Reusable evanescent wave DNA biosensor for rapid, highly sensitive, and selective detection of mercury ions

Mercury ions (Hg(2+)) are a highly toxic and ubiquitous pollutants requiring rapid and sensitive on-site detection methods in the environment and foods. Herein, we report an envanescent wave DNA-based biosensor for rapid and very sensitive Hg(2+) detection based on a direct structure-competitive detection mode. In this system, a DNA probe covalently immobilized onto a fiber optic sensor contains a short common oligonucleotide sequences that can hybidize with a fluorescently labeled complementary DNA. The DNA probe also comprises a sequence of T-T mismatch pairs that binds with Hg(2+) to form a T-Hg(2+)-T complex by folding of the DNA segments into a hairpin structure. With a structure-competitive mode, a higher concentration of Hg(2+) leads to less fluorescence-labeled cDNA bound to the sensor surface and thus to lower fluorescence signal. The total analysis time for a single sample, including the measurement and surface regeneration, was under 6 min with a Hg(2+) detection limit of 2.1 nM. The high specificity of the sensor was demonstrated by evaluating its response to a number of potentially interfering metal ions. The sensor's surface can be regenerated with a 0.5% SDS solution (pH 1.9) over 100 times with no significant deterioration of performance. This platform is potentially applicable to detect other heavy metal ions or small-molecule analytes for which DNA/aptamers can be used as specific sensing probes.     

Carbon nanoparticle for highly sensitive and selective fluorescent detection of mercury(II) ion in aqueous solution

In this article, carbon nanoparticles (CNPs) were used as a novel fluorescent sensing platform for highly sensitive and selective Hg(2+) detection. To the best of our knowledge, this is the first example of CNPs obtained from candle soot used in this type of sensor. The general concept used in this approach is based on that adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by CNP via π-π stacking interactions between DNA bases and CNP leads to substantial dye fluorescence quenching; however, in the presence of Hg(2+), T-Hg(2+)-T induced hairpin structure does not adsorb on CNP and thus retains the dye fluorescence. A detection limit as low as 10nM was achieved. The present CNP-based biosensor for Hg(2+) detection exhibits remarkable specificity against other possible metal ions. Furthermore, superior selectivity performance was observed when Hg(2+) detection was carried out in the presence of a large amount of other interference ions. Finally, in order to evaluate its potential practical application, Hg(2+) detection was conducted with the use of lake water other than pure buffer and it is believed that it holds great promise for real sample analysis upon further development.     

Signal amplification architecture for electrochemical aptasensor based on network-like thiocyanuric acid/gold nanoparticle/ssDNA

A novel electrogenerated chemiluminescence (ECL) biosensor for highly sensitive and selective detection of mercury ion was developed on the basis of mercury-specific oligonucleotide (MSO) served as a molecular recognition element and the ruthenium(II) complex (Ru1) as an ECL emitting species. The biosensor was fabricated on a glassy carbon electrode coated with a thin layer of single wall carbon nanotubes, where the ECL probe, NH(2)-(CH(2))(6)-oligo(ethylene oxide)(6)-MSO?Dend-Ru1, was covalently attached. The Dend-Ru1 pendant was prepared by covalent coupling Ru1 with the 4th generation polyamidoamine dendrimer (Dend), in which each dendrimer contained 35 Ru1 units so that a large amplification of ECL signal was obtained. Upon binding of Hg(2+) to thymine (T) bases of the MSO, the T-Hg-T structure was formed, and the MSO changed from its linear shape to a "hairpin" configuration. Consequently, the Dend-Ru1 approached the electrode surface resulting in the increase of anodic ECL signal in the presence of the ECL coreactant tri-n-propylamine. The reported biosensor showed a high reproducibility and possessed long-term storage stability (92.3% initial ECL recovery over 30 day's storage). An extremely low detection limit of 2.4 pM and a large dynamic range of 7.0 pM to 50 nM Hg(2+) were obtained. An apparent binding constant of 1.6 × 10(9)M(-1) between Hg(2+) and the MSO was estimated using an ECL based extended Langmuir isotherm approach involving multilayer adsorption. Determination of Hg(2+) contents in real water samples was conducted and the data were consistent with the results from cold vapor atomic fluorescence spectroscopy.     

Design of a highly specific and noninvasive biosensor suitable for real-time in vivo imaging of mercury (II) uptake

Mercury is a ubiquitous pollutant that when absorbed is extremely toxic to a wide variety of biochemical processes. Mercury (II) is a strong, "invisible" poison that is rapidly absorbed by tissues of the intestinal tract, kidneys, and liver upon ingestion. In this study, a novel fluorescence-based biosensor is presented that allows for the direct monitoring of the uptake and distribution of the metal under noninvasive in vivo conditions. With the introduction of a cysteine residue at position 205, located in close proximity to the chromophore, the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensor for this metal ion. The mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH. Absorbance and fluorescence properties with respect to the metal concentration exhibit sigmoidal binding behavior with a detection limit in the low nanomolar range. Time-resolved binding studies indicate rapid subsecond binding of the metal to the protein. The crystal structures obtained of mutant eGFP205C indicate a possible access route of the metal into the core of the protein. To our knowledge, this engineered protein is a first example of a biosensor that allows for noninvasive and real-time imaging of mercury uptake in a living cell. A major advantage is that its expression can be genetically controlled in many organisms to enable unprecedented studies of tissue specific mercury uptake.     

A G-quadruplex based label-free fluorescent biosensor for lead ion

Many Pb(2+) biosensors based on Pb(2+)-specific RNA-cleaving DNAzyme have been developed in the past years. However, many of them have limited practical use because of high cost (e.g., enzymes), complicated processing and the use of unstable molecules (e.g., RNA). In this study, a novel label-free fluorescent biosensor for Pb(2+) was proposed based on Pb(2+)-induced allosteric G-quadruplex (PS2.M). In the presence of K(+), N-methyl mesoporphyrin IX (NMM) could bind to K(+)-stabilized G-quadruplexes, giving rise to high fluorescence. On addition of Pb(2+), Pb(2+) competitively binded to K(+)-stabilized G-quadruplexes to form more compact DNA folds. The Pb(2+)-stabilized G-quadruplexes did not bind to NMM, which resulted in fluorescence decrease. This allowed us to utilize PS2.M for quantitative analysis of Pb(2+) using the NMM-G-quadruplex system by convenient "mix-and-detect" protocol. The fluorescence emission ratio (F(0)/F) showed a good linear response toward Pb(2+) over the range from 5.0 nM to 1.0 μM with a limit of detection of 1.0 nM. This proposed biosensor was simple and cost efficiency in design and in operation with high sensitivity and selectivity. We validated the practicality of this biosensor for the determination of Pb(2+) in lake water samples.     

Label-free G-quadruplex-specific fluorescent probe for sensitive detection of copper(II) ion

An effective G-quadruplex-based probe has been constructed for rapid and sensitive detection of Cu(2+). In this probe, an anionic porphyrin, protoporphyrin IX (PPIX) served as a reference signal, which binds to G-quadruplex specifically and the fluorescence intensity increases sharply. While, in the presence of Cu(2+), the G-quadruplex can catalyze the related Cu(2+) insertion into the protoporphyrin, the fluorescent intensity is decreased. The fluorescence of the response ligand could be selectively quenched in the presence of Cu(2+) and not interfered by other metal ions. The probe provided an effective platform for reliable detection of Cu(2+) with a detection limit as low as 3.0nM, the high sensitivity was attributed to the strong metalation of PPIX with Cu(2+) catalyzed by G-quadruplex (PS5.M). Linear correlations were obtained over the logarithm of copper ion concentration in the range from 8×10(-9)M to 2×10(-6)M (R=0.998). The G-quadruplex-based probe also could be used to detect Cu(2+) in real water samples. Additionally, these striking properties endow the G-quadruplex-ligand with a great promise for analytical applications.     

DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu(+) and Cu(2+) ions

The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu(+) and Cu(2+), with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu(2+) (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.     

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

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

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated.     

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

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

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

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

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

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

Oligonucleotide-functionalized silver nanoparticle extraction and laser-induced fluorescence for ultrasensitive detection of mercury(II) ion

This study describes the development of a simple, sensitive, and selective detection system for Hg(2+) ion by combining nanoparticle extraction, fluorescent dye labeling, and flow injection analysis (FIA) detection. Repeats of 33 thymine nucleotides-functionalized silver nanoparticles (T(33)-AgNPs) specifically capture Hg(2+) from aqueous solution through the coordination between T(33) and Hg(2+). Meanwhile, Hg(2+) ion drives a T(33) conformational change from a random coil to a folded structure. The T(33)-Hg(2+)complexes adsorbed on the NP surface were collected from the initial sample by centrifugation, and they were then detached from the NP surface by addition of H(2)O(2). The T(33)-Hg(2+) complexes preferentially bind to SYBR Green I (SG), enhancing the SG fluorescence. By contrast, SG fluoresces only weakly in the presence of T(33) alone. The extraction efficiency of Hg(2+) was highly dependent on polythymine length, the concentration of T(33)-AgNPs, and the incubaton time of T(33)-AgNPs with Hg(2+). Under optimal extraction and labeling conditions, FIA detection showed the limit of detection (at a signal-to-noise ratio of three) for Hg(2+)of 3 pM. The selectivity of our analytical system is more than 1000-fold for Hg(2+) over any metal ions. We validated the applicability of this system for the determination of Hg(2+) concentrations in tap water.     

A heavy metal biotrap for wastewater remediation using poly-gamma-glutamic acid

Poly-gamma-glutamic acid (gamma-PGA) obtained from Bacillus licheniformis ATCC 9945 was evaluated as a potential biosorbent material for use in the removal of heavy metals from aqueous solution. Copper (Cu(2+)) was chosen as the model heavy metal used in these studies since it is extensively used by electroplating and other industries, has been the model for many other similar studies, and can be easily assayed through a number of convenient methods. Cu(2+)-gamma-PGA binding parameters under varying conditions of pH, temperature, ionic strength, and in the presence of other heavy metal ions were determined for the purified biopolymer using a specially designed dialysis apparatus. Applying the Langmuir adsorption isotherm model showed that gamma-PGA had a copper capacity approaching 77.9 mg/g and a binding constant of 32 mg/L (0.5 mM) at pH 4.0 and 25 degrees C. Cu(2+)-gamma-PGA adsorption was relatively temperature independent between 7 and 40 degrees C, while an increase in ionic strength led to a decrease in metal ion binding. Cd(2+) and Zn(2+) ions compete with Cu(2+) for binding sites on the gamma-PGA biopolymer. Metal uptake by gamma-PGA was further tested using a tangential flow filtration apparatus in a diafiltration mode in which metal was continually processed through a dilute solution of gamma-PGA without allowing for equilibrium to be established. The circulating polymer solution was able to complex metal as well as successfully prevent passage of unbound copper ions present in solution through the membrane. Using 500 mL of a 0.2% gamma-PGA solution, up to 97% of a 50 mg/L copper sulfate solution processed at a flow rate of 115 mL/min was retained by the polymer. For a 10 mg/L solution of Cu(2+) as copper sulfate, filtrate concentrations of Cu(2+) never rose above 0.6 mg/L while processing 2.5 L of dilute copper sulfate.     

CdTe nanocrystal-based electrochemical biosensor for the recognition of neutravidin by anodic stripping voltammetry at electrodeposited bismuth film

CdTe quantum dots (QDs)-based electrochemical sensor for recognition of neutravidin, as a model protein, using anodic stripping voltammetry at electrodeposited bismuth film is presented. This biosensor involves the immobilization of the captured QDs conjugates which was dissolved with 1M HCl solution to release cadmium ions and metal components were quantified by anodic stripping voltammetry after a 3-min accumulation at -1.2V on bismuth-film electrode (BiFE) of the biotin, served as recognition element, onto the gold surface in connection with a cysteamine self-assembled monolayer. The modification procedure was characterized by electrochemical impedance spectroscopy and atomic force microscopy. We exploit QDs as labels for amplifying signal output and monitoring the extent of competition process between CdTe-labeled neutravidin and the target neutravidin for the limited binding sites on biotin. As expected for the competitive mechanism, the recognition event thus yields distinct cadmium stripping voltammetric current peak, whose response decreases upon increasing the level of target neutravidin concentrations. Under optimal conditions, the voltammetric response is highly linear over the range of 0.5-100 ngL(-1) neutravidin and the limit of detection is estimated to be 0.3 ngL(-1) (5 nM). Unlike earlier two-step sandwich bioassays, the present protocol relies on a one-step competitive assay, which is more accurate and sensitive, showing great promise for rapid, simple and cost-effective analysis of protein.     

Single-layer CVD-grown graphene decorated with metal nanoparticles as a promising biosensing platform

A new approach to the development of a single-layer graphene sensor decorated with metal nanoparticles is presented. Chemical vapor deposition is used to grow single layer graphene on copper. Decoration of the single-layer graphene is achieved by electroless deposition of Au nanoparticles using the copper substrate as a source of electrons. Transfer of the decorated single-layer graphene on glassy carbon electrodes offers a sensitive platform for biosensor development. As a proof of concept, 10 units of glucose oxidase were deposited on the surface in a Nafion matrix to stabilize the enzyme as well as to prevent interference from ascorbic acid and uric acid. Amperometric linear response calibration in the μmoll(-1) is obtained. The presented methodology enables highly sensitive platforms for biosensor development, providing a scalable roll-to-roll production with a much more reproducible scheme when compared to the graphene biosensors reported previously based on drop-cast of multi-layer graphene suspensions.     

Paper supported immunosensor for detection of antibiotics

Paper supports were used to develop a simple, inexpensive, fast and sensitive electrochemical immunosensor for the analysis of antibiotic residues in milk samples, where single-walled carbon nanotubes (SWNTs) and a simple dip-dry coating method were employed to prepare the highly sensitive biosensor. Well-dispersed SWNTs were impregnated with an antibody against neomycin to obtain a composite coating solution, followed by dipping the filtration paper in the solution to fabricate the sensitive biosensor which had high electrical conductivity. Based on the impedance change in the entire paper supported biosensor with increased concentrations of neomycin, the limit detection of the optimized method was 0.04 ng mL(-1) and a linear detection range from 0.2 to 125 ng mL(-1), well below the European Union regulations for neomycin in this matrix. This paper supported biosensor was applied to determine neomycin in milk samples after a simple sample treatment, with spiked recoveries which ranged from 93.25 to 110.47%. A variety of antibiotic residues in milk samples could be determined following similar sensor preparation.     

Carboxymethylcellulose–gelatin–superoxidase dismutase electrode for amperometric superoxide radical sensing

A novel, highly sensitive superoxide dismutase biosensor for the direct and simultaneous determination of superoxide radicals was developed by immobilization of superoxide dismutase within carboxymethylcellulose-gelatin on a Pt electrode surface. The parameters affecting the performance of the biosensor were investigated. The response of the CMC-G-SOD biosensor was proportional to O (2) (·-) concentration and the detection limit was 1.25 × 10(-3) mM with a correlation coefficient of 0.9994. The developed biosensor exhibited high analytical performance with wider linear range, high sensitivity and low response time. The biosensor retained 89.8% of its sensitivity after use for 80 days. The support system enhanced the immobilization of superoxide dismutase and promoted the electron transfer of superoxide dismutase minimizing its fouling effect. The biosensor was quite effective not only in detecting O (2) (·-) , but also in determining the antioxidant properties of acetylsalicylic acid-based drugs and the anti-radical activity of healthy and cancerous human brain tissues.     

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.     

Highly sensitive biosensor based on bionanomultilayer with water-soluble multiwall carbon nanotubes for determination of phenolics

A highly sensitive biosensor was developed based on bionanomultilyer with water-soluble carbon nanotubes (CNTs). The water-soluble poly(allylamine hydrochloride)-wrapped multiwall carbon nanotubes (PAH-MWNTs) can be obtained for the first time relying on the function of barbiturates, which provides a useful avenue for CNT application in material science and biosensor technology. Based on this, the PAH-MWNTs/horseradish peroxidase (HRP) bionanomultilayer was prepared via layer-by-layer (LBL) assembly. Electrochemical impedance spectroscopy, atomic force microscopy and UV-vis spectra were adopted to monitor the uniform LBL assembly of the homogeneous bionanomultilayer. The bionanomultilayer was used to construct a phenolic biosensor. Under the optimal conditions, the biosensor presented a linear response for catechol from 0.1 to 20.4muM, with a detection limit of 0.06muM. A series of phenolics were detected by the bionanomultilayer biosensor. The introduced MWNTs in the biosensor provided a suitable microenvironment to retain the HRP activity and acted as a transducer for amplifying the electrochemical signal of the product of the enzymatic reaction. So the developed bionanomultilayer biosensor exhibited a fast, sensitive and stable detection.