Using electrospray ionization mass spectrometry to explore the interactions among polythymine oligonucleotides, ethidium bromide, and mercury ions

We have used electrospray ionization mass spectrometry (ESI-MS) and fluorescence and circular dichroism (CD) spectroscopy to explore the binding of ethidium bromide (EthBr) to non-self-complementary polythymine (polyT) strands in the absence and presence of Hg²⁺ ions. In the gas phase, ESI-MS revealed that Hg²⁺ ions have greater affinity, through T-Hg²⁺-T coordination, toward polyT strands than do other metal ions. These findings are consistent with our fluorescence and CD results obtained in solution; they revealed that more T₃₃-EthBr-Hg²⁺ complexes existed upon increasing the concentrations of Hg²⁺ ions (from 0 to 50 μM). Surprisingly, the ESI-MS data indicated that the Hg²⁺ concentration dependence of the interaction between T₃₃ and EthBr is biphasic. Our ESI-MS data revealed that the T₃₃-EthBr-Hg²⁺ complexes formed with various stoichiometries depending on their relative concentrations of the components and the length of the DNA strand. When the concentrations of T₃₃/EthBr/Hg²⁺ were 5/5/2. 5 μM and 5/10/7. 5 μM, 1:1:1 and 1:1:2 T₃₃-EthBr-Hg²⁺ complexes were predominantly formed, respectively. Thus, Hg²⁺-induced DNA conformational changes clearly affect the interactions between DNA and EthBr.     

Control of the Fluorescence of DNA‐templated Silver Nanoclusters by Adenosine Triphosphate and Mercury(II)

Several DNA templates with the sequence 5′‐T _n TAACCCCTAACCCCT ‐3′ (n = 0, 15, 30, and 45) were used to prepare DNA template–silver nanoclusters (DNA –Ag NCs ). The T _n sequence acts as a recognition element for Hg²⁺, while the rest of the sequence acts as a template for DNA –Ag NCs . At pH 3.0, the fluorescence intensity of DNA –Ag NCs is enhanced by ATP , and the enhanced fluorescence is quenched by Hg²⁺. The length of polyT shows a slight effect on the sensitivity for the detection of Hg²⁺ but almost no effect on the optical properties of DNA –Ag NCs . The fluorescence response of DNA –Ag NCs (T₁₅‐DNA –Ag NCs ) vs. Hg²⁺ concentration shows two linear ranges over 10–100 and 100–1000 nM , mainly because of the fluorescence quenching due to DNA conformational changes through T–Hg²⁺–T coordination and the formation of an amalgam with Ag NCs , respectively. The sensitivity of the T₁₅‐DNA –Ag NC probe was validated through the analysis of Hg²⁺ in spiked pond water. Based on the switch‐on and switch‐off fluorescence properties of T₁₅‐DNA –Ag NCs , an IMPLICATION logic gate was fabricated using the concentrations of ATP and Hg²⁺ as inputs and the fluorescence intensity at 585 nm as output.     

Resonance Scattering Detection of Trace Hg2+ Using Aptamer‐modified AuPd Nanoalloy Probe as Catalyst

The 5 nm AuPd nanoalloy in mole ratio of Au:Pd=32:1 was prepared, using sodium citrate as the stabilizing agent and NaBH₄ as the reductant. The AuPd nanoalloy was modified by the aptamer to prepare an aptamer‐ AuPd (AptAuPd) probe for resonance scattering (RS) detection of 5.0–1250 nmol/L Hg²⁺. The AptAuPd‐Hg²⁺ aptamer reaction solution was filtrated by membrane, and the AptAuPd in the filtrate exhibited strong catalytic effect on the slow NiP particle reaction between NiCl₂ and NaH₂PO₂, and the NiP particles showed a RS peak at 508 nm. The RS intensity decreased when Hg²⁺ concentration increased. The decreased RS intensity was linear to Hg²⁺ concentration in the range of 0.5–1250 nmol/L. The RS assays were used to determine Hg²⁺ in real samples, with good results.     

Chelate Complexes with Methylmercury and Phenylmercury Cations

The complex formation of Hg²⁺, CH₃Hg⁺ and C₆H₅Hg⁺ with eight substituted quinolines, α,α′-bipyridyl and 1, 10-phenanthroline has been investigated in water and 75(v)% dioxane by pH and pHg methods. Hg²⁺ forms mercurated products with 8-hydroxyquinolines, if the 5- or 7-positions are unsubstituted. The formation of chelates by CH₃Hg⁺ and C₆H₅Hg⁺ is postulated.     

Fluorescence detection of mercury(II) and lead(II) ions using aptamer/reporter conjugates

We have developed a fluorescence technique for the detection of Hg²⁺ and Pb²⁺ ions using polythymine (T₃₃)/benzothiazolium-4-quinolinium dimer derivative (TOTO-3) and polyguanine (G₃₃)/terbium ions (Tb³⁺) conjugates, respectively. Hg²⁺ ions induce T₃₃ to form folded structures, leading to increased fluorescence of the T₃₃/TOTO-3 conjugates. Because Pb²⁺ ions compete with Tb³⁺ ions to form complexes with G₃₃, the extent of formation of the G₃₃-Tb³⁺ complexes decreases upon increasing the Pb²⁺ concentration, leading to decreased fluorescence at 545 nm when excited at 290 nm. To minimize interference from Hg²⁺ ions during the detection of Pb²⁺ ions, we conducted two-step fluorescence measurements; prior to addition of the G₃₃/Tb³⁺ probe, we recorded the fluorescence of a mixture of the T₃₃/TOTO-3 conjugates and Hg²⁺ ions. The fluorescence signal obtained was linear with respect to the Hg²⁺ concentration over the range 25.0-500 nM (R² = 0.99); for Pb²⁺ ions, it was linear over the range 3.0-50 nM (R² = 0.98). The limits of detection (at a signal-to-noise ratio of 3) for Hg²⁺ and Pb²⁺ ions were 10.0 and 1.0 nM, respectively. Relative to other techniques for the detection of Hg²⁺ and Pb²⁺ ions in soil and water samples, our present approach is simpler, faster, and more cost-effective.     

Aptamer-modified AuRe Nanoalloy Probe for Trace Hg2+ Using Resonance Scattering as Detection Technique

The AuRe nanoalloy particles in molar ratio of 9:1 were prepared by sodium borohydride procedure, and modified by single strand DNA (ssDNA) to prepare an aptamer AuRe nanoprobe (AuRessDNA) for Hg²⁺. In the pH 7.0 Na₂HPO₄‐NaH₂PO₄ buffer solution and in the presence of NaCl, Hg²⁺ interacted with AuRessDNA to form double‐stranded T‐Hg²⁺‐T mismatched and release AuRe nanoparticles that aggregate to large AuRe nanoparticles clusters causing the resonance scattering (RS) peak red shifting and the RS intensity enhanced linearly. On those grounds, 0.067–33.3 nmol·L^?1 Hg²⁺ can be detected rapidly by the aptamer‐modified AuRe nanoparticles RS assay, with a detection limit of 0.04 nmol·L^?1 Hg²⁺. If the aggregated AuRe particles were removed by membrane filtration, the excess AuRessDNA in the filtration solution exhibits catalytic effect on the new Te particle reaction between Na₂TeO₄ and SnCl₂. As the concentration of Hg²⁺ increased, the AuRessDNA nanoparticles in the filtrate solution decreased, the RS intensity at 734 nm decreased linearly. The Hg²⁺ concentration (c) in the range of 0.00133–0.267 nmol·L^?1 was linear to the decreased RS intensity (ΔI_734nm), with a regression equation of ΔI= ?786.4c?4.4, a correlation coefficient of 0.9975, and a detection limit of 0.9 pmol·L^?1 Hg²⁺. This method was applied to the detection of Hg²⁺ in water samples, with satisfactory results.     

Metal-Triggered DNA Folding by Different Mechanisms

Metal-mediated base pairs by the interaction between metal ions and artificial bases in oligonucleotides has been widely used in DNA nanotechnology and biosensing technique. Using isothermal titration calorimetry, circular dichroism spectroscopy and fluorescence spectroscopy, the folding process of T-C-rich oligonucleotides (TCO) induced by Hg²⁺ and Ag⁺ with the synthetic sequence d(T₆C₆T₆C₆T₆C₆T₆) was studied and analyzed. Although thermodynamic data predict that TCO should initially fold into a relatively stable hairpin through two possible pathways of conformational transitions whether Hg²⁺ or Ag⁺ were added at first, the mechanisms and final products between the two are entirely different from isothermal titration calorimetry outcomes. When Hg²⁺ were added first, the haipin was formed through T-Hg-T structure with further stabilization by C-Ag-C after Ag⁺ addition. However, it is proposed that an unusual metal-base pair for Ag⁺ binding is generated instead classical C-Ag-C when Ag⁺ was injected first. Moreover, further confirmation of this unconventional metal-base pair T-Ag-C was verified by circular dichroism and fluorescence spectroscopy.     

Spectral Studies on the Interaction between Mercuric Ion and ApoCopC

The interaction between mercuric ion and apoCopC in the absence or presence of cupric ion was investigated through difference UV spectra in Hepes buffer （10 mmol·L^-1） at pH 7.4. The results suggest that mercuric ion can bind to C- and N-terminal binding sites of apoCopC, and the conditional binding constants were calculated to be kN=（6.79± 1.12）× 10^6 mol^-1·L and kc=（3.06±0.05）× 10^5 mol^-1·L. Using urea as a chemical agent, the conformational stabilities of apoCopC and HgN^2＋ -CopC-Hgc^2＋ were monitored by fluorescence spectrum in Hepes buffer （50 mmol·L^-1） at pH 7.4. The free energy of stabilization is （14.69±0.85） and （16.66±0.55） kJ.mol^-1, respectively. HgN^2＋ -CopC-Hgc^2＋ is more stable than apoCopC.     

Total nucleotide analysis of Hydra DNA and RNA by MEKC with LIF detection and 32P‐postlabeling

The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity – fluorescence labeling of nucleotides followed by CE‐LIF detection and ³²P‐postlabeling. DNA of Hydra was digested either to deoxyribonucleoside‐5′‐monophosphates or deoxyribonucleoside‐3′‐monophosphates selectively conjugated with the fluorescent dye 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE‐LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5‐methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the ³²P‐postlabeling method.     

Electrochemical aptamer sensor for highly sensitive detection of mercury ion with Au/Pt@carbon nanofiber‐modified electrode

In this paper, an electrochemical aptamer sensor was proposed for the highly sensitive detection of mercury ion (Hg²⁺). Carbon nanofiber (CNF) was prepared by electrospinning and high‐temperature carbonization, which was used for the loading of platinum nanoparticles (PtNPs) by the hydrothermal method. The Pt@CNF nanocomposite was modified on the surface of carbon ionic liquid electrode (CILE) to obtain Pt@CNF/CILE, which was further decorated by gold nanoparticles (AuNPs) through electrodeposition to get Au/Pt@CNF/CILE. Self‐assembling of the thiol‐based aptamer was further realized by the formation of Au‐S bond to get an electrochemical aptamer sensor (Aptamer/Au/Pt@CNF/CILE). Due to the specific binding of aptamer probe to Hg²⁺ with the formation of T‐Hg²⁺‐T structure, a highly sensitive quantitative detection of Hg²⁺ could be achieved by recording the changes of current signal after reacting with Hg²⁺ within the concentration range from 1.0 × 10^?15 mol/L to 1.0 × 10^?6 mol/L and the detection limit of 3.33 × 10^?16 mol/L (3σ). Real water samples were successfully analyzed by this method.     

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 (Ba²⁺, Ca²⁺, K⁺, Mg²⁺, Na⁺, NH₄⁺, Sr²⁺ and the [Ru(NH₃)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(NH₃)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.     

A capillary electrophoresis system with dual capacitively coupled contactless conductivity detection and electrospray ionization tandem mass spectrometry

A commercial system that is comprised of a CE coupled to an ESI triple quadrupole mass spectrometer was equipped with two capacitively coupled contactless conductivity detectors (C⁴Ds). The first C⁴D was positioned inside the original cartridge, and the second C⁴D was positioned as close as possible to the ESI probe entrance by using a 3D‐printed support. The C⁴Ds electropherograms were matched to the ESI‐MS electropherogram by correcting their timescales by the factor L_T/L_D, where L_T and L_D are the total capillary length and the length until the C⁴D, respectively. A general approach for method development supporting the simultaneous conductivity and MS detection is discussed, while application examples are introduced. These examples include the use of C⁴D as a simple device that dismiss the use of an EOF marker, a low‐selectivity detector that continuously provide information about unexpected features of the sample, and even a detector that can be more sensitive than ESI‐MS. The C⁴D used in this setup proved to have a smaller contribution to the peak broadening than ESI‐MS, which allowed that a C⁴D, positioned at 12 cm from the inlet of an 80‐cm‐long capillary, could be used to foresee position and shape of the peaks being formed 6.8 times slower at the ESI‐MS electropherogram.     

Label-free aptamer-based colorimetric detection of mercury ions in aqueous media using unmodified gold nanoparticles as colorimetric probe

We report a simple and sensitive aptamer-based colorimetric detection of mercury ions (Hg²⁺) using unmodified gold nanoparticles as colorimetric probe. It is based on the fact that bare gold nanoparticles interact differently with short single-strand DNA and double-stranded DNA. The anti-Hg²⁺ aptamer is rich in thymine (T) and readily forms T–Hg²⁺–T configuration in the presence of Hg²⁺. By measuring color change or adsorption ratio, the bare gold nanoparticles can effectively differentiate the Hg²⁺-induced conformational change of the aptamer in the presence of a given salt with high concentration. The assay shows a linear response toward Hg²⁺ concentration through a five-decade range of 1 × 10⁻⁴ mol L⁻¹ to 1 × 10⁻⁹ mol L⁻¹. Even with the naked eye, we could identify micromolar Hg²⁺ concentrations within minutes. By using the spectrometric method, the detection limit was improved to the nanomolar range (0.6 nM). The assay shows excellent selectivity for Hg²⁺ over other metal cations including K⁺, Ba²⁺, Ni²⁺, Pb²⁺, Cu²⁺, Cd²⁺, Mg²⁺, Ca²⁺, Zn²⁺, Al³⁺, and Fe³⁺. The major advantages of this Hg²⁺ assay are its water-solubility, simplicity, low cost, visual colorimetry, and high sensitivity. This method provides a potentially useful tool for the Hg²⁺ detection.     

Polythymine Oligonucleotide‐Modified Gold Electrode for Voltammetric Determination of Mercury(II) in Aqueous Solution

Polythymine oligonucleotide (PTO)‐modified gold electrode (PTO/Au) was developed for selective and sensitive Hg²⁺ detection in aqueous solutions. This modified electrode was prepared by self‐assembly of thiolated polythymine oligonucleotide (5′‐SH‐T₁₅‐3′) on the gold electrode via Au? S bonds, and then the surface was passivated with 1‐mercaptohexanol solution. The proposed electrode utilizes the specific binding interactions between Hg²⁺ and thymine to selectively capture Hg²⁺, thereby reducing the interference from coexistent ions. After exchanging the medium, electrochemical reduction at ?0.2 V for 60 s, voltammetric determination was performed by differential pulse voltammetry using 10 mM HEPES; pH 7.2, 1 M NaClO₄ as supporting electrolyte. This electrode showed increasing voltammetric response in the range of 0.21 nM Hg²⁺, with a relative standard deviation of 5.32% and a practical detection limit of 60 pM. Compared with the conventional stripping approach, the modified electrode exhibits good sensitivity and selectivity, and is expected to be a new type of green electrode.     

Dulplex analysis of mercury and silver ions using a label-free fluorescent aptasensor

Label-free Hg²⁺ aptamer was used as a sensing element and the PicoGreen dye was specific to ultra-sensitive double-stranded DNA (dsDNA), which achieved novel fluorescence assay for detection of both mercury and silver ions. In this aptasensor, Hg²⁺ bound to thymidine (T) to form T–Hg^2+-T base pairs and Ag⁺ specifically interacted with C–C mismatches to produce C–Ag⁺–C base pairs. The conformation changes prevented the aptamer from binding to its complementary sequences to form dsDNA and caused a fluorescence intensity decrease with PicoGreen. The change in the fluorescence intensity made it possible to detect both Hg²⁺ and Ag⁺ in a dose-dependent manner. The sensing system could detect as low as 5 × 10^–8 mol/L of Hg²⁺ and 9.3 × 10^–10 mol/L of Ag⁺. The fluorescent intensity changes in the system were specific for Hg²⁺ and Ag⁺, making this simple and cost-effective method extremely valuable in its future applications in monitoring Hg²⁺ and Ag⁺ pollution in environmental analysis.     

Probing the mechanism of the interaction between l‐cysteine‐capped‐CdTe quantum dots and Hg2+ using capillary electrophoresis with ensemble techniques

A good understanding of the mechanism of interaction between quantum dots (QDs) and heavy metal ions is essential for the design of more effective sensor systems. In this work, CE was introduced to explore how l ‐cysteine‐capped‐CdTe QDs (l ‐cys‐CdTe QDs) interacts with Hg²⁺. The change in electrophoretic mobility can synchronously reflect the change in the composition and property of QDs. The effects of the free and capping ligands on the system are discussed in detail. ESI‐MS, dynamic light scattering (DLS), zeta potential, and fluorescence (FL) were also applied as cooperative tools to study the interaction mechanism. Furthermore, the interaction mechanism, which principally depended on the concentration of Hg²⁺, was proposed reasonably. At the low concentration of Hg²⁺, the formation of a static complex between Hg²⁺ and the carboxyl and amino groups of l ‐cys‐CdTe QDs surface was responsible for the FL quenching. With the increase of Hg²⁺ concentration, the capping l ‐cys was stripped from the surface of l ‐cys‐CdTe QDs due to the high affinity of Hg²⁺ to the thiol group of l ‐cys. Our study demonstrates that CE can reveal the mechanism of the interaction between QDs and heavy metal ions, such as FL quenching.     

Interaction study of a lysozyme‐binding aptamer with mono‐ and divalent cations by ACE

Binding between an aptamer and its target is highly dependent on the conformation of the aptamer molecule, this latter seeming to be affected by a variety of cations. As only a few studies have reported on the interactions of monovalent or divalent cations with aptamers, we describe herein the use of ACE in its mobility shift format for investigating interactions between various monovalent (Na⁺, K⁺, Cs⁺) or divalent (Mg²⁺, Ca²⁺, Ba²⁺) cations and a 30‐mer lysozyme‐binding aptamer. This study was performed in BGEs of different natures (phosphate and MOPS buffers) and ionic strengths. First, the effective charges of the aptamer in 30 mM ionic strength phosphate and MOPS (pH 7.0) were estimated to be 7.4 and 3.6, respectively. Then, corrections for ionic strength and counterion condensation effects were performed for all studies. The effective mobility shift was attributed not only to these effects, but also to a possible interaction with the buffer components (binary or ternary complexes) as well as possible conformational changes of the aptamer. Finally, apparent binding constants were calculated for divalent cations with mathematical linearization methods, and the influence of the nature of the BGE was evidenced.     

Highly Specific Recognition of Guanosine Using Engineered Base-Excised Aptamers

Purines and their derivatives are highly important molecules in biology for nucleic acid synthesis, energy storage, and signaling. Although many DNA aptamers have been obtained for binding adenine derivatives such as adenosine, adenosine monophosphate, and adenosine triphosphate, success for the specific binding of guanosine has been limited. Instead of performing new aptamer selections, we report herein a base-excision strategy to engineer existing aptamers to bind guanosine. Both a Na⁺-binding aptamer and the classical adenosine aptamer have been manipulated as base-excising scaffolds. A total of seven guanosine aptamers were designed, of which the G16-deleted Na⁺ aptamer showed the highest bindng specificity and affinity for guanosine with an apparent dissociation constant of 0.78 mm . Single monophosphate difference in the target molecule was also recognizable. The generality of both the aptamer scaffold and excised site were systematically studied. Overall, this work provides a few guanosine binding aptamers by using a non-SELEX method. It also provides deeper insights into the engineering of aptamers for molecular recognition.     

Determination of mercury species by the diffusive gradient in thin film technique and liquid chromatography – atomic fluorescence spectrometry after microwave extraction

A diffusive gradient in thin films technique (DGT) was combined with liquid chromatography (LC) and cold vapor atomic fluorescence spectrometry (CV-AFS) for the simultaneous quantification of four mercury species (Hg²⁺, CH₃Hg⁺, C₂H₅Hg⁺, and C₆H₅Hg⁺). After diffusion through an agarose diffusive layer, the mercury species were accumulated in resin gels containing thiol-functionalized ion-exchange resins (Duolite GT73, and Ambersep GT74). A microwave-assisted extraction (MAE) in the presence of 6 M HCl and 5 M HCl (55 °C, 15 min) was used for isolation of mercury species from Ambersep and Duolite resin gels, respectively. The extraction efficiency was higher than 95.0% (RSD 3.5%). The mercury species were separated with a mobile phase containing 6.2% methanol + 0.05% 2-mercaptoethanol + 0.02 M ammonium acetate with a stepwise increase of methanol content up to 80% in the 16th min on a Zorbax C18 reverse phase column. The LODs of DGT–MAE–LC–CV-AFS method were 38 ng L⁻¹ for CH₃Hg⁺, 13 ng L⁻¹ for Hg²⁺, 34 ng L⁻¹ for C₂H₅Hg⁺ and 30 ng L⁻¹ for C₆H₅Hg⁺ for 24 h DGT accumulation at 25 °C.     

Highly Sensitive Electrochemical Bioassay for Hg(II) Detection Based on Plasma-Polymerized Propargylamine and Three-Dimensional Reduced Graphene Oxide Nanocomposite

In this study, a nanocomposite consisting of three-dimensional reduced graphene oxide (3D-rGO) and plasma-polymerized propargylamine (3D-rGO@PpPG) was prepared and used as a highly sensitive and selective DNA sensor for detecting Hg²⁺. Given the high density of amino groups in the resultant 3D-rGO@PpPG nanocomposite, thymine-rich and Hg²⁺-targeted DNA was preferentially immobilized on the fabricated sensor surface via the strong electrostatic interaction between DNA strands and the amino-functionalized nanocomposites, followed by detecting Hg²⁺ through T–Hg²⁺–T coordination chemistry between DNA and Hg²⁺. The results of electrochemical measurements revealed that the anchored amount of DNA strands anchored on the 3D-rGO@PpPG nanofilm surface affects the determination of Hg²⁺ in aqueous solution. It showed high sensitivity and selectivity toward Hg²⁺ within concentrations ranging from 0.1 to 200 nM and displayed a low detection limit of 0.02 nM. The new strategy proposed also provides high selectivity of Hg²⁺ against other interfering metal ions, good stability, and repeatability. The excellent applicability of the developed sensor confirms the potential use of plasma-modified nanofilms for the detection of heavy metal ions in real environmental samples and water.