Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.     

Highly sensitive, selective, and rapid fluorescence Hg2+ sensor based on DNA duplexes of poly(dT) and graphene oxide

Coupling T base with Hg(2+) to form stable T-Hg(2+)-T complexes represents a new direction in detection of Hg(2+). Here a graphene oxide (GO)-based fluorescence Hg(2+) analysis using DNA duplexes of poly(dT) that allows rapid, sensitive, and selective detection is first reported. The Hg(2+)-induced T(15)-(Hg(2+))(n)-T(15) duplexes make T(15) unable to hybridize with its complementary A(15) labelled with 6'-carboxyfluorescein (FAM-A(15)), which has low fluorescence in the presence of GO. On the contrary, when T(15) hybridizes with FAM-A(15) to form double-stranded DNA because of the absence of Hg(2+), the fluorescence largely remains in the presence of GO. A linear range from 10 nM to 2.0 μM (R(2) = 0.9963) and a detection limit of 0.5 nM for Hg(2+) were obtained under optimal experimental conditions. Other metal ions, such as Al(3+), Ag(+), Ca(2+), Ba(2+), Mg(2+), Zn(2+), Mn(2+), Co(2+), Pb(2+), Ni(2+), Cu(2+), Cd(2+), Cr(3+), Fe(2+), and Fe(3+), had no significant effect on Hg(2+) detection. Moreover, the sensing system was used for the determination of Hg(2+) in river water samples with satisfactory results.     

Influence of Cd2+, Hg2+ and Pb2+ on (+)-catechin binding to bovine serum albumin studied by fluorescence spectroscopic methods

The effect of heavy metal ions, Cd(2+), Hg(2+) and Pb(2+) on (+)-catechin binding to bovine serum albumin (BSA) has been investigated by spectroscopic methods. The results indicated that the presence of heavy metal ions significantly affected the binding modes and binding affinities of (+)-catechin to BSA, and the effects depend on the types of heavy metal ion. One binding mode was found for (+)-catechin with and without Cd(2+), while two binding modes - a weaker one at low concentration and a stronger one at high concentration were found for (+)-catechin in the presence of Hg(2+) and Pb(2+). The presence of Cd(2+) decreased the binding affinities of (+)-catechin for BSA by 20.5%. The presence of Hg(2+) and Pb(2+) decreased the binding affinity of (+)-catechin for BSA by 8.9% and 26.7% in lower concentration, respectively, and increased the binding affinity of (+)-catechin for BSA by 5.2% and 9.2% in higher concentration, respectively. The changed binding affinity and binding distance of (+)-catechin for BSA in the presence of Cd(2+), Hg(2+) and Pb(2+) were mainly because of the conformational change of BSA induced by heavy metal ions. However, the quenching mechanism for (+)-catechin to BSA was based on static quenching combined with non-radiative energy transfer irrespective of the absence or presence of heavy metal ions.     

Label-free and amplified electrochemical detection of cytokine based on hairpin aptamer and catalytic DNAzyme

In this work, by incorporating a specific DNAzyme sequence into a hairpin aptamer probe, we describe a label-free and sensitive method for electrochemical detection of cytokines using recombinant human IFN-γ as the model analyte. The hairpin aptamer probes are immobilized on a gold electrode through self-assembly. The presence of IFN-γ opens the hairpin structure and forms the hemin/G-quadruplex peroxidase-mimicking DNAzyme with subsequent addition of hemin. The peroxidase-mimicking DNAzyme catalyzes the electro-reduction of H(2)O(2) and amplifies the current response for IFN-γ detection, which enables the monitoring of IFN-γ at the sub-nanomolar level. The proposed sensor also shows high selectivity towards the target analyte. Our strategy thus opens new opportunities for label-free and amplified detection of different types of cytokines.     

Simple detection of trace Pb2+ by enrichment on cerium phosphate membrane filter coupled with color signaling

A Pb(2+) selective membrane filter was fabricated from the fibrous CeO(H(2)PO(4))(2).2H(2)O (CeP) crystals by blending with cellulose fiber. Enrichment of ppb level of Pb(2+) was achieved simply by filtration of aqueous sample solution through the membrane filter. Pb(2+) was strongly retained on the membrane filter by accommodation into the interlayer gallery of a CeP crystal. Visual detection of the enriched Pb(2+) was achieved by subsequent color signaling as PbS deposit upon treatment of the membrane filter with 3% Na(2)S solution. The analytical procedure and sample treatment conditions were optimized with respect to pH of the sample solution, filtration rate and masking of interfering ions. Detection of 20 ppb of Pb(2+) was not interfered by the presence of 1000-fold of Ca(2+), Mg(2+), and up to 100-fold of Fe(3+)and Cu(2+) by masking with 1 x 10(-3) mol dm(-3) of iminodiacetic acid (IDA). Most anions including phosphate (20 000 times) did not interfere with the determination of Pb(2+). The present simple method was applied to the determination of Pb(2+) in real samples like mine valley water.     

Selective colorimetric sensing of Cu2+ using triazolyl monoazo derivative

Although the high sensitivity, high selectivity and fast response make emission (fluorescence) based technique as one of the most promising tool for developing the chemosensors for metal ions, the past few years have witnessed a demand for the absorption based chemosensors for paramagnetic heavy metal ions, especially Cu(2+). Being paramagnetic, Cu(2+) leads to the low signal outputs ("turn-off") caused by decreased emission which may sometimes give false positive response, rendering the emission based technique less reliable for analytical purposes. Herein, we report synthesis and characterization of a hetarylazo derivative, characterized by a strong charge-transfer band which gets attenuated convincingly in the presence of Cu(2+) leading to distinct naked-eye color change (yellow to purple), and to a lesser extent in the presence of Cd(2+), Zn(2+), Co(2+), Pb(2+), Fe(2+), Ni(2+), Fe(3+) and Hg(2+) for which the naked eye sensitivity was comparatively (w.r.t. Cu(2+)) much less. No response was observed for the other metal ions including Li(+), Na(+), K(+), Mg(2+), Ca(2+), Ba(2+), Mn(2+), Ag(+), Zn(2+), Cd(2+), Pb(2+), and lanthanides Ce(3+), La(3+), Pr(3+), Eu(3+), Nd(3+), Lu(3+), Yb(3+), Tb(3+), Sm(3+), Gd(3+). The proposed sensing mechanism has been ascribed to the stabilization of LUMO after complexation with Cu(2+) and a 1:1 stoichiometry has been deduced.     

Adsorption of Pb2+, Zn2+, and Cd2+ from waters by amorphous titanium phosphate

In the current study, amorphous titanium phosphate (TiP) was prepared as an adsorbent for heavy metals from waters. Uptake of Pb(2+), Zn(2+), and Cd(2+) onto TiP was assayed by batch tests; a polystyrene-sulfonic acid exchanger D-001 was selected for comparison and Ca(2+) was chosen as a competing cation due to its ubiquitous occurrence in waters. The pH-titration curve of TiP implied that uptake of heavy metals onto TiP is essentially an ion-exchange process. Compared to D-001, TiP exhibits more preferable adsorption toward Pb(2+) over Zn(2+) and Cd(2+) even in the presence of Ca(2+) at different levels. FT-IR analysis of the TiP samples laden with heavy metals indicated that the uptake of Zn(2+) and Cd(2+) ions onto TiP is mainly driven by electrostatic interaction, while that of Pb(2+) ions is possibly dependent upon inner-sphere complex formation, except for the electrostatic interaction. Moreover, uptake of heavy metals onto TiP approaches equilibrium quickly and the exhausted TiP particles could be readily regenerated by HCl solution.     

检测痕量Hg~(2+)的DNA电化学生物传感器

通过自组装方法将修饰有二茂铁基团的富T序列DNA分子(DNA-Fc)固定在金电极表面,得到了一种基于DNA修饰电极的电化学汞离子(Hg2+)传感器.当溶液中有Hg2+存在时,Hg2+可与修饰电极上DNA的T碱基发生较强的特异结合,形成T-Hg2+-T发卡结构,使DNA分子构象发生改变,其末端具有电化学活性的二茂铁基团远离电极表面,电化学响应随之发生变化.示差脉冲伏安法(DPV)结果显示:DNA末端二茂铁基团的还原峰在0.26V(vs饱和甘汞电极(SCE))附近,峰电流随溶液中Hg2+浓度的增加而降低;Hg2+浓度范围在0.1nmol·L-1-1μmol·L-1时,电流相对变化率与Hg2+浓度的对数呈现良好的线性关系.该修饰电极对Hg2+的检测限为0.1nmol·L-1,可作为痕量Hg2+检测的电化学生物传感器.干扰实验也表明,该传感器对Hg2+具有良好的特异性与灵敏度.     

Molecular view of the anomalous acidities of Sn2+, Pb2+, and Hg2+

Experimental results taken from both the condensed and gaseous phase show that, when associated with water, the three dications Sn(2+), Pb(2+), and Hg(2+) exhibit a facile proton-transfer reaction. In the gas phase, no stable [M.(H(2)O)(n)](2+) ions are observed; but instead the cations appear to undergo rapid hydrolysis to give ions of the form M(+)OH(H(2)O)(n-1). A series of ab initio calculations have been undertaken on the structures and proton-transfer reaction profiles associated with the complexes [M.(H(2)O)(2,4)](2+), where M is one of Sn, Pb, Hg, and Ca. The latter has been used as a reference point both in terms of comparisons with previous calculations, and the fact that Ca(2+) is a very weak acid. The calculations show that for Sn(2+), Pb(2+), and Hg(2+), the only barriers to proton transfer are those associated with the movement of water molecules. In the gas phase, these barriers could be overcome through energy gained during ion formation, and in the condensed phase the thermal motion of water molecules would be sufficient. In contrast, the calculations show that for Ca(2+) it is the proton-transfer step that provides the most significant reaction barrier. Proton transfer in Sn(2+) and Pb(2+) is further assisted by distortions in the geometries of [M.(H(2)O)(2,4)](2+) complexes due to voids created by the 5s(2) (6s(2)) inert lone pair. For Hg(2+), ease of proton transfer is derived partly from the high degree of covalent bonding found in both the reactants and products.     

Pb2+ as modulator of protein-membrane interactions

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb(2+) on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb(2+) with higher affinity than its natural cofactor, Ca(2+). To gain insight into the coordination geometry of protein-bound Pb(2+), we determined the crystal structures of apo and Pb(2+)-bound C2α at 1.9 and 1.5 ? resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb(2+) ions coexist within a single protein molecule. Using protein-to-membrane F?rster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb(2+) displaces Ca(2+) from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb(2+) associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb(2+) on the PKCα activity.     

Elongated thrombin binding aptamer: a G-quadruplex cation-sensitive conformational switch

Aptamer-based biosensors offer promising perspectives for high performance, specific detection of proteins. The thrombin binding aptamer (TBA) is a G-quadruplex-forming DNA sequence, which is frequently elongated at one end to increase its analytical performances in a biosensor configuration. Herein, we investigate how the elongation of TBA at its 5'?end affects its structure and stability. Circular dichroism spectroscopy shows that TBA folds in an antiparallel G-quadruplex conformation with all studied cations (Ba(2+), Ca(2+), K(+), Mg(2+), Na(+), NH(4)(+), Sr(2+) and the [Ru(NH(3))(6)](2+/3+) redox marker) whereas other structures are adopted by the elongated aptamers in the presence of some of these cations. The stability of each structure is evaluated on the basis of UV spectroscopy melting curves. Thermal difference spectra confirm the quadruplex character of all conformations. The elongated sequences can adopt a parallel or an antiparallel structure, depending on the nature of the cation; this can potentially confer an ion-sensitive switch behavior. This switch property is demonstrated with the frequently employed redox complex [Ru(NH(3))(6)](3+), which induces the parallel conformation at very low concentrations (10 equiv per strand). The addition of large amounts of K(+) reverts the conformation to the antiparallel form, and opens interesting perspectives for electrochemical biosensing or redox-active responsive devices.     

Colorimetric test kit for Cu2+ detection

A coumarin-based colorimetric chemosensor 1 was designed and synthesized. It exhibits good sensitivity and selectivity for the copper cation over other cations such as Zn(2+), Cd(2+), Pb(2+), Co(2+), Fe(2+), Ni(2+), Ag(+), and alkali and alkaline earth metal cations both in aqueous solution and on paper-made test kits. The change in color is very easily observed by the naked eye in the presence of Cu(2+) cation, whereas other metal cations do not induce such a change. The quantitative detection of Cu(2+) was preliminarily examined.     

Amplified surface plasmon resonance based DNA biosensors, aptasensors, and Hg2+ sensors using hemin/G-quadruplexes and Au nanoparticles

Thiolated nucleic acid hairpin nanostructures that include in their stem region a "caged" G-quadruplex sequence, and in their single-stranded loop region oligonucleotide recognition sequences for DNA, adenosine monophosphate (AMP), or Hg(2+) ions were linked to bare Au surfaces or to Au nanoparticles (NPs) linked to Au surfaces. The opening of the hairpin nanostructures associated with the bare Au surface by the complementary target DNA, AMP substrate, or Hg(2+) ions, in the presence of hemin, led to the self-assembly of hemin/G-quadruplexes on the surface. The resulting dielectric changes on the surface exhibited shifts in the surface plasmon resonance (SPR) spectra, thus providing a readout signal for the recognition events. A similar opening of the hairpin nanostructures, immobilized on the Au NPs associated with the Au surface, by the DNA, AMP, or Hg(2+) led to an ultrasensitive SPR-amplified detection of the respective analytes. The amplification originated from the coupling between the localized surface plasmon associated with the NPs and the surface plasmon wave, an effect that cooperatively amplifies the SPR shifts that result from the formation of the hemin/G-quadruplexes. The different sensing platforms reveal impressive sensitivities and selectivities toward the target analytes.     

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.     

Pb(2+)-introduced activation of horseradish peroxidase (HRP)-mimicking DNAzyme

A novel nucleic acid hairpin structure composed of Pb(2+)-dependent DNAzyme and HRP-mimicking DNAzyme was developed. This hairpin structure can be used as a sensor for the detection of Pb(2+) based on colorimetry.     

Nanoparticle-enhanced sensitivity of a nanogap-interdigitated electrode array impedimetric biosensor

Interdigitated electrode (IDE) arrays with nanometer-scale gaps have been utilized to enhance the sensitivity of affinity-based detection. The geometry of nanogap IDEs was first optimized on the basis of simulations of the electric field and current density. It was determined that the gap (G) between the electrodes was the most important geometric parameter in determining the distribution and strength of the electric field and the current density compared to the width (W) and height (H) of the IDEs. Several devices were materialized and analyzed for their sensitivity to the electrochemical environment using faradic electrochemical impedance spectroscopy (EIS) as the detection technique. Nanogap optimized IDEs were then employed as biosensors for the label-free, affinity-based detection of antitissue transglutaminase antibodies (αtTG-Abs), a biomarker for the detection of autoimmune disorder celiac sprue, triggered by ingesting gluten. The label-free biosensor assay was found to be less sensitive compared to on-chip ELISA. Gold nanoparticles (GNPs) were then employed to improve the sensitivity of the nanogap IDE-based biosensor. With GNPs, the transducer sensitivity increased by 350% over that of label-free detection. The suitability of nanogap IDEs as biosensor transducers for EIS in label-free and GNP-labeled formats was established. The immunobiosensor assay detection sensitivity with the GNPs was found comparable to ELISA.     

钾离子浓度依赖的铅离子稳定G-四链体构型转化

已有研究普遍认为铅离子(Pb²⁺)诱导富G适体链形成的G-四链体(Pb²⁺-G4)比钾离子(K⁺)诱导富G适体链形成的G-四链体(K⁺-G4)更为稳定,因而Pb²⁺可以置换K⁺-G4中的K⁺,而且K⁺的存在不影响Pb²⁺-G4的稳定性。有趣的是本研究发现K⁺ (20 μmol∙L⁻¹–1 mmol∙L⁻¹)不仅可以诱导10 µmol∙L⁻¹ Pb²⁺稳定的T2TT(Pb²⁺-T2TT,杂合G4结构)发生构型转换,甚至还可取代Pb²⁺-T2TT中的Pb²⁺,形成K⁺稳定的T2TT (K⁺-T2TT,平行G4结构),最终转化形成的K⁺-G4结构与单独K⁺诱导富G适体链形成K⁺-G4的构型基本一致。随后,进一步考察了另外7条富G适体链,发现这一转化过程具有一定的普适性。该研究结果为理解G4构型转化以及内嵌离子交换提供了新的视角,也为拓展G4在生化分析和生物领域的应用提供了新的理论基础。     

Hairpin Assembly Amplified Electrochemical Biosensor for Highly Sensitive and Specific Detection of DNA

In this report, a simple electrochemical biosensor has been developed for highly sensitive and specific detection of DNA based on hairpin assembly amplification. In the presence of target DNA, the biotin‐labelled hairpin H1 is opened by hybridizing with target DNA through complementary sequences. Then the opened hairpin H1 assembles with the hairpin H2 to displace the target DNA, generating H1‐H2 complex. The displaced target DNA could trigger the next cycle of hairpins assembly, resulting in the generation of numerous H1‐H2 complexes. Subsequently, the H1‐H2 complex hybridizes with the capture probe immobilized on the electrode. Finally, the streptavidin alkaline phosphatase (ST‐ALP) binds to biotin in the capture probe‐H1‐H2 complex and catalyzes the substrate α‐naphthol (α‐NP) to produce electrochemical signal. To make a more fascinating hairpin assembly amplification strategy in signal amplification, mismatched base sequences are designed in hairpin H2 to decrease non‐specific binding of the hairpin substrates. The developed biosensor achieves a sensitivity of 20 pM with a linear range from 25 pM to 25 nM, and shows high selectivity toward single‐base mismatch. Thus, the proposed electrochemical biosensor might have the potential for early clinical diagnosis and therapy.     

Bis[1,1'-N,N'-(2-picolyl)aminomethyl]ferrocene as a redox sensor for transition metal ions

The compound bis[1,1'-N,N'-(2-picolyl)aminomethyl]ferrocene, L(1), was synthesized. The protonation constants of this ligand and the stability constants of its complexes with Ni(2+), Cu(2+), Zn(2+), Cd(2+) and Pb(2+) were determined in aqueous solution by potentiometric methods at 25 degrees C and at ionic strength 0.10 mol dm(-3) in KNO(3). The compound L(1) forms only 1:1 (M:L) complexes with Pb(2+) and Cd(2+) while with Ni(2+) and Cu(2+) species of 2 [ratio] 1 ratio were also found. The complexing behaviour of L(1) is regulated by the constraint imposed by the ferrocene in its backbone, leading to lower values of stability constants for complexes of the divalent first row transition metals when compared with related ligands. However, the differences in stability are smaller for the larger metal ions. The structure of the copper complex with L(1) was determined by single-crystal X-ray diffraction and shows that a species of 2:2 ratio is formed. The two copper centres display distorted octahedral geometries and are linked through the two L(1) bridges at a long distance of 8.781(10) Angstrom. The electrochemical behaviour of L(1) was studied in the presence of Ni(2+), Cu(2+), Zn(2+), Cd(2+) and Pb(2+), showing that upon complexation the ferrocene-ferrocenium half-wave potential shifts anodically in relation to that of the free ligand. The maximum electrochemical shift ([capital Delta]E(1/2)) of 268 mV was found in the presence of Pb(2+), followed by Cu(2+)(218 mV), Ni(2+)(152 mV), Zn(2+)(111 mV) and Cd(2+)(110 mV). Moreover, L(1) is able to electrochemically and selectively sense Cu(2+) in the presence of a large excess of the other transition metal cations studied.     

Determination of Lead(II) by a Nitrocellulose Membrane Fluorescent Biosensor Based on G-Quadruplex Conformational Changes

A simple, label-free fluorescence method was developed for the sensitive determination of lead(II) using a nitrocellulose membrane biosensor. The surface of the nitrocellulose membrane was modified by glutaraldehyde to conjugate streptavidin, followed by the immobilization of a DNA probe via a biotin modifier. The biotinylated DNA probe can fold into a G-quadruplex structure in the presence of potassium ion that selectively binds to N-methyl mesoporphyrin IX and yields a strong fluorescence signal. The presence of lead(II) can induce a conformational change of the G-quadruplex to a more compact structure, which results in the release of potassium ion and N-methyl mesoporphyrin IX with a concomitant reduction of the fluorescence signal. The biosensor displayed a detection limit as low as 10 nM with excellent selectivity for lead(II) over other metal ions. The developed biosensor was employed for the determination of lead(II) in spiked river water.