Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg²⁺) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg²⁺, the specific coordination between Hg²⁺ and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H₂O₂ in the presence of dissolved O₂. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H₂O₂, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg²⁺ detection with the dynamic concentration range spanning from 1.0 ng L⁻¹ to 10 mg L⁻¹ Hg²⁺ and a detection limit of 0.5 ng L⁻¹ (2.5 pM) at the 3S_blank level, and it also demonstrated excellent selectivity against other interferential metal ions.     

利用对G-四链体环部的构型调节进行传感器的设计

对鸟嘌呤碱基G重复序列之间连接环结构对G-四链体形成的影响进行了研究。发现在连接环较长,DNA链不易形成G-四链体的情况下,可以通过将环序列设计成双链结构的方式促进G-四链体的重新形成。这就为传感器的设计提供了一个新途径,即可以利用目标分子对环部双链的调节作用控制G-四链体DNA酶的活性。为证明这一点,在双链区域引入T-T碱基错配,破坏双链结构使DNA链不能形成G-四链体。Hg2+对T-T错配的稳定作用可以促进双链结构的形成,DNA链重新折叠成G-四链体,得到的G-四链体与氯化血红素(Hemin)结合后形成具有过氧化物酶活性的G-四链体DNA酶,据此构建了Hg2+传感器。利用此传感器可在10~700 nmol/L范围内实现Hg2+的定量检测,检出限为8.7 nmol/L。在此基础上,利用半胱氨酸可以将Hg2+从T-Hg2+-T碱基对上竞争下来的能力,设计了一种半胱氨酸的检测方法。此方法可以在20~600 nmol/L范围内实现半胱氨酸的定量检测,检出限为14 nmol/L。     

Simultaneous Determination of Cadmium,Lead, Copper and Mercury Ions Using Organofunctionalized SBA‐15 Nanostructured Silica Modified Graphite–Polyurethane Composite Electrode

A new sensor has been developed for the simultaneous detection of cadmium, lead, copper and mercury, using differential pulse and square wave anodic stripping voltammetry (DPASV and SWASV) at a graphite–polyurethane composite electrode with SBA‐15 silica organofunctionalized with 2‐benzothiazolethiol as bulk modifier. The heavy metal ions were preconcentrated on the surface of the modified electrode at ?1.1 V vs. SCE where they complex with 2‐benzothiazolethiol and are reduced to the metals, and are then reoxidized. Optimum SWASV conditions lead to nanomolar detection limits and simultaneous determination of Cd²⁺, Pb²⁺, Cu²⁺ and Hg²⁺ in natural waters was achieved.     

Sensitive electrochemical DNA-based biosensors for the determination of Ag<Superscript>+</Superscript> and Hg<Superscript>2+</Superscript> ions and their application in analysis of amalgam filling

In the present paper, we used single-stranded poly-T (100% thymine bases) and poly-C (100% cytosine bases) nucleic acids as DNA probes for selective and sensitive individual electrochemical determination of Hg²⁺ and Ag⁺_, respectively, on the multi-walled carbon nanotube paste electrodes (MWCNTPEs) using [Fe(CN)₆]^3?/4? as electroactive labels. In the presence of Hg²⁺ and Ag⁺, the probe–Hg²⁺/Ag⁺ interactions through T–Hg²⁺–T and C–Ag⁺–C complexes formation could cause the formation of a unimolecular hybridized probe. This structure of probe led to its partial depletion from electrode surface and facilitation of electron transfer between [Fe(CN)₆]^3?/4? redox couple and electrode surface, resulting in the enhanced differential pulse voltammetry (DPV) oxidation current of [Fe(CN)₆]^3?/4? at the probe-modified electrode surface. We applied the difference in the oxidation peak currents of [Fe(CN)₆]^3?/4? before and after Hg²⁺/Ag⁺–DNA probe bonding (?I) for electrochemical determination of these heavy metal ions. Detection limits were 8.0?×?10^?12 M and 1.0?×?10^?11 M for Hg²⁺ and Ag⁺ ions determination, respectively. The biosensors were utilized to determine the weight percent of toxic metals, i.e., silver and mercury in dental amalgam filling composition. The results of their practical applicability in analysis of the amalgam sample were satisfactory.     

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.     

Detection of Metal Ions (Cu2+, Hg2+) and Cocaine by Using Ligation DNAzyme Machinery

The Cu²⁺‐dependent ligation DNAzyme is implemented as a biocatalyst for the colorimetric or chemiluminescence detection of Cu²⁺ ions, Hg²⁺ ions, or cocaine. These sensing platforms are based on the structural tailoring of the sequence of the Cu²⁺‐dependent ligation DNAzyme for specific analytes. The tethering of a subunit of the hemin/G‐quadruplex DNAzyme to the ligation DNAzyme sequence, and the incorporation of an imidazole‐functionalized nucleic‐acid sequence, which acts as a co‐substrate for the ligation DNAzyme that is tethered to the complementary hemin/G‐quadruplex subunit. In the presence of different analytes, Cu²⁺ ions, Hg²⁺ ions, or cocaine, the pretailored Cu²⁺‐dependent ligation DNAzyme sequence stimulates the respective ligation process by combining the imidazole‐functionalized co‐substrate with the ligation DNAzyme sequence. These reactions lead to the self‐assembly of stable hemin/G‐quadruplex DNAzyme nanostructures that enable the colorimetric analysis of the substrate through the DNAzyme‐catalyzed oxidation of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid), ABTS^2?, by H₂O₂ into the colored product ABTS^.?, or the chemiluminescence detection of the substrate through the DNAzyme‐catalyzed oxidation of luminol by H₂O₂. The detection limits for the sensing of Cu²⁺ ions, Hg²⁺ ions, and cocaine correspond to 1 nM , 10 nM and 2.5 μM , respectively. These different sensing platforms also reveal impressive selectivities.     

Ionic liquids dispersive liquid–liquid microextraction and HPLC‐atomic fluorescence spectrometric determination of mercury species in environmental waters

An ionic liquid (IL) based dispersive liquid–liquid microextraction combined with HPLC hydride generation atomic fluorescence spectrometry method for the preconcentration and determination of mercury species in environmental water samples is described. Four mercury species (MeHg⁺, EtHg⁺, PhHg⁺, and Hg²⁺) were complexed with dithionate and the neutral chelates were extracted into IL drops using dispersive liquid–liquid microextraction. Variables affecting the formation and extraction of mercury dithizonates were optimized. The optimum conditions found were as follows: IL‐type and amount, 0.05 g of 1‐octyl‐3‐methylimidazolium hexafluorophosphate; dispersive solvents type and amount, 500 μL of acetone; pH, 6; extraction time, 2 min; centrifugation time, 12 min; and no sodium chloride addition. Under the optimized conditions, the detection limits of the analytes were 0.031 μg/L for Hg²⁺, 0.016 μg/L for MeHg⁺, 0.024 μg/L for EtHg⁺, and 0.092 μg/L for PhHg⁺, respectively. The repeatability of the method, expressed as RSD, was between 1.4 and 5.2% (n = 10), and the average recoveries for spiked test were 96.9% for Hg²⁺, 90.9% for MeHg⁺, 90.5% for EtHg⁺, 92.3% for PhHg⁺, respectively. The developed method was successfully applied for the speciation of mercury in environmental water samples.     

Highly selective detection of mercury (II) using a G-rich oligonucleotide-based fluorescence quenching method

We provide a highly sensitive and selective assay to detect Hg²⁺ in aqueous solutions using single fluorescence-labeled G-quadruplex at room temperature. The mechanism is that AS1411 converted to G-quadruplex in the presence of potassium ion, and then, by this technique utilizing the high binding capacity of T–Hg²⁺–T makes the fluorescence dye come closer to GGG of AS1411 to causing fluorescence signal quenching by photoinduced electron transfer energy transfer. At physiological pH, the detection limit can be as low as 10 nM, with high selectivity toward Hg²⁺ ions over a lot of metal ions. The linear correlation existed between the fluorescence intensity and the concentration of Hg²⁺ over the range of 0–250 nM (R = 0.9920) in real sample. Accordingly, we expect this G-quadruplex-based sensor will be a potential application for detection of environmentally toxic mercury.     

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.     

A signal amplification by QDs used for ferrocene-labeled sandwich aptasensor for determination of Hg2+ in water samples

In this paper, the design of a novel sandwich-type electrochemical aptasensor was reported for an ultrasensitive mercury ion (Hg²⁺) detection in water samples, which labeled with two-labeled aptamer (Apt) sequences. The used Apts were Apt1 and Apt2 as the capture and signal probe, respectively. The Apt1 probe was immobilized on the poly(4-aminobenzoic acid) (p-ABA) and quantum dots (QDs) film as the platform, as well as the Apt2 reporter was labeled with ferrocene. In the presence of Hg²⁺, the strong coordination complex has been formed between the specific thymine of the Apt1, Hg²⁺, as well as the thymine of the Apt as T–Hg²⁺–T adduct. The QDs and p-ABA were applied for increasing the conductivity of platform and suitable binding of the recognition elements. Under the optimized conditions, the constructed aptasensor illustrated either a wide linear relationship between the logarithm of Hg²⁺ concentration and current, from 0.05 to 100 nM and also an excellent low limit of detection of 0.01 nM. The quality of carefully choosing, an excellent stability and specificity sensitivity of the designed aptasensor, was investigated by spiked tap water samples as real sample. Moreover, the aptasensor exhibits the good reproducibility as well as has high selectivity for the other cations. The recoveries of the Hg²⁺ assay of the tap water samples were acquired satisfactorily which imply the generated aptasensor can use Hg²⁺ measurement in the real laboratories.

     

Eco‐friendly Sensors Developed by Herbal Based Silver Nanoparticles for Electrochemical Detection of Mercury (II) Ion

In our study, the single‐use & eco‐friendly electrochemical sensor platform based on herbal silver nanoparticles (AgNPs) was developed for detection of mercury (II) ion (Hg²⁺). For this purpose, the surface of pencil graphite electrode (PGE) was modified with AgNPs and folic acid (FA), respectively. The concentrations of AgNPs and FA were firstly optimized by differential pulse voltammetry (DPV) to obtain an effective surface modification of PGE. Each step at the surface modification process was characterized by using cyclic voltammetry (CV) and electrochemical impedence spectroscopy (EIS). The limit of detection (LOD) for Hg²⁺ was estimated and found to be 8.43 μM by CV technique. The sensor presented an excellent selectivity for Hg²⁺ against to other heavy metal ions such as Ca²⁺, Cd²⁺, Cr³⁺, Cu²⁺, Mg²⁺, Ni²⁺, Pb²⁺, Zn²⁺, Co²⁺ and Mn²⁺. Moreover, a rapid, selective and sensitive detection of Hg²⁺ was successfully performed in the samples of tap water within 1 min.     

Ultrasensitive and rapid screening of mercury(II) ions by dual labeling colorimetric method in aqueous samples and applications in mercury-poisoned animal tissues

Rapid and ultrasensitive detection of trace heavy metal mercury(II) ions (Hg²⁺) are of significant importance due to the induced serious risks for environment and human health. This presented article reports the gold nanoparticle-based dual labeling colorimetric method (Dual-COLO) for ultrasensitive and rapid detection of Hg²⁺ using the specific thymine–Hg²⁺–thymine (T–Hg²⁺–T) as recognition system and the dual labeling strategy for signal amplification. Both qualitative and quantitative detections of Hg²⁺ are achieved successfully in aqueous samples. More importantly, the achieved detection limit of 0.005 ng mL⁻¹ (0.025 nM) without any instruments is very competitive to other rapid detection methods even ICP-MS based methods. This Dual-COLO method is also applied directly for real water sample monitoring and, more importantly, applied in analysis of mercury poisoned animal tissues and body fluidic samples, indicating a potentially powerful and promising tool for environmental monitoring and food safety control.     

One-step Bioluminescence ATPase Assay for the Evaluation of Neurotoxic Effects of Metal Ions

Summary. Membrane-bound ATPases, such as Na,K-ATPase and nucleotide triphospho-diphosphohydrolase (NTPDase), being one of the first targets of a toxic action are generally considered as good markers for estimating toxicity. A bioluminescence assay was applied for fast and sensitive evaluation of heavy metals effect on the rat brain synaptosomal membrane ATPase activity. The assay consists of ATP-consuming reaction catalyzed by synaptic plasma membrane ATPases coupled to the luminescent firefly luciferase reaction, which consumes residual ATP after the course of ATPase reaction. The bioluminescence ATPase assay was applied to study the effect of heavy and transitional metals (Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺) on rat brain ATPase activity after assay optimization. All metals applied inhibited synaptic membrane ATPase activity in a concentration dependent manner. The IC ₅₀ values (Hg²⁺ < Cu²⁺ < Cd²⁺ < Pb²⁺) obtained with the bioluminescence assay were highly correlated with those obtained by the spectrophotometric method. The fast bioluminescence ATPase assay with small sample and substrate requirements could be adjusted for high-throughput environmental and pharmacological screening.     

Amplified Fluorescence Turn‐On Assay for Mercury(II) Detection and Quantification based on Conjugated Polymer and Silica Nanoparticles

An amplified fluorescence turn‐on assay for mercury(II) detection and quantification was developed. This method makes use of specific thymine/mercury(II)/thymine coordination to capture Fl‐labeled DNA onto NP surface. Addition of a cationic conjugated polymer leads to an amplified Fl signal in solution. A sigmoidal Hg²⁺ working curve is obtained at fixed [NP] with a detection limit of 0.1 × 10⁻⁶ M . However, by reducing [Hg²⁺] and [NP] simultaneously, while maintaining [Hg²⁺]:[DNA duplex] = 3:1, a linear calibration curve is observed with a detection limit of 5 × 10⁻⁹ M . The CCP‐assisted mercury(II) assay shows potential applications in environmental mercury detection and for industrial process control.

     

Highly Sensitive Fluorometric Hg2+ Biosensor with a Mercury(II)‐Speci?c Oligonucleotide (MSO) Probe and Water‐Soluble Graphene Oxide (WSGO)

A highly sensitive and selective, "turn‐on" and simple Hg²⁺ biosensor is reported by using water‐soluble graphene oxide (WSGO) and dye‐labeled mercury(II)‐specific oligonucleotide (MSO) probe. The probe is rich of thymine (T) and can readily form the stem‐loop structure which consists of the T‐Hg²⁺‐T configuration. In the absence of Hg²⁺, the probe exists as a random coil conformation which can be readily adsorbed on the surface of WSGO by strong noncovalent binding of bases, as a result, the fluorescence of the dye labeled on the terminus of the MSO is strongly quenched by the efficient electron/energy transfer from the dye to WSGO. Upon addition of Hg²⁺, the formation of the T‐Hg²⁺‐T structure releases the MSO from the surface of WSGO, resulting in a restoration of the fluorescence of dye‐labeled MSO probe. Based on this observation, a highly sensitive and selective Hg²⁺ sensor is developed, which can work with "turn‐on" mode in aqueous solutions at room temperature. By using the fluorometric method, the limit of detection for Hg²⁺ can reach picomolar range (187 pmol·L^?1), and it is demonstrated that the biosensor is highly selective and only minimally perturbed by a wide range of non‐specific metal ions.     

Chemiluminescent Detection of HIV DNA Based on Allosteric Activation of Peroxidase-Mimicking DNAzyme

Homogeneous chemiluminescent method for HIV DNA detection based on allosteric activation of peroxidase-mimicking DNAzyme (PMDNAzyme) was developed. The probes used in the assay contain PMDNAzyme fragment and the additional oligonucleotide sequence complementary to HIV DNA. The interaction of PMDNAzyme fragment and the additional oligonucleotide sequence results in changes in G-quadruplex structure of the PMDNAzyme and decreases peroxidase-like activity of the probe. In the presence of HIV DNA such interaction was destroyed due to the formation of stable duplex between the additional fragment of the probe and DNA-analyte. Consequently, some reorganizations in G-quadruplex structure of the probe are observed, which are accompanied by enhancement of catalytic activity of the PMDNAzyme. The mechanism of the DNA-dependent activation of PMDNAzyme containing probes was confirmed by CD spectroscopy as well as modeling of the probes and their complexes with DNA target. The calibration curves for HIV DNA determination allowed estimating the analytical parameters of the assay. The detection limit value and the linear range were shown to be 0.3 nM and 0.3–15 nM, respectively. The assay sensitivity was high (190000 nM^–1). The values of coefficient of variation (CV) measured within the working range varied less than 4%, which indicates the high accuracy of the proposed assay.     

Crystal Structure of Metallo DNA Duplex Containing Consecutive Watson–Crick‐like T–HgII–T Base Pairs

The metallo DNA duplex containing mercury‐mediated T–T base pairs is an attractive biomacromolecular nanomaterial which can be applied to nanodevices such as ion sensors. Reported herein is the first crystal structure of a B‐form DNA duplex containing two consecutive T–Hg^II–T base pairs. The Hg^II ion occupies the center between two T residues. The N3‐Hg^II bond distance is 2.0 Å. The relatively short Hg^II‐Hg^II distance (3.3 Å) observed in consecutive T–Hg^II–T base pairs suggests that the metallophilic attraction could exist between them and may stabilize the B‐form double helix. To support this, the DNA duplex is largely distorted and adopts an unusual nonhelical conformation in the absence of Hg^II. The structure of the metallo DNA duplex itself and the Hg^II‐induced structural switching from the nonhelical form to the B‐form provide the basis for structure‐based design of metal‐conjugated nucleic acid nanomaterials.     

Chemiluminescent Determination of MicroRNA-141 Using Target-Dependent Activation of the Peroxidase-Mimicking DNAzyme

A chemiluminescent method was developed for microRNA-141 (miRNA-141) detection based on the target-dependent activation of peroxidase-mimicking DNAzyme. The structure of the probes was optimized which allowed the development of a sensitive method for miRNA-141. Under the optimized conditions, the detection limit and the linear range were 100?pM and 0.1–50?nM, respectively. The sensitivity of the assay was 270,000?nM^?1. The values of coefficient of variation measured within the working range varied less than 2%, which indicates excellent precision for the proposed method.     

Ultrasensitive detection of lead ion based on target induced assembly of DNAzyme modified gold nanoparticle and graphene oxide

In this paper, we report a novel colorimetric strategy for the detection of small molecules by using Pb²⁺ ion as an example. In this strategy, DNAzyme duplex modified gold nanoparticles (GNPs) are designed to be unable to interact with graphene oxide (GO). However, in the presence of Pb²⁺, the substrate strand of the DNAzyme is cleaved at its cleavage site, resulting in the disassembly of the DNAzyme duplex modified GNPs into three parts, i.e., the 3′- and 5′-fragments of substrate strand and the DNAzyme strand modified GNPs. By taking advantage of the efficient cross-linking effect of ssDNA-GNPs to GO, colorimetric sensor for the detection of the metal ion can be fabricated with a detection limit of 100 pM, which is much lower than the previous reports. This colorimetric method has also been used for the determination of Pb²⁺ in the tap water of the local city and the water from a reservoir with satisfactory results, so it may have potential applications in the future.     

Oligonucleotides-based biosensors with high sensitivity and selectivity for mercury using electrochemical impedance spectroscopy

A novel electrochemical biosensor with high sensitivity and selectivity for mercuric ion detection, based on DNA self-assembly electrode, is designed. Thiol functionalized poly-T oligonucleotides were used as gold electrode modifier through formation of Au–S bond between DNA and gold electrode. In presence of Hg²⁺ ions, the specific coordination between Hg²⁺ and thymine bases can change parallel ss-DNA from linear to hairpin structures, which can cause the release of partial DNA molecules from the surface of the electrode. The density of DNA on the surface of electrode correlated with the concentration of mercury in the solution and can be monitored by electrochemical impedance spectroscopy. The limit of detection of this method is pM level of mercuric ions which is far below the upper limit of Hg²⁺ mandated by United States Environmental Protection Agency (EPA), 2 ppb (10 nM). In addition, this method showed excellent selectivity. A series of divalent metal ions, including Ni²⁺, Co²⁺, Mg²⁺, Zn²⁺, Ba²⁺ and Cd²⁺, have little interference with the detection of Hg²⁺.