Metal-dependent global folding and activity of the 8-17 DNAzyme studied by fluorescence resonance energy transfer

The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ > Zn2+ >Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.     

基于脱氧核酶的新型铅离子荧光探针

将荧光猝灭基团修饰的17E脱氧核酶(17E DNAzyme)与荧光基团修饰的底物链通过6个脱氧核苷酸相连, 得到了一种新型的对Pb2+敏感的荧光探针. 由于DNAzyme与底物链发生分子内杂交, 荧光基团与猝灭基团相互靠近, 导致荧光猝灭. 当Pb2+存在时, DNAzyme被激活, 底物链被切断后释放出荧光基团标记的DNA片段, 从而产生明显的荧光信号. 据此可在常温下快速检测Pb2+, 检测下限为10 nmol/L. 在Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Mg2+和Ni2+等多种二价金属离子中, 除Zn2+, Mn2+和Cd2+略有干扰外, 其它几种金属离子均无响应, 表明该荧光探针对Pb2+具有良好的选择性.     

Functionalized gold nanoparticles as phosphorescent nanomaterials and sensors

Ligand-capped gold nanoparticles were synthesized by capping monothiol derivatives of 2,2'-dipyridyl onto the surface of Au nanoparticles (Au-BT). The average size of the metal core is around 4 nm, with a shell of approximately 340 bipyridine ligands around the Au nanoparticle. The high local concentration of the chelating ligands ( approximately 5 M) around the Au nanoparticle makes these particles excellent ion sponges, and their complexation with Eu(III)/Tb(III) ions yields phosphorescent nanomaterials. Absorption spectral studies confirm a 1:3 complexation between Eu(III)/Tb(III) ions and bipyridines, functionalized on the surface of Au nanoparticles. The red-emitting Au-BT:Eu(III) complex exhibits a long lifetime of 0.36 ms with six line-like emission peaks, whereas the green-emitting Au-BT:Tb(III) complex exhibits a lifetime of 0.7 ms with four line-like emission peaks. These phosphorescent nanomaterials, designed by linking BT:Eu(III) complexes to Au nanoparticles, were further utilized as sensors for metal cations. A dramatic decrease in the luminescence was observed upon addition of alkaline earth metal ions (Ca(2+), Mg(2+)) and transition metal ions (Cu(2+), Zn(2+), Ni(2+)), resulting from an isomorphous substitution of Eu(III) ions, whereas the luminescence intensity was not influenced by the addition of Na(+) and K(+) ions. Direct interaction of bipyridine-capped Au nanoparticles with Cu(2+) ions brings the nanohybrid systems closer, leading to the formation of three-dimensional superstructures. Strong interparticle plasmon interactions were observed in these closely spaced Au nanoparticles.     

基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复;同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.     

Investigation of Ion-Binding Properties of Synthetic Polyelectrolytes with The Tb[III] Luminescence Probe

The binding properties of trivalent metal ions to polyelectrolytes were investigated through the use of Tb(III) luminescence studies. The condensation of Tb(III) with the homopolymers poly(acrylic acid) and poly(methacrylic acid) was studied in detail. In addition, the 1 : 1 copolymers of maleic acid with ethylene, isobutene, and 2,4,4-trimethyl-1-pentene were also examined. The emission intensity of the 305 nm Tb(III) hypersensitive excitation band was found to correlate with the size of the alkyl group on the polymer chains. Tb(III) luminescence lifetime studies indicated that the metal ion binding site was equivalent over a wide range of Tb(III)/polymer ratios. The number of solvent molecules coordinated by Tb(III) in the various polymer complexes was determined and found to range between 3.5 and 4 molecules of water of hydration.     

The importance of stereochemically active lone pairs for influencing Pb(II) and As(III) protein binding

The toxicity of heavy metals, which is associated with the high affinity of the metals for thiolate rich proteins, constitutes a problem worldwide. However, despite this tremendous toxicity concern, the binding mode of As(III) and Pb(II) to proteins is poorly understood. To clarify the requirements for toxic metal binding to metalloregulatory sensor proteins such as As(III) in ArsR/ArsD and Pb(II) in PbrR or replacing Zn(II) in δ-aminolevulinc acid dehydratase (ALAD), we have employed computational and experimental methods examining the binding of these heavy metals to designed peptide models. The computational results show that the mode of coordination of As(III) and Pb(II) is greatly influenced by the steric bulk within the second coordination environment of the metal. The proposed basis of this selectivity is the large size of the ion and, most important, the influence of the stereochemically active lone pair in hemidirected complexes of the metal ion as being crucial. The experimental data show that switching a bulky leucine layer above the metal binding site by a smaller alanine residue enhances the Pb(II) binding affinity by a factor of five, thus supporting experimentally the hypothesis of lone pair steric hindrance. These complementary approaches demonstrate the potential importance of a stereochemically active lone pair as a metal recognition mode in proteins and, specifically, how the second coordination sphere environment affects the affinity and selectivity of protein targets by certain toxic ions.     

Enhanced metal ion selectivity of 2,9-di-(pyrid-2-yl)-1,10-phenanthroline and its use as a fluorescent sensor for cadmium(II)

The metal ion complexing properties of the ligand DPP (2,9-di-(pyrid-2-yl)-1,10-phenanthroline) were studied by crystallography, fluorimetry, and UV-visible spectroscopy. Because DPP forms five-membered chelate rings, it will favor complexation with metal ions of an ionic radius close to 1.0 A. Metal ion complexation and accompanying selectivity of DPP is enhanced by the rigidity of the aromatic backbone of the ligand. Cd2+, with an ionic radius of 0.96 A, exhibits a strong CHEF (chelation enhanced fluorescence) effect with 10(-8) M DPP, and Cd2+ concentrations down to 10(-9) M can be detected. Other metal ions that cause a significant CHEF effect with DPP are Ca2+ (10(-3) M) and Na+ (1.0 M), whereas metal ions such as Zn2+, Pb2+, and Hg2+ cause no CHEF effect with DPP. The lack of a CHEF effect for Zn2+ relates to the inability of this small ion to contact all four donor atoms of DPP. The structures of [Cd(DPP)2](ClO4)2 (1), [Pb(DPP)(ClO4)2H2O] (2), and [Hg(DPP)(ClO4)2] (3) are reported. The Cd(II) in 1 is 8-coordinate with the Cd-N bonds to the outer pyridyl groups stretched by steric clashes between the o-hydrogens on these outer pyridyl groups and the central aromatic ring of the second DPP ligand. The 8-coordinate Pb(II) in 2 has two short Pb-N bonds to the two central nitrogens of DPP, with longer bonds to the outer N-donors. The coordination sphere around the Pb(II) is completed by a coordinated water molecule, and two coordinated ClO4(-) ions, with long Pb-O bonds to ClO4(-) oxygens, typical of a sterically active lone pair on Pb(II). The Hg(II) in 3 shows an 8-coordinate structure with the Hg(II) forming short Hg-N bonds to the outer pyridyl groups of DPP, whereas the other Hg-N and Hg-O bonds are rather long. The structures are discussed in terms of the fit of large metal ions to DPP with minimal steric strain. The UV-visible studies of the equilibria involving DPP and metal ions gave formation constants that show that DPP has a higher affinity for metal ions with an ionic radius close to 1.0 A, particularly Cd(II), Gd(III), and Bi(III), and low affinity for small metal ions such as Ni(II) and Zn(II). The complexes of several metal ions, such as Cd(II), Gd(III), and Pb(II), showed an equilibrium involving deprotonation of the complex at remarkably low pH values, which was attributed to deprotonation of coordinated water molecules according to: [M(DPP)(H2O)]n+ <==> [M(DPP)(OH)](n-1)+ + H+. The tendency to deprotonation of these DPP complexes at low pH is discussed in terms of the large hydrophobic surface of the coordinated DPP ligand destabilizing the hydration of coordinated water molecules and the build-up of charge on the metal ion in its DPP complex because of the inability of the coordinated DPP ligand to hydrogen bond with the solvent.     

Heterodinuclear metal arrangement in a flat macrocycle with two chemically-equivalent metal chelating sites

A phenanthroline-based macrocycle 1 has been newly developed which has two chemically equivalent metal chelating sites within the spatially restricted cavity for dinuclear metal arrangement. The macrocycle 1 reacts with Zn(CF(3)CO(2))(2) or ZnCl(2) to form homodinuclear Zn(II)-complexes. A single-crystal X-ray structural analysis of the resulting Zn(2)1(CF(3)CO(2))(4) determined the complex structure in which two Zn(II) ions are bound by two phenanthroline sites and two CF(3)CO(2)(-) ions bind to each Zn(II) ion in a tetrahedral geometry. Similarly, a homodinuclear Cu(I)-macrocycle was formed from 1 and Cu(CH(3)CN)(4)BF(4). Notably, from 1 and an equimolar mixture of Cu(CH(3)CN)(4)BF(4) and Zn(CF(3)CO(2))(2), a heterodinuclear Cu(I)-Zn(II)-macrocycle was exclusively formed in high yield (>90%) because of the relatively low stability of the dinuclear Cu(I)-macrocycle. A heterodinuclear Ag(I)-Zn(II)-macrocycle was similarly formed with fairly high selectivity from a mixture of Ag(I) and Zn(II) ions. Such selective heterodinuclear metal arrangement was not observed with other combinations of M-Zn(II) (M = Li(I), Mg(II), Pd(II), Hg(II), La(III), and Tb(III)).     

Lanthanide luminescence sensing of copper and mercury ions using an iminodiacetate-based Tb(III)-cyclen chemosensor

The design, synthesis and photophysical evaluation of 1.Tb.Na, a Tb(III)-cyclen-based sensor, possessing a phenyl iminodiacetate-based receptor, for the selective detection of Cu(II) and Hg(II) ions in water is demonstrated. Sensitisation of the Tb(III) ⁵D₄ excited state was achieved by excitation of the phenyl receptor, which in water gave rise to a characteristic time-delayed and line-like Tb(III) emission. The Tb(III) emission was shown to be pH independent over the physiological pH window. The changes in the Tb(III) emission were monitored by carrying out metal titrations using various groups I, II and transition metal ions. Of these, only the titrations of Cu(II) and Hg(II) gave rise to modulations in the Tb(III) emission; resulting in quenching in the Tb(III) emission by ca. 65% and 40%, respectively.     

Gold nanorods-based FRET assay for sensitive detection of Pb2+ using 8-17DNAzyme

In this paper, we have reported a sensitive assay for fluorescence "turn-on" detection of Pb(2+) in aqueous solutions based on FRET between gold nanorods (GNRs) and the FAM-labeled substrate strand of 8-17DNAzyme. The fluorescence of the FAM-labeled substrate strand is quenched when 8-17DNAzyme is adsorbed on GNRs surface through electrostatic interaction. In the presence of lead ions, the fluorescence is restored due to the decrease of FRET efficiency caused by the specific cleavage of the FAM-labeled substrate strand by the enzyme, which weakens the electrostatic interaction between the GNRs and short FAM-labeled DNA fragment. The interference of eleven common metal ions has been tested, indicating that Pb(2+) can be selectively detected. This method exhibits a high sensitivity for Pb(2+) with a detection limit of 61.8 pM and a linear range from 0.1 nM to 100 nM. It is a simple, sensitive, and selective method for Pb(2+) detection. Moreover, this sensing system obtained satisfying results for Pb(2+) detection in tap water samples.     

Sensitization of the 4f-Luminescence of Terbium with Copper Ions and Its Analytical Application

The intense sensitization of terbium luminescence with copper(II) ions was observed in the Tb–Cu mixed-metal complex of 1.5-bis(2-hydrazinocarbophenoxy)-3-oxapentane (HCPhO) with the ratio Tb : Cu : HCPhO = 1 : 1 : 1. Other d-block metals produced no sensitizing effect. The effect observed was used for the luminescence determination of trace copper in the presence of 1000-fold amounts of Mn(II), Fe(III), Co(II), Ni(II), Zn(II), and Cd(II). The accuracy of determining copper was verified by the analysis of Certified Reference Materials of zinc- and nickel-based alloys.     

DNAzyme based electrochemical sensors for trace uranium

We have developed a uranyl-specific DNAzyme that was immobilized on the surface of a gold electrode to give a highly sensitive and selective biosensor for uranyl ion. The typical DNAzyme system consisted of the RNA (rA) as the substrate (ADNA), and the other strand is the enzyme (TDNA) with a ferrocene (Fc). The presence of uranyl ion induces the cleavage of the DNA substrate strand at the rA position to form two fragments. The Fc unit thereby is released from the surface of the electrode, and this results in a decreased peak current. This electrochemical biosensor has a dynamic range from 2 nM to 14 nM of uranyl ion, with a detection limit at 1 nM. It exhibits high sensitivity and excellent selectivity over other metal ions, and thus represents a promising technique for simple, fast, on-site, and real-time electrochemical sensing of UO₂(II) ion. It also serves as a guide in choosing different methods for designing electrochemical sensors for other metal ions.

'Thiacalix[4]aniline' as a highly specific extractant for Au(III) and Pd(II) ions

Thiacalix[4]aniline (4), a cyclic tetramer of p-tert-butylaniline bridged with four sulfides, extracted Au(III) and Pd(II) ions specifically from acidic solutions among 41 metal ions including soft metal ions such as Hg(II), Cd(II), Zn(II), Pb(II), and Cu(II).     

Preparation and analytical properties of a chelating resin functionalized with 1-hydrazinophthalazine ligand

A poly[styrene-co-(divinylbenzene)] resin (XAD-4) functionalized with 1-hydrazinophthalazine ligand has been prepared and its analytical properties investigated. The pH dependence of sorption of metal ion on the resin has been determined for Cu(II), Ni(II), Co(II), Zn(II), Cd(II), Pb(II), Fe(III) and Cr(III). Trace amounts of these metal ions were quantitatively retained on the resin and recovered by eluting with 1 mol l(-1) hydrochloric acid. The resin was found to be selective for Fe(III) and its separation from other metal ions was carried out effectively. Metal ions concentrations were determined using AAS.     

Sorption behaviour of lanthanum(III), neodymium(III), terbium(III), thorium(IV) and uranium(VI) on Amberlite XAD-4 resin functionalized with bicine ligands

The complexing properties (capacity, pH effect, breakthrough curve) of a chelating resin, containing bicine ligands, were investigated for La(III), Nd(III), Tb(III), Th(IV) and U(VI). Trace amounts of these metal ions were quantitatively retained on the resin and recovered by eluting with 1 M hydrochloric acid. The capacity of the resin for La(III), Nd(III), Tb(III), Th(IV) and U(VI) was found to be 0.35, 0.40, 0.42, 0.25 and 0.38 mmol g(-1), respectively. Separation of U(VI) and Th(IV) from Ni(II), Zn(II), Co(II) and Cu(II) in a synthetic solution was carried out.     

Incorporation of a DNAzyme into Au-coated nanocapillary array membranes with an internal standard for Pb(ii) sensing

A Pb(ii)-specific DNAzyme has been successfully incorporated into Au-coated polycarbonate track-etched (PCTE) nanocapillary array membranes (NCAMs) by thiol-gold immobilization. Incorporation of the DNAzyme into the membrane provides a substrate-bound sensor using a novel internal control methodology for fluorescence-based detection of Pb(ii). A non-cleavable substrate strand, identical to the cleavable DNAzyme substrate strand except the RNA-base is replaced by the corresponding DNA-base, is used for ratiometric comparison of intensities. The cleavable substrate strand is labeled with fluorescein, and the non-cleavable strand is labeled with a red fluorophore (Cy5 or Alexa 546) for detection after release from the membrane surface. This internal standard based ratiometric method allows for real-time monitoring of Pb(ii)-induced cleavage, as well as standardizing variations in substrate size, solution detection volume, and monolayer density. The result is a Pb(ii)-sensing structure that can be stored in a prepared state for 30 days, regenerated after reaction, and detect Pb(ii) concentrations as low as 17 nM (3.5 ppb).     

Adsorption of heavy metal ions onto dithizone-anchored poly (EGDMA-HEMA) microbeads

The dithizone-anchored poly (EGDMA-HEMA) microbeads were prepared for the removal of heavy metal ions (i.e. cadmium, mercury, chromium and lead) from aqueous media containing different amounts of these ions (25-500 ppm) and at different pH values (2.0-8.0). The maximum adsorptions of heavy metal ions onto the dithizone-anchored microbeads from their solutions was 18.3, Cd(II); 43.1, Hg(II); 62.2, Cr(III) and 155.2 mg g(-1) for Pb(II). Competition between heavy metal ions (in the case of adsorption from mixture) yielded adsorption capacities of 9.7, Cd(II); 28.7, Hg(II); 17.6, Cr(III) and 38.3 mg g(-1) for Pb(II). The same affinity order was observed under non-competitive and competitive adsorption, i.e. Cr(III)>Pb(II)>Hg(II)>Cd(II). The adsorption of heavy metal ions increased with increasing pH and reached a plateaue value at around pH 5.0. Heavy metal ion adsorption from artificial wastewater was also studied. The adsorption capacities are 4.3, Cd(II); 13.2, Hg(II); 7.2, Cr(III) and 16.4 mg g(-1) for Pb(II). Desorption of heavy metal ions was achieved using 0.1 M HNO(3). The dithizone-anchored microbeads are suitable for repeated use (for more than five cycles) without noticeable loss of capacity.     

Using silica modified by Tiron for metal preconcentration and determination in natural waters by inductively coupled plasma atomic emission spectrometry

A new adsorbent is synthesized on the basis of silica consecutively modified by polyhexamethylene guanidine and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) for the group preconcentration of Fe(III), Al(III), Cu(II), Pb(II), Zn(II), and Mn(II) followed by determination by inductively coupled plasma atomic emission spectrometry. The adsorbent in the batch mode quantitatively (recovery 98?99%) extracts Fe(III), Al(III) and Cu(II) ions at pH 4.0 and Fe(III), Al(III), Cu(II), Pb(II), Zn(II), and Mn(II) ions at pH 7.0; the time of attainment of an adsorption equilibrium does not exceed 10 min. Consecutive preconcentration at pH 4.0 and 7.0 in the batch and dynamic modes ensures the quantitative separation of Fe(III), Al(III), and Cu(II) from Pb(II), Zn(II), and Mn(II) and their separate determination. The quantitative desorption of metals was attained with 0.5?1.0 M HNO₃ (5 or 10 mL). In preconcentration from 200 mL of solution with 5 mL of a desorbing solution, the preconcentration coefficient was equal to 40. The developed procedure was used for the determination of metal ions in river waters of Krasnoyarsk Krai. The results obtained were verified by the added?found method.     

DNAzyme-based fluorescent microarray for highly selective and sensitive detection of lead(II)

A facile microarray-based fluorescent sensor for the detection of lead (II) was developed based on the catalytic cleavages of the substrates by a DNAzyme upon its binding to Pb(2+). The release of the fluorophore labelled substrates resulted in the decrease of fluorescence intensity. The sensor had a quantifiable detection range from 1 nM to 1 μM and a selectivity of >20 fold for Pb(2+) over other metal ions.     

Effect of alkali metal ions on the intrazeolitic migration of Tb(III) ions in zeolite Y

The luminescence spectra of TbLi-Y, TbNa-Y and TbK-Y zeolites treated at different temperatures are reported. It is found that the intrazeolitic migration temperature of Tb(III) ions to hexagonal prisms is influenced by the type of the alkali metal ion present together with Tb(III) to neutralize the negative framework of zeolite Y.