismatched base pair at a molar ratio of 1:1 with a binding constant of 10(6) M(-1), which is significantly higher than that for nonspecific metal ion-DNA interactions. Furthermore, the higher-order structure of the duplex was not significantly distorted by the Hg(II) ion binding. Our results support the idea that the T-Hg-T base pair could eventually lead to progress in potential applications of metal-mediated base pairs in nanotechnology.     

Binding of metal ions by pyrimidine base pairs in DNA duplexes

Pyrimidine base pairs in DNA duplexes selectively capture metal ions to form metal ion-mediated base pairs, which can be evaluated by thermal denaturation, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. In this critical review, we discuss the metal ion binding of pyrimidine bases (thymine, cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA duplexes. Thymine-thymine (T-T) and cytosine-cytosine (C-C) base pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the metallo-base pairs, T-Hg(II)-T and C-Ag(I)-C, are formed in DNA duplexes. The metal ion binding properties of the pyrimidine-pyrimidine pairs can be changed by small chemical modifications. The binding selectivity of a metal ion to a 5-fluorouracil-5-fluorouracil pair in a DNA duplex can be switched by changing the pH of the solution. Two silver ions bind to each thiopyrimidine-thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized. Oligonucleotides containing these bases are commercially available and can readily be applied in many scientific fields (86 references).     

Electropolymerized surface ion imprinting films on a gold nanoparticles/single-wall carbon nanotube nanohybrids modified glassy carbon electrode for electrochemical detection of trace mercury(II) in water

Electrochemical detection of Hg(II) using a electropolymerized ion imprinting poly(2-mercaptobenzothiazole) films at the surface of gold nanoparticles/single-walled carbon nanotube nanohybrids modified glassy carbon electrode (PMBT/AuNPs/SWCNTs/GCE) is described for the first time. The Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE sensor exhibits larger binding to functionalized capacity, larger affinity, faster binding kinetics and higher selectivity to template Hg(II). The differential pulse anodic stripping voltammetry (DPASV) response of the Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE sensor to Hg(II) is ca. 3.7- and 10.5-fold higher than that at the non-imprinted PMBT/AuNPs/SWCNTs/GCE and the imprinted PMBT/AuNPs/GCE, respectively, and the detection limit for Hg(II) is 0.08 nM (S/N = 3, which is well below the guideline value given by the World Health Organization) and a sensitivity of 0.749 μA nM⁻¹ was obtained. Excellent wide linear range (0.4–96.0 nM) and good repeatability (relative standard deviation of 2.6%) were obtained for Hg(II). The interference experiments show that Ag(I), Pb(II), Cd(II), Zn(II) and Cu(II) had little or no influence on the Hg(II) signal. These values, particularly the high sensitivity and excellent selectivity in contrast to the values reported previously in the area of electrochemical Hg(II) detection, demonstrate the analytical performance of the Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE toward Hg(II) is superior to the existing electrodes and could be used for efficient determination of Hg(II) in natural water samples.     

Influence of Mg2+ and Cd2+ on the interaction between sparfloxacin and calf thymus DNA

Mg(2+) and Cd(2+) have different binding capacity to sparfloxacin, and have different combination modes with calf thymus DNA. Selecting these two different metal ions, the influence of them on the binding constants between SPFX and calf thymus DNA, as well as the related mechanism have been studied by using absorption and fluorescence spectroscopy. The result shows that Cd(2+) has weak binding capacity to SPFX in the SPFX-Cd(2+) binary system, but can decrease the binding between SPFX and DNA obviously in SPFX-DNA-Cd(2+) ternary system. Mg(2+) has strong binding capacity to SPFX. It can increase the binding between SPFX and DNA at concentrations <0.01 mM, and decrease the binding between them at concentrations >0.01 mM. Referring to the different modes of Mg(2+) and Cd(2+) binding to DNA, the mechanism of the influence of metal ions on the binding between SPFX and DNA has been proposed. SPFX can directly bind to DNA by chelating DNA base sites. If a metal ion at certain concentration mainly binds to DNA bases, it can decrease the binding constants between SPFX and DNA through competing with SPFX. While if a metal ion at certain concentration mainly binds to phosphate groups of DNA, it can increase the binding constants by building a bridge between SPFX and DNA. The influence direction of metal ions on the binding between quinolone and DNA relays on their binding ratio of affinity for bases to phosphate groups on DNA. Our result supports Palumbo's conclusion that the binding between SPFX and the phosphate groups is the precondition for the combination between SPFX and DNA, which is stabilized through stacking interactions between the condensed rings of SPFX and DNA bases.     

聚胸腺嘧啶单链DNA-CuNCs荧光增强法用于汞离子的高灵敏检测

基于Hg~(2+)与DNA中胸腺嘧啶(T)结合的高度特异性和DNA铜纳米簇的荧光增强性质,构建了一种简便、灵敏检测汞离子的新方法.当Hg~(2+)存在时,聚T单链DNA(P1)通过T-Hg~(2+)-T特异性结合形成双链DNA,Cu~(2+)经抗坏血酸钠还原后生成的中间体Cu+与双链DNA螺旋结构间的氢键部分有强的结合力,促使Cu0附着聚集在双链DNA上形成铜纳米簇,导致体系荧光增强,从而实现对汞离子的高灵敏检测.体系荧光强度与Hg~(2+)浓度的对数值成正比,对Hg~(2+)检测的线性范围为1.0 nmol/L~10μmol/L,检出限达0.4 nmol/L,对湖水样品中Hg~(2+)检测的回收率达到97.2%~106.6%.与传统方法相比,该方法具有无需标记、检出限低及选择性好等优点,可用于环境水体中汞离子的测定.     

Sequence-specific self-stitching motif of short single-stranded DNA on a single-walled carbon nanotube

The DNA-single-walled carbon nanotube (SWCNT) hybrid molecule has attracted significant attention recently for its ability to disperse and sort SWCNTs according to their chirality. Key for utilizing their unique properties is an understanding of the structure of DNA adsorbed on the SWCNT surface, which we study here using molecular simulations. Using replica exchange molecular dynamics (REMD), we explore equilibrium structures formed by single strands of 12-mer oligonucleotides, of varying sequence, adsorbed on a (6,5)-SWCNT. We find a consistent motif in which the DNA strand forms a right-handed helical wrap around the SWCNT, stabilized by "stitches" (hydrogen bonding between distant bases) to itself. Variability among equilibrium populations of DNA self-stitched structures was observed and shown to be directly influenced by DNA sequence and composition. Competition between conformational entropy and hydrogen bonding between bases is predicted to be responsible for the formation of random versus stitched configurations.     

基于T-T碱基错配的荧光传感器特异性检测汞离子

在本文中,我们研制了一种基于T-T碱基错配特异性键合汞离子的荧光传感器用于汞离子的检测。该传感器由两条分别标记了荧光基团（F）和淬灭基团（Q）的DNA探针组成,并且含有两对用于结合汞离子的T-T错配碱基。当汞离子存在时,两条探针之间形成T-Hg2+-T结构,作用力增强,从而拉近了荧光基团与淬灭基团之间的距离,发生能量转移,使荧光信号在一定程度上被淬灭。在优化的条件下,我们使用该传感器对汞离子进行检测,动力学响应范围为50nM到1000nM,线性相关方程为y= 5281.13 - 1650.56 lg[Hg2+] ( R2 = 0.985),检测下限为79nM。此外,我们还考察了该传感器的选择性,当用其它干扰离子（浓度都为1.0µM）代替待测离子进行实验时,没有发生明显的荧光淬灭,说明该传感器具有较高的选择性。该传感器的构建为汞离子的检测提供了一条快速、简便的新途径。     

Layer-by-layer fabrication and characterization of DNA-wrapped single-walled carbon nanotube particles

Carbon nanotubes have been proposed as support materials for numerous applications, including the development of DNA sensors. One of the challenges is the immobilization of DNA or other biological molecules on the sidewall of carbon nanotubes. This paper introduces a new fabrication of DNA-carbon nanotubes particles using the layer-by-layer (LBL) technique on single-walled carbon nanotubes (SWCNTs). Poly(diallyldimethylammonium) (PDDA), a positively charged polyelectrolyte, and DNA as a negatively charged counterpart macromolecule are alternatively deposited on the water-soluble oxidized SWCNTs. Pure DNA/PDDA/SWCNTs particles can be prepared and separated by simple unltracentrifugation. The characterization of DNA/PDDA/SWCNTs particles was carried out by scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible spectroscopy, Raman spectroscopy, and thermogravimetric analysis (TGA). An electrode modified by the DNA/PDDA/SWCNTs particles shows a dramatic change of the electrochemical signal in solutions of tris(2,2'-bipyridyl)ruthenium(II) ((Ru(bpy)(3)2+) as a reporting redox probe. A preliminary application of the DNA-modified carbon nanotubes in the development of DNA sensors used in the investigation of DNA damage by nitric oxide is presented.     

Lower rim 1,3-di{4-antipyrine}amide conjugate of calix[4]arene: synthesis, characterization, and selective recognition of Hg2+ and its sensitivity toward pyrimidine bases

The structurally characterized lower rim 1,3-di{4-antipyrine}amide conjugate of calix[4]arene (L) exhibits high selectivity toward Hg(2+) among other biologically important metal ions, viz., Na(+), K(+), Ca(2+), Mg(2+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ag(+) as studied by fluorescence, absorption, and ESI MS. L acts as a sensor for Hg(2+) by switch-off fluorescence and exhibits a lowest detectable concentration of 1.87 ± 0.1 ppm. The complex formed between L and Hg(2+) is found to be 1:1 on the basis of absorption and fluorescence titrations and was confirmed by ESI MS. The coordination features of the mercury complex of L were derived on the basis of DFT computations and found that the Hg(2+) is bound through an N(2)O(2) extending from both the arms to result in a distorted octahedral geometry with two vacant sites. The nanostructural features such as shape and size obtained using AFM and TEM distinguishes L from its Hg(2+) complex and were different from those of the simple mercuric perchlorate. L is also suited to sense pyrimidine bases by fluorescence quenching with a minimum detection limit of 1.15 ± 0.1 ppm in the case of cytosine. The nature of interaction of pyrimidine bases with L has been further studied by DFT computational calculations and found to have interactions through a hydrogen bonding and NH-π interaction between the host and the guest.     

Colorimetric assay for mercury (II) based on mercury-specific deoxyribonucleic acid-functionalized gold nanoparticles

A colorimetric nanoprobe-mercury-specific DNA-functionalized gold nanoparticles (Au-MSD) was developed for sensing Hg(2+). The new mercury-sensing concept relies on measuring changes in the inhibition of "non-crosslinking" aggregation of Au-MSD-induced by the folding of mercury-specific DNA strand through the thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination. In the absence of Hg(2+), a high concentration of MgCl(2) (50 mM) results in a rapid aggregation of Au-MSD because of the removal of charge repulsion. When Hg(2+) is present, the particles remain stable due to the folding of MSD functionalized on the particle surface. The assay enables the colorimetric detection of Hg(2+) in the concentration range of 0.1-10 μM Hg(2+) ions with a detection limit of 60 nM, and allows for the selective discrimination of Hg(2+) ions from the other competitive metal ions. Toward the goal for practical applications, the sensor was further evaluated by monitoring Hg(2+) in fish tissue samples.     

Control over surface DNA density on gold nanoparticles allows selective and sensitive detection of mercury(II)

We have developed a new highly selective and sensitive technique for the detection of Hg(2+) using DNA-functionalized gold nanoparticles (Au NPs) and OliGreen. This system is the first that allows the detection of Hg(2+) based on the release of DNA molecules, induced by conformational changes on Au NP surfaces, and its sensitivity is highly dependent upon surface DNA density. When Hg(2+) ions interact with the thymidine units of the DNA molecules bound to the Au NPs through Au-S bonds, the conformations of these DNA derivatives change from linear to hairpin structures, causing the release of some of the DNA molecules from the surface of the Au NPs into the bulk solution to react with OliGreen. The fluorescence of OliGreen-DNA complexes increased with increasing concentration of Hg(2+), and Hg(2+) could be detected at concentrations as low as 25 nM. A linear correlation existed between the fluorescence intensity and the concentration of Hg(2+) over the range 0.05-2.5 microM (R(2) = 0.98). This simple and cost-effective probe was applied to determine the spiked Hg(2+) in the pond samples; the recoveries (96-102%) suggested low matrix interference and high sensitivity.     

A simple and sensitive fluorescent sensing platform for Hg²+ ions assay based on G-quenching

In this work, a novel fluorescence biosensor was demonstrated for detection of Hg(2+) ions with relatively high selectivity and sensitivity. The sensing scheme was based on G-quenching induced by Hg(2+) ions. In the presence of Hg(2+) ions, the single-stranded signal probe which has carboxylfluorescein (FAM) and guanine segment at its 5' and 3' ends, respectively, folded into duplex-like structure via the Hg(2+)-mediated coordination of T-Hg(2+)-T base pairs. It brought guannine segment close to the dye and caused a remarkable decrease of fluorescence signal. The sensor showed a sensitive response to Hg(2+) ions in a concentration range from 0.5 to 10 μM, and a detection limit of 0.5 nM was given. This homogeneous system required only a single-labeled oligonucleotide, operated by concise procedures, and possessed comparable sensitivity as previous approaches. Furthermore, the sensor exhibits a great perspective for future practical applications.     

Oligonucleotide-functionalized gold nanoparticles-enhanced QCM-D sensor for mercury(II) ions with high sensitivity and tunable dynamic range

A quartz crystal microbalance with dissipation monitoring (QCM-D) sensor was developed for highly sensitive and specific detection of mercury(II) ions (Hg(2+)) with a tunable dynamic range, using oligonucleotide-functionalized gold nanoparticles (GNPs) for both frequency and dissipation amplification. The fabrication of the sensor employed a 'sandwich-type' strategy, and formation of T-Hg(2+)-T structures in linker DNA reduced the hybridization of the GNPs-tagged DNA on the gold electrode, which could be used as the molecular switch for Hg(2+) sensing. This QCM-D mercury sensor showed a linear response of 10-200 nM, with detection limits of 4 nM and 7 nM for frequency and dissipation measurements, respectively. Moreover, the dynamic range of the sensor could be tuned by simply altering the concentration of linker DNA without designing new sensors in the cases where detection of Hg(2+) at different levels is required. This sensor afforded excellent selectivity toward Hg(2+) compared with other potential coexisting metal ions. The feasibility of the sensor was demonstrated by analyzing Hg(2+)-spiked tap- and lake-water samples with satisfactory recoveries. The proposed approach extended the application of the QCM-D system in metal ions sensing, and could be adopted for the detection of other analytes when complemented with the use of functional DNA structures.     

Designing,Structural Elucidation and Comparison of the Cleavage Ability of Metal Complexes Containing Tetradentate Schiff Bases 1

New N₂O₂ donor type Schiff bases have been designed and synthesized by condensing acetylaceto-4-aminoantipyrine/acetoacetanilido-4-aminoantipyrine with 2-amino benzoic acid in ethanol. Solid metal complexes of the Schiff bases with Cu(II), Ni(II), Co(II), Mn(II), Zn(II), VO(IV), Hg(II) and Cd(II) metal ions were synthesized and characterized by elemental analyses, magnetic susceptibility, molar conduction, FAB Mass, IR, UV-Vis., ¹H NMR, and ESR spectral studies. The data show that the complexes have a composition of the ML type. The UV-Vis., magnetic susceptibility, and ESR spectral data of the complexes suggest a square planar geometry around the central metal ion, except for VO(IV) complexes, which have square-pyramidal geometry. The redox behavior of copper and vanadyl complexes has been studied by cyclic voltammetry. The nuclease activity of the above metal complexes shows that the complexes cleave DNA through redox chemistry. In the presence of H₂O₂, all the complexes are capable of cleaving calf thymus DNA plasmids, in order to compare the cleavage efficiency of all metal complexes in the two different ligand environments. In this assay, Cu(II), Ni(II), Co(II), and Zn(II) exhibit more cleavage efficiency than other metal ions. This article was submitted by the authors in English.     

Selective and sensitive ratiometric detection of Hg(II) ions using a simple amino acid based sensor

Synthesis of a novel pyrene derivative sensor (Py-Met) based on amino acid and its fluorescent behavior for Hg(II) in water was investigated. Upon Hg(II) binding, the Py-Met-bearing sulfonamide group exhibited a considerable excimer emission at 480 nm along with a decrease of monomer emission at 383 nm. Py-Met allows a selective and sensitive ratiometric detection of Hg(II) without any interference from other metal ions.     

Visual scanometric detection of DNA through silver enhancement regulated by gold-nanoparticle aggregation with a molecular beacon as the trigger

A convenient and label-free scanometric approach for DNA assay was designed by integrating a metal-ion-mediated conformational molecular beacon (MB) and silver-signal amplification regulated by gold-nanoparticle (AuNP) aggregation. The strategy was based on displacing the interaction between the target DNA sequence and a competitor Hg(2+) ion with a link DNA sequence. In the absence of the target DNA sequence, a link DNA sequence interacted with the Hg(2+) ions, thus forming an inactive cyclic conformation of the MB. This result led to the poor aggregation of polyadenosine-functionalized AuNPs (A-AuNP). In the presence of a target DNA sequence with a stronger affinity than that of the competitor, hybridization between the link DNA and target DNA sequences turned on the trigger. The polythymidine end of the resulting linear duplex structure could react with A-AuNP, thus leading to a cross-linking aggregation. This aggregation weakened AuNP-catalyzed silver enhancement on a spot substrate. Further, by using scanometric detection, the concentration of the target DNA sequence could be conveniently read out within a linear range from 1.0 to 30 nM. Interestingly, in the same amount of Hg(2+) ions, one-base mismatched DNA showed only 22% of the relative gray-scale intensity for the target DNA sequence at the same concentration, thus indicating good specificity. The designed approach, with the help of the ion-mediated conformational MB, was simple, cost effective, adaptable, and convenient and provided significant potential applications in clinical analysis.     

稀土及其配合物对蛇毒磷脂酶A_2活性的影响

分别研究了三价稀土离子 (La3 + ,Eu3 + ,Dy3 + ,Yb3 + )、二乙三胺五乙酸及其衍生物二乙三胺五乙酸 -双二甲酰胺 ,二乙三胺五乙酸 -双 (异烟肼 )与稀土离子的配合物以及Tb -谷氨酰胺配合物对蛇毒磷脂酶A2 活性的影响 .浓度低于 <3μmol/L的稀土离子可以激活磷脂酶A2 ,浓度大于 5 μmol/L后稀土离子对酶活性表现出抑制作用 ;外源Ca2 + 离子的加入可以缓解稀土离子对酶活性的抑制作用 ,表明稀土离子和钙离子是竞争性地结合在酶的活性部位 ;稀土离子和二乙三胺五乙酸及其衍生物的配合物对酶活性没有明显影响 ;Tb -谷氨酰胺在浓度大于 10 μmol/L后开始抑制酶的活性     

Synthesis of Nucleobase‐Functionalized Carbon Nanotubes and Their Hybridization with Single‐Stranded DNA

For the first time ssDNA (25‐aptamer of mixed dA, dT, dG, and dC) was wrapped around functionalized single‐walled carbon nanotubes (SWCNTs), whose external surfaces were attached to multiple triazole‐(ethylene glycol)‐dA ligands. This method of hybridization involved the formation of hydrogen bonds between dT of ssDNA and dA of functionalized SWCNTs. It deviates from the reported π–π stacking between the nucleobases of DNA and the external sidewalls of nanotubes. The structural properties of the functionalized SWCNTs and its ssDNA complex were characterized by spectroscopic (including CD and Raman), thermogravimetric, and microscopic (TEM) methods. The results thus obtained establish a new platform of DNA delivery by use of nanotubes as a new vehicle with great potential in biomedical applications and drug development.     

Visual and on-site detection of mercury(II) ions on lateral flow strips using DNA-functionalized gold nanoparticles

A test strip for detection of Hg(2+) in aqueous solution based on the DNA-functionalized gold nanoparticles (DNA-AuNPs) was developed and evaluated. When Hg(2+) ions were introduced, the biotinylated DNA(2) hybridized with thiolated DNA(1) functionalized on the AuNPs (DNA(1)-AuNPs) to form mismatch complexes through thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination. The formed mismatch complexes and excess DNA(1)-AuNPs could be captured on the test line formed by streptavidin and the control line formed by DNA(3)-BSA, respectively. Two red lines appeared due to the accumulation of AuNPs, enabling visual detection of Hg(2+) with a detection limit of about 6 nM. The assay results can be obtained within 5 min. The results show that the test strip has excellent sensitivity and selectivity for detection of Hg(2+); thus it holds a great potential for rapid, on-site and real time detection of Hg(2+).