Highly sensitive oligonucleotide-based fluorometric detection of mercury(II) in aqueous media

This paper describes a highly sensitive and selective Hg(2+) sensor using a label free Hg(2+) specific probe (5'-18T-3') and an intercalation dye SYBR Green I (SG). The Hg(2+) specific probe is composed of thymines (T) and readily forms T-Hg(2+)-T complexes in the presence of Hg(2+). This specific T-Hg(2+)-T formation affects the hybridization of the Hg(2+) specific probe and the intercalation of SG. Upon treatment of 1 nM 5'-18T-3' with different amount of Hg(2+) (0.1-10nM), which is followed by hybridization with 1 nM 5'-18T-3' and incubation with 1 microL of SG, the solution fluorescence gave a linear response (R=0.996) to the concentration of Hg(2+). The detection limit for Hg(2+) was 0.5 nM (0.1 ppb). The overall test only takes few minutes and very little interference is observed from non-specific metal ions. This approach may find potential applications in monitoring the Hg(2+) concentration in drinking water.     

Oligonucleotide-based fluorogenic sensor for simultaneous detection of heavy metal ions

In this study, we report a new fluorogenic sensor based on fluorescence resonance energy transfer (FRET) for detection of heavy metal ions in aqueous solution. The method showed the advantage of being simple, highly sensitive and selective, and rapid. The donor (CdTe QDs) and acceptor (TAMRA or Cy5) are brought into close proximity to one another due to Hg(2+) and Ag(+) form strong and stable T-Hg(2+)-T complexes and C-Ag(+)-C complexes, which quenches the fluorescent intensity of CdTe QDs and enables the energy transfer from donor to acceptor. This sensor showed high sensitivity and selectivity when only one kind of ion (Ag(+) or Hg(2+)) exists. Furthermore, the assay can also simultaneously detect Ag(+) and Hg(2+) in water media with the limit of detection (LOD) of 2.5 and 1.8 nM, separately, which satisfactorily meets the sensitivity demands of Environmental Protection Agency (EPA) and World Health Organization (WHO). This assay also exhibits excellent selectivity toward Ag(+) and Hg(2+). Therefore, this method is of great practical and theoretical importance for detecting heavy metal ions in aqueous solution.     

A highly sensitive and selective optical sensor for Pb(2+) by using conjugated polymers and label-free oligonucleotides

The detection of Pb(2+) with DNA-based biosensor is usually susceptible to severe interference from Hg(2+) because of the T-Hg(2+)-T interaction between Hg(2+) and T residues. In this study, we developed a rapid, sensitive, selective and label-free sensor for the detection of Pb(2+) in the presence of Hg(2+) based on the Pb(2+)-induced G-quadruplex formation with cationic water-soluble conjugated polymer (PMNT) as a "polymeric stain" to transduce optical signal. We selected a specific sequence oligonucleotide, TBAA (5'-GGAAGGTGTGGAAGG-3'), which can form a G-quadruplex structure upon the addition of Pb(2+). This strategy provided a promising alternative to Pb(2+) determination in the presence of Hg(2+) instead of the universal masking agents of Hg(2+) (such as CN(-), SCN(-)). Based on this observation, a simple "mix-and-detect" optical sensor for the detection of Pb(2+) was proposed due to the distinguishable optical properties of PMNT-ssDNA and PMNT-(G-quadruplex) complexes. By this method, we could identify micromolar Pb(2+) concentrations within 5min even with the naked eye. Furthermore, the detection limit was improved to the nanomolar range by the fluorometric method. We also successfully utilized this biosensor for the determination of Pb(2+) in tap water samples.     

Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+). In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 microM Hg(2+). When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.     

Colorimetric detection of mercury, lead and copper ions simultaneously using protein-functionalized gold nanoparticles

A simple, cost-effective and rapid colorimetric method for any or all of Hg(2+), Pb(2+) and Cu(2+) detection using papain-functionalized gold nanoparticles (P-AuNPs) has been developed. Papain is a protein with seven cystein residues, which can selectively bind with Hg(2+), Pb(2+) and Cu(2+). We functionalized gold nanoparticles with papain. The P-AuNPs could be used to simultaneously detect Hg(2+), Pb(2+) and Cu(2+), and showed different responses to the three ions in an aqueous solution based on the aggregation-induced color change of gold nanoparticles. The P-AuNPs displayed the most obvious response to mercury ions in water in contrast to lead and copper ions, and the real water sample analysis verified the conclusion. The sensitivity of the detection system was influenced by the pH of the P-AuNPs solution, the concentration of P-AuNPs and the size of gold nanoparticles, and we found that larger gold nanoparticles contributed to more sensitive results. The detection system can detect as low as 200 nM Hg(2+), Pb(2+) or Cu(2+) using 42 nm gold nanoparticles. We expect our approach to have wide-ranging applications in the developing region for monitoring water quality in some areas.     

Oligonucleotide probes applied for sensitive enzyme-amplified electrochemical assay of mercury(II) ions

We developed a novel electrochemical sensor for Hg(2+) detection using two mercury-specific oligonucleotide probes and streptavidin-horseradish peroxidase (HRP) enzymatic signal amplification. The two mercury-specific oligonucleotide probes comprised a thiolated capture probe and a biotinated signal probe. The thiolated capture probe was immobilized on a gold electrode. In the presence of Hg(2+), the thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction between the mismatched T-T base pairs directed the biotinated signal probe hybridizing to the capture probe and yielded a biotin-functioned electrode surface. HRP was then immobilized on the biotin-modified substrate via biotin-streptavidin interaction. The immobilized HRP catalyzed the oxidation of hydroquinone (H(2)Q) to benzoquinone (BQ) by hydrogen peroxide (H(2)O(2)) and the generated BQ was further electrochemically reduced at the modified gold electrode, producing a readout signal for quantitative detection of Hg(2+). The results showed that the enzyme-amplified electrochemical sensor system was highly sensitive to Hg(2+) in the concentration of 0.5 nM to 1 μM with a detection limit of 0.3 nM, and it also demonstrated excellent selectivity against other interferential metal ions.     

A sugar-aza-crown ether-based fluorescent sensor for Cu2+ and Hg2+ ions

A sugar-aza-crown ether (SAC)-based fluorescent sensor 4 was prepared. It contains a pyrene as the fluorophore and its fluoroionophoric properties toward transition metal ions were investigated. Chemosensor 4 exhibits highly selective recognition toward Cu(2+) and Hg(2+) ions among a series of tested metal ions in methanol solution. The association constants for 4*Cu(2+) and 4*Hg(2+) in methanol solution were calculated to be 7.4×10(1)M(-1) and 4.4×10(3)M(-1), respectively. Chemosensor 4 formed complexes with the Cu(2+) or Hg(2+) ion at a 1:1 ligand-to-metal ratio with a detection limit of 1.3×10(-4)M Cu(2+) and 1.26×10(-5)MHg(2+), respectively.     

Hg(2+) affects the intracellular free Ca(2+) oscillatory pattern and the correlated membrane conductance changes in Mg(2+)-stimulated oocytes of the prawn Palaemon serratus

The impact of mercuric ions (Hg(2+)) on prawn oocytes was examined. Prawn oocytes constitute an unusual system in that they are activated at spawning by seawater Mg(2+), which mediates correlated dynamic changes in intracellular free calcium concentration [(Ca(2+))(i)] and membrane conductance associated with the meiosis resumption. Using a voltage clamp technique and intracellular calcium measurements, we observed that treatment with Hg(2+) (5, 10, and 20 microM) resulted in simultaneous impairments of both (Ca(2+))(i) and membrane current responses to external Mg(2+). Treatment with Hg(2+) also resulted in a gradual dose-dependent slow increase in the baseline level of both (Ca(2+))(i) and membrane conductance, independent of stimulation with external Mg(2+). The effect of Hg(2+) on (Ca(2+))(i) and membrane conductance changes resulted from a block of the signal transduction pathway at some point before the InsP(3) receptor channel involved in Ca(2+) release from the endoplasmic reticulum (ER) stocks. The Hg(2+)-dependent gradual increase in both (Ca(2+))(i) and membrane conductance baseline levels may potentially result from a slow permeabilization of the ER membrane, resulting in Ca(2+) leaking into the cytosol. Indeed, this effect could be blocked with the cell permeable Hg(2+) competitor dithiothreitol, which was able to displace Hg(2+) from its intracellular target regardless of whether external Ca(2+) was present or not.     

Ultrasensitive and selective detection of copper (II) and mercury (II) ions by dye-coded silver nanoparticle-based SERS probes

A simple and distinctive method for the ultrasensitive detection of Cu(2+) and Hg(2+) based on surface-enhanced Raman scattering (SERS) using cysteine-functionalized silver nanoparticles (AgNPs) attached with Raman-labeling molecules was developed. The glycine residue in a silver nanoparticle-bound cysteine can selectively bind with Cu(2+) and Hg(2+) and form a stable inner complex. Silver nanoparticles co-functionalized with cysteine and 3,5-Dimethoxy-4-(6'-azobenzotriazolyl)phenol (AgNP conjugates) can be used to detect Cu(2+) and Hg(2+) based on aggregation-induced SERS of the Raman tags. The addition of SCN(-) to the analyte can successfully mask Hg(2+) and allow for the selective detection of Cu(2+). This SERS-based assay showed an unprecedented limit of detection (LOD) of 10pM for Cu(2+) and 1pM for Hg(2+); these LODs are a few orders of magnitude more sensitive than the typical colorimetric approach based on the aggregation of noble nanoparticles. The analysis of real water samples diluted with pure water was performed and verified this conclusion. We envisage that this SERS-based assay may provide a general and simple approach for the detection of other metal ions of interest, which can be adopted from their corresponding colorimetric assays that have already been developed with significantly improved sensitivity and thus have wide-range applications in many areas.     

High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA. We report six X-ray structures of the substrate-free and the UDP-bound enzyme. Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+). The substrate-free BGT structure differs by a domain movement from one previously determined in another space group. Further domain movements are seen in the complex with UDP and the four UDP-metal complexes. Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form. Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis.     

Highly sensitive and selective photoelectrochemical DNA sensor for the detection of Hg²⁺ in aqueous solutions

A "turn-on" photoelectrochemical sensor for Hg(2+) detection based on thymine-Hg(2+)-thymine interaction is presented by using a thymine-rich oligonucleotide film and a double-strand DNA intercalator, Ru(bpy)(2)(dppz)(2+) (bpy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c]phenazine) as the photocurrent signal reporter. The presence of Hg(2+) induces the formation of a double helical DNA structure which provides binding sites for Ru(bpy)(2)(dppz)(2+). The double helical structure was confirmed by circular dichroism and fluorescence measurements. Under the optimized conditions, a linear relationship between photocurrent and Hg(2+) concentration was obtained over the range of 0.1 nM to 10 nM Hg(2+), with a detection limit of 20 pM. Interference by 10 other metal ions was negligible. Analytical results of Hg(2+) spiked into tap water and lake water by the sensor were in good agreement with mass spectrometry data. With the advantages of high sensitivity and selectivity, simple sensor construction, low instrument cost and low sample volume, this method is potentially suitable for the on-site monitoring of Hg(2+) contamination.     

Construction and characterization of a photosynthetic bacterium genetically engineered for Hg2+ uptake

A recombinant photosynthetic bacterium, Rhodopseudomonas palustris, was constructed to simultaneously express mercury transport system and metallothionein for Hg(2+) removal from heavy metal wastewater. The effects of essential process parameters, including pH, ionic strength and presence of co-ions on Hg(2+) uptake were evaluated. The results showed that compared with wild type R. palustris, recombinant strain displayed stronger resistance to toxic Hg(2+), and its Hg(2+) binding capacity was enhanced threefolds. In the range of pH 4-10, recombinant R. palustris maintained effective accumulation of Hg(2+). The presence of 10 mg L(-1) Mg(2+), Ca(2+), Zn(2+) or Ni(2+) did not significantly influence Hg(2+) bioaccumulation by recombinant R. palustris from solutions containing 0.2 mg L(-1) Hg(2+), while Na(+) and Cd(2+) posed serious adverse effect on Hg(2+) uptake. Furthermore, EDTA treatment experiment confirmed that different from wild type R. palustris that mainly absorbed Hg(2+) on the cell surface, recombinant R. palustris transported most of the bound Hg(2+) into the cells.     

Multi-step analysis of Hg2+ ion inhibition of jack bean urease

We performed a multi-step analysis of the inhibition of jack bean urease by Hg(2+) ions that included residual activity measurements after incubation of the enzyme with the metal ion, reactivation of Hg(2+)-inhibited urease, protection of urease with thiol reagents prior to incubation with Hg(2+), progress curve analysis, and spectroscopic assay of thiol groups in urease-Hg(2+) complexes with a cysteine selective agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Hg(2+) ions were found to form stable complexes with urease that could rapidly be reversed only by the treatment with dithiotreitol, and not by dilution or dialysis. The residual activity data interpreted in terms of the Hill equation revealed the multisite Hg(2+) inhibition of urease, and along with the DTNB thiol-assay they demonstrated the involvement in the reaction with Hg(2+) of six cysteine residues per enzyme subunit, including the active-site flap cysteine. The molar ratios of the inhibitor and enzyme imply that the inhibition consists of the formation of RSHgX complexes, X being a water molecule or an anion. The time-dependent Hg(2+) inhibitory action on urease determined in the system without enzyme preincubation was best described by slow-binding mechanism with the steady-state inhibition constant K(i) = 1.9 nM (+/-10%).     

Surface plasmon resonance sensing detection of mercury and lead ions based on conducting polymer composite

A new sensing area for a sensor based on surface plasmon resonance (SPR) was fabricated to detect trace amounts of mercury and lead ions. The gold surface used for SPR measurements were modified with polypyrrole-chitosan (PPy-CHI) conducting polymer composite. The polymer layer was deposited on the gold surface by electrodeposition. This optical sensor was used for monitoring toxic metal ions with and without sensitivity enhancement by chitosan in water samples. The higher amounts of resonance angle unit (ΔRU) were obtained for PPy-CHI film due to a specific binding of chitosan with Pb(2+) and Hg(2+) ions. The Pb(2+) ion bind to the polymer films most strongly, and the sensor was more sensitive to Pb(2+) compared to Hg(2+). The concentrations of ions in the parts per million range produced the changes in the SPR angle minimum in the region of 0.03 to 0.07. Data analysis was done by Matlab software using Fresnel formula for multilayer system.     

Ultrasensitive and dual functional colorimetric sensors for mercury (II) ions and hydrogen peroxide based on catalytic reduction property of silver nanoparticles

The method provides an innovative dual functional sensors for mercury (II) ions and hydrogen peroxide. The addition of H(2)O(2) to the mixture of silver nanoparticles (AgNPs) and Hg(2+) induced color changes of the solution within several seconds even at 2.0 nM Hg(2+). Other metallic ions could not induce color change even at 10 μM. Of importance, this probe was not only successfully applied to detect Hg(2+), but also it could be used to sense H(2)O(2) at a concentration as low as 50 nM (by naked-eye). The outstanding sensitivity and selectivity property for Hg(2+) and H(2)O(2) resulted from the AgNPs mediated reduction of Hg(2+) to elementary Hg in the presence of H(2)O(2), causing the aggregation and colorimetric response of AgNPs. This sensitive and selective colorimetric assay opens up a fresh insight of development facile and fast detection methods for metal ions and biomolecules using the special catalytic reactivity of AgNPs.     

Magnesium binding to DM-nitrophen and its effect on the photorelease of calcium

The effect of Mg(2+) on the process of Ca(2+) release from the caged Ca(2+) compound DM-nitrophen (NP) was studied in vitro by steady light UV photolysis of NP in the presence of Ca(2+) and Mg(2+). Ca(2+) release during photolysis and its relaxation/recovery after photolysis were monitored with the Ca(2+)-sensitive dye fura-2. Mg(2+) speeds the photorelease of Ca(2+) during photolysis and slows the relaxation of Ca(2+) to new steady-state levels after photolysis. Within the context of a model describing NP photolysis, we determined the on and off rates of Mg(2+) binding to unphotolyzed NP (k(on) = 6.0 x 10(4) M(-1) s(-1); k(off) = 1.5 x 10(-1) s(-1)). Furthermore, to fully account for the slow postphotolysis kinetics of Ca(2+) in the presence of Mg(2+) we were forced to add an additional photoproduct to the standard model of NP photolysis. The additional photoproduct is calculated to have a Ca(2+) affinity of 13.3 microM and is hypothesized to be produced by the photolysis of free or Mg(2+)-bound NP; photolysis of Ca(2+)-bound NP produces the previously documented 3 mM Ca(2+) affinity photoproduct.     

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ～1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.     

The cytoplasmic C-terminal fragment of polycystin-1 regulates a Ca2+-permeable cation channel

The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca(2+)-permeable cation current, which shifted oocyte reversal potential (E(rev)) by +33 mV. Whole cell currents were sensitive to inhibition by La(3+), Gd(3+), and Zn(2+), and partially inhibited by SKF96365 and amiloride. Currents were not activated by bath hypertonicity, but were inhibited by acid pH. Outside-out patches pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold increased NP(o) of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes also exhibited elevated NP(o) of unitary calcium currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and (45)Ca(2+) flux experiments. Both Ca(2+) conductance and influx were inhibited by La(3+). Mutation of candidate phosphorylation sites within PKD1(115-226) abolished the cation current. We conclude that the C-terminal cytoplasmic tail of PKD1 up-regulates inward current that includes a major contribution from Ca(2+)-permeable nonspecific cation channels. Dysregulation of these or similar channels in autosomal dominant polycystic kidney disease may contribute to cyst formation or expansion.     

Detection of heavy metal ions using protein-functionalized microcantilever sensors

Microcantilevers functionalized with metal-binding protein, AgNt84-6, are demonstrated to be sensors for the detection of heavy metal ions like Hg(2+) and Zn(2+). AgNt84-6, a protein that has the ability to bind multiple atoms of Ni(2+), Zn(2+), Co(2+), Cu(2+), Cd(2+) and Hg(2+) was attached to the gold-coated side of silicon nitride cantilevers via linker groups. Upon exposure to 0.1 mM HgCl(2) and 0.1 mM ZnCl(2) solutions, the microcantilevers underwent bending corresponding to an expanding gold side. Exposure to a 0.1 mM solution of MnCl(2) solution did not result in a similar bending indicating a weak or no interaction of Mn(2+) ions with the AgNt84-6 protein. The microcantilever bending data were consistent with data from electrophoresis carried out on SDS-PAGE gels containing metal ions that showed protein interaction with Zn(2+) ions but not with Mn(2+) ions. Thus, we demonstrate that microcantilever bending can be used to discriminate between metal ions that bind and do not bind to AgNt84-6 protein in real time.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).