Peroxidase Mimetics Research Articles

Carbon dots-stabilized Cu4O3 for a multi-responsive nanozyme with exceptionally high activity

A novel nanocomposite of carbon dots-stabilized paramelaconite (CDs@Cu4O3) is firstly reported and investigated as a stimuli-responsive nanozyme. The CDs@Cu4O3 is a more robust catalyst without incurring the burden of easy denaturation or decomposition and behaves as oxidase and peroxidase mimetics. The activity of CDs@Cu4O3 is up to 3.6 times higher than that of pure cuprous oxide nanoparticles and can be boosted by multiple kinds of external stimuli including of light irradiation, temperature, pH and oxidizing agent. We find that carbon dots play the key roles in facilitating the generation of Cu4O3 and sustaining Cu2+/Cu+ redox couples that are required for the catalytic reaction to proceed quickly. This nanocomposite endowed with ease of preparation, versatility and high stability is expected to be wide uses for biotechnology, environmental treatment, and organic synthesis.

Porphyrins as Colorimetric and Photometric Biosensors in Modern Bioanalytical Systems.

Advances in porphyrin chemistry have provided novel materials and exciting technologies for bioanalysis such as colorimetric sensor array (CSA), photo‐electrochemical (PEC) biosensing, and nanocomposites as peroxidase mimetics for glucose detection. This review highlights selected recent advances in the construction of supramolecular assemblies based on the porphyrin macrocycle that provide recognition of various biologically important entities through the unique porphyrin properties associated with colorimetry, spectrophotometry, and photo‐electrochemistry.

A label-free fluorescence biosensor based on a bifunctional MIL-101(Fe) nanozyme for sensitive detection of choline and acetylcholine at nanomolar level

A label-free and high sensitive fluorescence biosensor for the detection of choline and acetylcholine (ACh) was developed based on the MIL-101(Fe) nanozyme with dual functions of mimetic peroxidase and fluorescent emission. This fluorescence sensing strategy involves the reaction of acetylcholinesterase (AChE) catalyzing the hydrolysis of ACh into choline that is in turn oxidized by choline oxidase (ChOx) to generate H2O2, and the as-produced H2O2 is decomposed into highly reactive hydroxyl radicals under the catalysis of MIL-101(Fe) nanozyme, thereby the non-fluorescent organic ligand terephthalic acid of MIL-101(Fe) is oxidized by hydroxyl radicals to form a highly fluorescent 2-hydroxy terephthalic acid. By virtue of this finding, the label-free detection of choline and ACh via multienzyme cascade reaction was realized with low detection limits of 20.0 nM and 8.9 nM, respectively. Moreover, the practicality of the developed bifunctional MIL-101(Fe)-based sensing strategy was successfully validated through the detection of choline in milk and ACh in human plasma. This proposed assay not only exhibited high sensitivity and selectivity towards choline and ACh, but also simplified the sensing systems, reduced the detection cost, and avoided the potential risks caused by the fluorescent reagents, which opened a green avenue for the analysis of complex biological samples.

Well-water-dispersed N-trimethyl chitosan/Fe3O4 hybrid nanoparticles as peroxidase mimetics for quick and effective elimination of bacteria

Fe3O4 nanoparticles, used as peroxidase mimetics, exhibit splendid future in the biomedical field. However, the functionalization on Fe3O4 nanoparticles always goes with the loss of superparamagnetism and decrease in peroxidase-activity. Here, we synthesized green polyethylene glycol (PEG)-functionalized magnetic/N-trimethyl chitosan (CS) hybrid nanoparticles (Fe3O4@PAA/TMC/PEG NPs) with improved water dispersibility, superparamagnetism, high saturation magnetization and well peroxidase-like activity. The functionalized coating was divided in two steps, one involved a cross-linked PEG/PAA/CS middle layer to protect the nanocrystal Fe3O4 from oxidization, the other was a hydrophilic PEG/TMC outer layer improving the water dispersion, biocompatibility, as well as supplying positive quaternary ammonium groups for a potential increase of cell binding efficiency. The structure, composition and morphology of Fe3O4@PAA/TMC/PEG NPs were characterized by TEM, FT-IR spectroscopy, DLS, zeta potential measurement, respectively. Thermal performance was characterized by TGA, and the peroxidase-like mimics activity was tested by TMB·2HCl colour development experiments. The magnetic property of the as-prepared hybrid nanoparticles was first confirmed by VSM, and then proved by the bacterial pathogens adsorption, especially at ultralow pathogen concentration. Particularly, with an external magnet, the Fe3O4@PAA/TMC/PEG NPs, combined cationic quaternary ammonium groups and peroxidise-mimetic catalytic activity, were tested for antibacterial effect by plating method.

Detection mechanism and classification of design principles of peroxidase mimic based colorimetric sensors: A brief overview

Synthesis and characterization of enzyme mimics with characteristic stability and high catalytic efficiency is an interesting field for researchers. Especially, with the development of nanoscience and introducing of Fe 3 O 4 magnetic nanoparticles as peroxidase mimics in 2007, various nanomaterials such as noble metals, metal oxides, and carbon materials were introduced as enzyme mimics (nanozymes). Various nanomaterials exhibit peroxidase-like activity, hence, most of the nanozymes are peroxidase mimetics. Although the nanozyme based sensors were previously classified, the classifications have been focused on the type of nanozyme action. Therefore, the nanozyme based sensors were classified as peroxidase, hydrolase, and urease mimic-based sensors. However, heretofore, these sensors are not classified based on the detection mechanism and principles of system design. The aim of this review is the focus on the peroxidase mimic based colorimetric sensors as the most common nanozyme-based sensors and their classification based on principles of sensor design and review of the detection mechanism of the current mimic peroxidase based sensors. Moreover, some current challenges and future developments in this field are discussed.

Mimicking peroxidase active site microenvironment by functionalized graphene quantum dots

The development of high-efficiency peroxidase mimetics is highly desirable in view of high cost and low stability of natural enzymes. From the perspective of mimicking active site microenvironment at low cost, we herein report a novel histidine-functionalized graphene quantum dot (His-GQD)/hemin complex, which exhibits the highest catalytic rate for the peroxidase-based chromogenic reaction among the hemin-containing mimetics reported so far. Also, our peroxidase mimetic shows excellent tolerance to strongly acidic conditions and can function in a wide temperature range. Lineweaver-Burk plots and comprehensive electron paramagnetic resonance analysis reveal a ping-pong type catalytic mechanism for this mimetic. In addition, His-GQD/hemin demonstrates high efficiency and accuracy in detecting H2O2 and blood glucose. Our work provides an effective design of artificial enzymes for practical applications.

Colorimetric Assay of Bacterial Pathogens Based on Co3O4 Magnetic Nanozymes Conjugated with Specific Fusion Phage Proteins and Magnetophoretic Chromatography.

It is important to detect pathogens rapidly, sensitively, and selectively for clinical medicine, homeland security, food safety, and environmental control. We report here a specific and sensitive colorimetric assay that incorporated a bovine serum albumin-templated Co3O4 magnetic nanozyme (Co3O4 MNE) with a novel specific fusion phage protein and magnetophoretic chromatography to detect Staphylococcus aureus. The Co3O4 MNE was conjugated to S. aureus-specific fusion-pVIII (Co3O4 MNE@fusion-pVIII), screened from the S. aureus-specific phage AQTFLGEQD (the phage monoclone is denoted by the peptide sequence). The as-prepared triple-functional Co3O4 MNE@fusion-pVIII particles were capable of capturing S. aureus in sterile milk, which were then isolated from milk magnetically. Assisted by polyethylene glycol, the Co3O4 MNE@fusion-pVIII@S. aureus complex was separated from the free Co3O4 MNE@fusion-pVIII by magnetophoretic chromatography in an external magnetic field. After transferring the isolated Co3O4 MNE@fusion-pVIII@S. aureus complexes into a 96-well plate, diammonium salt of 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) and H2O2 were added to develop color because of the peroxidase mimetics activity of the Co3O4 MNE. A S. aureus concentration within 10-10,000 cfu/mL in milk can be detected (detection limit: 8 cfu/mL). The as-developed method is simple, cost-efficient, and sensitive, which is useful for rapidly diagnosing pathogenic bacteria and helpful to prevent disease outbreaks induced by pathogens in developing countries.

Seleninic Acid Potassium Salts as Water-Soluble Biocatalysts with Enhanced Bioavailability.

Organoselenium compounds are well-known glutathione peroxidase (GPx) mimetics that possess antioxidants/prooxidant properties and are able to modulate the concentration of reactive oxygen species (ROS), preventing oxidative stress in normal cells or inducing ROS formation in cancer cells leading to apoptosis. The purpose of this study was the synthesis of potent GPx mimics with antioxidant and anticancer activity along with improved bioavailability, as a result of good solubility in protic solvents. As a result of our research, glutathione peroxidase (GPx) mimetics in the form of water-soluble benzeneseleninic acid salts were obtained. The procedure was based on the synthesis of 2-(N-alkylcarboxyamido)benzeneselenenic acids, through the oxidation of benzisoselenazol-3(2H)-ones or analogous arenediselenides with an amido group, which were further converted to corresponding potassium salts by the treatment with potassium tert-butanolate. All derivatives were tested as potential antioxidants and anticancer agents. The areneseleninic acid salts were significantly better peroxide scavengers than analogous acids and the well-known organoselenium antioxidant ebselen. The highest activity was observed for the 2-(N-ethylcarboxyamido)benzeneselenenic acid potassium salt. The strongest cytotoxic effect against breast cancer (MCF-7) and human promyelocytic leukemia (HL-60) cell lines was found for 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid potassium salt and the 2-(N-ethylcarboxyamido)benzeneselenenic acid, respectively. The structure–activity correlations, including the differences in reactivity of benzeneseleninic acids and corresponding salts were evaluated.

A highly sensitive and adjustable colorimetric assay of hydrogen sulfide by signal amplification based on G-quadruplex-Cu2+ peroxidase mimetics.

This work reports the first example of a colorimetric H2S sensor constructed through G-quadruplex-Cu2+ (G4-Cu2+) peroxidase mimetics employing Cu2+ ions and G-rich DNA with signal amplification. In the hydrogen peroxide (H2O2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), the catalytic capacity of Cu2+ can be greatly improved in the presence of 22AG DNA, where 22AG DNA acts as a signal amplifier. However, G4-Cu2+ peroxidase mimetics lose their catalytic abilities after reacting with H2S. This is employed to develop a colorimetric assay of H2S without complex synthesis and instrumentation, with a linear range from 0.01 μM to 150 μM and a detection limit of 7.5 nM. The sensitivity of the sensor can also be adjusted by changing the concentration of Cu2+. Moreover, the developed sensor is successfully applied for the quantitative determination of H2S in human serum samples.

Modified Ti3C2 nanosheets as peroxidase mimetics for use in colorimetric detection and immunoassays.

Since being discovered in 2011, a large class of two-dimensional materials, labeled MXenes, has received increased research enthusiasm both theoretically and experimentally due to the unique physical, optical and electrical properties. Here, we prepared few-layered Ti3C2 nanosheets by a facile two-step liquid exfoliation method and, for the first time, demonstrated their intrinsic peroxidase-like activity in a Ti3C2-TMB-H2O2 system. The as-produced Ti3C2 nanosheets, especially after histidine modification, were characterized with excellent water dispersibility, large specific surface area, and high stability, which contribute to their much higher affinity to both substrates when compared to HRP. We have also established the catalytic mechanism whereby Ti3C2 nanosheets, where Ti switched spontaneously from an oxidized to reduced state, promoted the electron transfer from TMB to H2O2. Given the color reaction of Ti3C2 nanosheets, we have fabricated a colorimetric paper-based sensor integrated with a smartphone to detect glucose and an immunoassay to detect IR-β, enabling Ti3C2 nanosheets to be a powerful tool in the biodetection field.

Antigen-labeled mesoporous silica-coated Au-core Pt-shell nanostructure: a novel nanoprobe for highly efficient virus diagnosis

BackgroundAs an emerging research area of artificial enzymes, nanozyme, the catalytic nanomaterials with enzyme-like characteristics, have attracted enormous attention in research. Here, a nanozyme probe has been realized by utilizing antigen-labeled mesoporous silica-encapsulated Au-core Pt-shell (Au@Pt@SiO2) nanostructures for the diagnosis of rubella virus (RV). Pt nanoparticles have been suggested to act as potent peroxidase mimetics with high activities. However, smaller Pt nanoparticles are very easily aggregated, which has negative effects on the catalytic activities.ResultsIn this work, the use of gold nanorod as the support favours the well dispersion of the small Pt nanoparticles to improve the stability of them. Furthermore, the designed the silica shell could also isolate the recognition antigens from the surface reactive sites, retaining catalytic activity of the inner nanozyme. In addition, compared with antigen-labeled horseradish peroxidase (HRP), the antigen-labeled Au@Pt@SiO2 nanozyme was more stable and robust. A capture enzyme-linked immunosorbent assay (ELISA) for the determination of RV showed that the antigen-labeled Au@Pt@SiO2 nanozyme-based ELISA exhibited good sensitivity.ConclusionsThe highly sensitive peroxidase-like activity of antigen-labeled Au@Pt@SiO2 nanozyme, along with their catalytic stability and robustness, can facilitate their utilization in biochemical assays and clinical diagnosis.

A novel signal-off photoelectrochemical biosensor for M.SssI MTase activity assay based on GQDs@ZIF-8 polyhedra as signal quencher

DNA methylation catalyzed by M.SssI methyltransferases (MTase) has important roles in gene expression and other cellular activities, and relates to some diseases, especially cancers. Therefore, it is important to develop a sensitive sensing platform for M.SssI MTase activity assay. Here, taking zeolitic imidazolate framework-8 (ZIF-8) polyhedra as the carriers of graphene quantum dots (GQDs), GQDs-embedded ZIF-8 polyhedra (denoted as GQDs@ZIF-8 polyhedra) were successfully prepared and used as the multi-functional signal quencher to construct a novel signal-off photoelectrochemical (PEC) biosensor for M.SssI MTase activity assay. Firstly, the indium tin oxide (ITO) slice was modified with TiO2, poly(diallyldimethylammonium chloride) and CdTe quantum dots (QDs). The obtained electrode was used as the photoelectrode and labeled as ITO/TiO2/CdTe QDs. Then, single-stranded DNA (S1) was anchored on the photoelectrode surface via S–Cd bond. After hybridization between S1 and biotinylated single-stranded DNA (S2), the streptavidin (SA)-labeled GQDs@ZIF-8 polyhedra were introduced to the modified electrode via the specific reaction between biotin and SA. As the signal quencher, GQDs@ZIF-8 polyhedra could not only inhibit the photocurrent signal of the ITO/TiO2/CdTe QDs electrode due to the steric hindrance effect, but also act as peroxidase mimetics to catalyze precipitation reaction of 4-chloro-1-naphthol, resulting in the evident depression of the photocurrent signal. For the specially designed S1/S2 double-strand DNA, the decreased photocurrent was quantitatively correlated with the M.SssI MTase activity (linear response range, 0.005–150 U mL−1; detection limit, 0.004 U mL−1). The developed GQDs@ZIF-8 polyhedra and related PEC biosensor may have potential applications in clinical research and disease diagnosis.

ATP Mimics pH-Dependent Dual Peroxidase-Catalase Activities Driving H 2 O 2 Decomposition

Adenosine triphosphate (ATP) is produced mainly in the mitochondrion, and its primary task is to function as a ubiquitous energy currency to meet the cellular metabolic demands in biological system...

Nitrogen- and sulfur-doped carbon dots as peroxidase mimetics: colorimetric determination of hydrogen peroxide and glutathione, and fluorimetric determination of lead(II).

Fluorescent carbon dots co-doped with nitrogen and sulfur (N/S CDs) were prepared and found to display viable peroxidase mimicking activity. They have a blue fluorescence (with excitation/emission maxima at 340/456nm) with a quantum yield of 35%. The N/S CDs catalyze the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2, and this leads to the appearance of a blue solution with a absorption maximum at 654nm. A colorimetric method was developed for the determination of H2O2 that has a 1.75μM detection limit and a linear response in the 10-5 to 10-4M concentration range. The method can be extended to the enzymatic determination of glutathione with a 0.26μM detection limit and a working rangefrom 0.20 to 100μM. In addition, the CDs respond to lead(II) which is a quencher of the blue fluorescence at 456nm, with a detectionlimit of 11μM and a working range up to 100μM. Simultaneously, the color changes can be visually detected with absorbance signal changes from 10 to 100μM with limit of 3.9μM. A multiple detection system was worked out that allows monitoring of H2O2 and glutathione successively, and of lead(II). Graphical abstract (A) Schematic representation of the nitrogen & sulphur doped carbon dots with blue fluorescence, (B) the peroxidase-like activity in colorimetric detecting of H2O2 and GSH and (C) the illustration for the application of Pb2+ detection with fluorescence and colorimetric method.

Colorimetric assay of acetylcholinesterase inhibitor tacrine based on MoO2 nanoparticles as peroxidase mimetics.

Molybdenum dichalcogenides MoX2 (X=S, Se) have been found to possess intrinsic peroxidase-like activity. However, molybdenum oxides (MoO2) as peroxidase mimetics have not been exploited yet. Herein, MoO2 nanoparticles were synthesized by a simple hydrothermal method and found to possess the peroxidase-like activity for the first time. MoO2 nanoparticles could catalyze the oxidation of 3,3',5,5'-tetrametylbenzidine (TMB) by H2O2 to produce a blue-color product (oxTMB). The catalytic property and mechanism were investigated by stead-state kinetics experiment and free radicals scavenging experiment, respectively. Acetylcholinesterase (AChE) could catalyze the hydrolysis of acetylthiocholine chloride (ATCh) into thiocholine (TCh), which could reduce oxTMB to decrease the absorbance in solution. In the presence of AChE inhibitor tacrine, the generation of TCh was inhibited and the absorbance was preserved. Based on these properties, a colorimetric assay method was developed for AChE inhibitor tacrine. This work not only broadens the application of the peroxidase mimetics, but also overcome the disadvantages of traditional methods such as expensive, complex and vulnerable to background interference for colorimetric assay of AChE inhibitor.

MXene-Ti3C2/CuS nanocomposites: Enhanced peroxidase-like activity and sensitive colorimetric cholesterol detection.

Nanomaterials with enzyme-like activity have attracted much attention recently. Herein, we report the synthesis of a new type of 2D MXene-Ti3C2/CuS nanocomposites with peroxidase-like activity using a simple hydrothermal approach. Significantly, compared with the individual MXene-Ti3C2 nanosheets or CuS nanoparticles, the MXene-Ti3C2/CuS nanocomposites show a synergistically enhanced peroxidase-like activity and can be used as an efficient mimetic peroxidase to catalyze the reaction of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), causing a blue color change. Kinetic studies reveal that the MXene-Ti3C2/CuS nanocomposites have a higher catalytic activity to TMB than their single components, and the catalytic reaction follows the ping-pong mechanism. The MXene-Ti3C2/CuS nanocomposites are used for the colorimetric determination of cholesterol with a linear range of 10-100 μM and a limit of detection (LOD) of 1.9 μM. Our results show that the MXene-Ti3C2/CuS nanocomposites based colorimetric cholesterol biosensor is cost-effective, sensitive, and selective, which has potential application in H2O2 and cholesterol detection and clinic medicine diagnostics.

In-situ generation of nanozymes by natural nucleotides: a biocatalytic label for quantitative determination of hydrogen peroxide and glucose

Four natural nucleotides including 5'-cytidine monophosphate (CMP), 5'-thymidine monophosphate (TMP), guanosine monophosphate (GMP) and 5'-adenosine monophosphate (AMP) were employed to modulate the coordination environment and the valence state of PtCl42-. This is the first report that natural nucleotides have the ability to produce highly active Pt nanoclusters. The latter are shown to act as peroxidase mimetics. Both the size distribution and the charge state of Pt-nucleotide nanozymes vary with the chemical structures of nucleotides, thereby contributing to distinct enzyme-like activities. By adopting Pt-CMP as a signal amplifier, a photometric assay was well-established for quantitative determination of glucose. The assay is based on the oxidation of glucose by glucose oxidase. The oxidation product (H2O2)is detected at 652nm via the Pt-CMP-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine with H2O2. Response is linear in the 5 to 100μMglucose concentration range, and the limit of detection is 0.12μM (atS/N= 3). The method excels by a low signal background, high sensitivity, and low consumption of energy and materials. Graphical abstract Peroxidase mimicking Pt nanoclusters were synthesized by employing natural nucleotides as both the reducing agent and the stabilization template.

Target-directed functionalized ferrous phosphate-carbon dots fluorescent nanostructures as peroxidase mimetics for cancer cell detection and ROS-mediated therapy

A novel kind of fluorescence nanozyme was developed for detection and treatment of cancer cells. The ferrous phosphate–carbon dots (Fe3(PO4)2·8H2O–CDs) nanohybrids were first synthesized through a facial co-precipitation method and then further modified with folic acid (FA) on the surface to finally obtain the Fe3(PO4)2·8H2O–CDs–FA hybrid nanoflowers (hNFs). The obtained hNFs were demonstrated to have excellent peroxidase-like activity by a colour reaction with H2O2. In light of superior catalytic activity, we applied the hNFs to develop a simple colorimetric detection strategy of cancer cells. This proposed assay enabled to distinguish as low as 25 HeLa cells by naked eye. Afterwards, the hNFs were utilized for reactive-oxygen species (ROS)-mediated cancer cell treatment considering the efficient generation of hydroxyl radicals through the considerable high catalytic activity. In vitro experiments exhibit that the hNFs caused the enhanced cytotoxicity to HeLa cells even with low dosages of exogenous H2O2 or endogenous H2O2 triggered by ascorbic acid, while no obvious damage was observed in normal cells with the same treatment, confirming that the selective killing of cancer cells could be realized via hNFs. Moreover, the fluorescent imaging capability of the hNFs was further used to testify its specific targeting ability and therapeutic effect to HeLa cells.

Photoelectrochemical biosensor for CEA detection based on SnS2-GR with multiple quenching effects of Au@CuS-GR

A novel signal on-off type photoelectrochemical (PEC) biosensing system was designed for sensitive detection of carcinoembryonic antigen (CEA) based on tin disulfide nanosheets loaded on reduced graphene cxide (SnS2-GR) as the photoactive material and gold nanoparticles coated on reduced graphene oxide-functionalized copper sulfide (Au@CuS-GR) for signal amplification. It's the first time for SnS2-GR was exploited as a sensing matrix. Here, the photocurrent signals of SnS2 were amplified attributed to the sensitization effect of graphene. As signal amplifier, Au@CuS-GR could quench the photocurrents of SnS2-GR not only through the p-n type semiconductor quenching effect as well as the steric hindrance effect, but also as peroxidase mimetics to catalyze the oxidation of 4-Chloro-1-naphthol (4-CN) to produce insoluble product on the electrode surface. Based on the multiple signal amplification ability of Au@CuS-GR, CEA was detected sensitively with a linear range from 0.1 pg mL−1 to 10 ng mL−1 and limit of detection down to 59.9 fg mL−1 (S/N = 3). Meanwhile, the PEC biosensor displayed excellent performance in the assay of human serum sample, showing good application prospects for various target analysis.

Fabrication of a covalent organic framework and its gold nanoparticle hybrids as stable mimetic peroxidase for sensitive and selective colorimetric detection of mercury in water samples

Gold nanoparticles (AuNPs) without surface capping agents are easily aggregated owing to their high surface energy, leading to an unexpected decrease of their catalytic activity. Herein, we report gold nanoparticles decorated covalent organic framework (COF) as mimetic peroxidase for colorimetric detection of mercury. 1,3,5-Tris-(4-formyl-phenyl)triazine (PT) and 4, 4′-azodianiline (Azo) was employed as the monomers to prepare a novel imine based COF PTAzo under solvothermal conditions. AuNPs are further decorated into the PTAzo to form COF-AuNPs hybrid via a citrate reducing method. The COF-AuNPs show high stability, and exhibits enhanced peroxidase mimetic activity in the presence of Hg2+. The Hg2+ concentration dependent peroxidase mimetic activity of COF-AuNPs enables the development of a sensitive and selective method for detection of Hg2+ in aqueous solution. The developed method gives good linearity in the range of 5–300 nM with the limit of detection of 0.75 nM. The results show that Hg2+-enhanced peroxidase mimetic activity of COF-AuNPs offers great potential for the detection of Hg2+ in real samples.