A DNAzyme cascade for the amplified detection of Pb(2+) ions or L-histidine

DNAzyme cascades activated by Pb(2+)- or L-histidine-dependent DNAzymes yield the horseradish peroxidase-mimicking catalytic nucleic acids that enable the colorimetric or chemiluminescence detection of Pb(2+) or L-histidine.     

Active species of horseradish peroxidase (HRP) and cytochrome P450: two electronic chameleons

The active site of HRP Compound I (Cpd I) is modeled using hybrid density functional theory (UB3LYP). The effects of neighboring amino acids and of environmental polarity are included. The low-lying states have porphyrin radical cationic species (Por(*)(+)). However, since the Por(*)(+) species is a very good electron acceptor, other species, which can be either the ligand or side chain amino acid residues, may participate in electron donation to the Por(*)(+) moiety, thereby making Cpd I behave like a chemical chameleon. Thus, this behavior that was noted before for Cpd I of P450 is apparently much more wide ranging than initially appreciated. Since chemical chameleonic behavior property was found to be expressed not only in the properties of Cpd I itself, but also in its reactivity, the roots of this phenomenon are generalized. A comparative discussion of Cpd I species follows for the enzymes HRP, CcP, APX, CAT (catalase), and P450.     

Improved activity of horseradish peroxidase (HRP) in ‘specifically designed’ ionic liquid

The ionic liquid (IL), tetrakis (2-hydroxyethyl) ammonium triflouromethanesulfonate is rationally designed for horseradish peroxidase (HRP) on the basis of its stability and activity in the presence of an excipient, tris(hydroxymethyl)aminoethane (TRIS) in different ILs. The activity of HRP in this tailor made IL is at least 30-240-fold higher than that in conventional ILs. Also, the activity is more than 10 times greater than that in methanol, a common organic solvent used for HRP.     

Spectrofluorimetric determination of hydrogen peroxide with 2-hydroxy-1-naphthaldehyde salicyloylhydrazone (HNSH) as the substrate for horseradish peroxidase (HRP).

The oxidation reaction of HNSH with H2O2 under the catalysis of HRP was studied in detail. The possible reaction mechanism was discussed. Under optimum experimental conditions, the oxidized product of HNSH had excitation and emission maxima at 296 and 414 nm, respectively. A study to prove the existence of -O-O-H in polyethylene glycols was carried out. The proposed method was successfully applied to the determination of -O-O-H in polyethylene glycols.     

FIA-near-infrared spectrofluorimetric trace determination of hydrogen peroxide using tricarchlorobocyanine dye (Cy.7.Cl) and horseradish peroxidase (HRP)

A new kind of near-infrared fluorescence agent, tricarbochlorocyanine dye (Cy.7.Cl), had been synthesized in house and used for near-infrared spectrofluorimetric determination of hydrogen peroxide (H₂O₂) by flow injection analysis (FIA) for the first time. The oxidation reaction of Cy.7.Cl with H₂O₂ occurred under the catalysis of horseradish peroxidase (HRP) and it was studied in detail. The possible reaction mechanism was discussed. Under optimal experimental conditions, fluorescence from Cy.7.Cl displayed excitation and emission maxima (ex/em) at 780 and 800 nm, respectively. The two linear working ranges were 1.86 × 10⁻⁷ to 4.11 × 10⁻⁷ mol L⁻¹ and 4.11 × 10⁻⁷ to 7.19 × 10⁻⁶ mol L⁻¹, respectively. The detection limit was 5.58 × 10⁻⁸ mol L⁻¹ of H₂O₂. The effect of interferences was studied. The proposed method was successfully applied to the determination of hydrogen peroxide in rainwater, serum and plant samples.     

Studies on the oxidation reaction of tyrosine (Tyr) with H2O2 catalyzed by horseradish peroxidase (HRP) in alcohol-water medium by spectrofluorimetry and differential spectrophotometry

An oxidation reaction of tyrosine (Tyr) with H(2)O(2) catalyzed by horseradish peroxidase (HRP) was studied by spectrofluorimetry and differential spectrophotometry in the alcohol(methanol, ethanol, 1-propanol and isopropanol)-water mutual solubility system. Compared with the enzymatic-catalyzed reaction in the water medium, the fluorescence intensities of the product weakened, even extinguished. Because the addition of alcohols made the conformation of HRP change, the catalytic reaction shifted to the side of polymerization and the polymer (A(n)H(2), n>or=3) exhibited no fluorescence. The four alcohols cannot deactivate HRP. Moreover isopropanol activated HRP remarkably.     

Spectrofluorimetric determination of both hydrogen peroxide and -O-O-H in polyethylene glycols (PEGs) using 2-hydroxy-1-naphthaldehyde thiosemicarbazon (HNT) as the substrate for horseradish peroxidase (HRP)

2-Hydroxy-1-naphthaldehyde thiosemicarbazon (HNT) had been synthesized and used as a new kind of substrate for horseradish peroxidase (HRP) in spectrofluorimetric determination of hydrogen peroxide (H(2)O(2)). The oxidation reaction of HNT with H(2)O(2) under the catalysis of HRP was studied in detail. The possible reaction mechanism was discussed. Under optimum experimental conditions, the oxidized product of HNT had excitation and emission maxima at 260 and 450 nm, respectively. The linear range of this method was 1.30 x 10(-9)-1.25 x 10(-5) mol l(-1) with a detection limit of 3.89 x 10(-10) mol l(-1). The effect of interferences, surfactants and organic solvents on the determination of H(2)O(2) had been investigated. A study to prove the existence of -O-O-H in PEGs was carried out. The proposed method was successfully applied to the determination of -O-O-H in polyethylene glycols.     

Redox thermodynamics of the Fe(3+)/Fe(2+) couple in horseradish peroxidase and its cyanide complex.

The thermodynamics of Fe3+ to Fe2+ reduction for the five-coordinate high-spin native form of horseradish peroxidase and for its six-coordinate low-spin cyanide adduct have been determined from variable-temperature UV-vis spectroelectrochemical experiments. In both cases, the DeltaH degrees 'rc and DeltaS degrees 'rc values are positive. Hence, the negative reduction potentials turn out to be the result of two opposing and partially compensating contributions: a large enthalpic term, which is the determinant of the negative E degrees ' values for both species, and a smaller, yet relevant, entropic contribution. The decrease in E degrees ' of the Fe3+/Fe2+ couple on cyanide binding turns out to be a fully entropic effect, unequivocally demonstrating the importance of entropic effects in determining the E degrees ' values of redox metal centers.     

The activation of molecular oxygen by horseradish peroxidase with sodium sulfite

Horseradish peroxidase (HRP) utilizes molecular oxygen (O2) with sodium sulfite (Na2SO3) to oxidize thioanisole and styrene at the exterior of the heme pocket.     

Catalytic spectrofluorimetric determination of superoxide anion radical and superoxide dismutase activity using N,N-dimethylaniline as the substrate for horseradish peroxidase (HRP)

The coupled reaction of N,N-dimethylaniline (DMA) with 4-aminoantipyrine (4-AAP) using superoxide anion radical (O2-) as oxidizing agent under the catalysis of horseradish peroxidase (HRP) was studied. Based on the reaction, O2- produced by irradiating Vitamin B2, (VB2) was spectrophotometricly determined at 554 nm. The linear range of this method was 1.8 x 10(-6)-1.2 x 10(-4) mol l(-1) with a detection limit of 5.3 x 10(-7) mol l(-1). The effect of interferences on the determination of O2- was investigated. The proposed method was successfully applied to the determination of superoxide dismutase (SOD) activity in human blood and mouse blood.     

Amide Group Coordination to the Pb(2+) Ion

The binary and ternary (2,2'-bipyridine) complexes of dipositive lead formed by N-carbonyl and N-sulfonyl amino acids, which are ligands containing the peptide and the sulfonamide group, respectively, were investigated in aqueous solution by NMR and differential pulse polarography, and some were also characterized crystallographically. N-Tosylglycine, N-tosyl-beta-alanine, and N-benzoylglycine behave as simple carboxylate ligands at acid pH, while around neutrality they switch to dianionic N,O-bidentate chelating ligands due to the involvement of the deprotonated amide nitrogen as an additional donor site. The same coordination behavior is maintained in the presence of 2,2'-bipyridine. The binary and ternary species formed in solution, and their stability constants were determined and compared with those of the homologous complexes of Pd(2+), Cu(2+), Cd(2+), and Zn(2+). The Pb(2+) ion is the only dipositive metal which is effective in promoting peptide nitrogen deprotonation in benzoylglycine. The molecular structures of [Pb(N-tosylglycinato-N,O)(H(2)O)] (1), [Pb(N-benzoylglycinato-O)(2)(H(2)O)(2)].2H(2)O (2), and [Pb(N-tosylglycinato-O)(2)(bpy)] (3) were determined by X-ray crystallography (O and N,O refer to the ligands binding as carboxylates and as N,O-chelating dianions, respectively). These compounds are all polymeric with six- to eight-coordinate metals showing distorted coordination geometries indicative of a stereochemically active metal lone pair. Polymerization is invariably determined by a bidentate chelate carboxylate group with one oxygen bridging between two metals, and in 2 and 3 it occurs through the formation of chains of Pb(2)O(2) square-planar rings. The binding set in 1, involving a deprotonated amide nitrogen and a sulfonic oxygen, is unprecedented for the Pb(2+) ion. This work provides new information on the solution and solid state chemistry of dipositive lead with ligands of biological interest, a research area that has received little attention in the past, although it is of great relevance for understanding the mechanisms of metal toxicity.     

Mesoporous silica hollow sphere (MSHS) for the bioelectrochemistry of horseradish peroxidase

In this work, novel mesoporous silica hollow spheres (MSHS) were chosen as an immobilization matrix, to construct a mediator-free third-generation HRP biosensor. UV-vis spectroscopy revealed that horseradish peroxidase (HRP) entrapped in MSHS could retain its native structure. FTIR spectroscopy and nitrogen adsorption-desorption isotherms indicated that HRP are intercalated into the mesopores. The direct electron transfer of HRP entrapped in MSHS was observed. A pair of stable and well-defined redox peaks of HRP with a formal potential of about −0.150 V (vs. Ag/AgCl) in 0.1 M pH 7.0 phosphate-buffered solution (PBS) were obtained. The biosensor exhibited a fast amperometric response to H₂O₂ with a linear range of 3.9 × 10⁻⁶ to 1.4 × 10⁻⁴ M (R = 0.997, N = 20). The detection limit was 1.2 × 10⁻⁶ M based S/N = 3.     

Attachment of horseradish peroxidase (HRP) onto the poly(styrene/acrolein) latexes and onto their derivatives with amino groups on the surface; activity of immobilized enzyme

The polystyrene (P(S)), poly(styrene/acrolein) (P(SA)), and polyacrolein (P(A)) latexes, with varied fraction of polyacrolein in the surface layer (f _A=0, 0.50, 0.63, 0.84, 1.00), were used for the attachment of horseradish peroxidase. Surfaces of latexes were modified by reaction with ethylenediamine. In this step the aldehyde groups from polyacrolein were blocked and the primary amino groups were introduced. The carbohydrate portion of HRP was oxidized in the reaction leading to formation of aldehyde groups. The adsorption and covalent immobilization of HRP onto the P(S), P(SA), and P(A) latexes and of the oxidized HRP (HRP-OX) onto the modified latex particles, with amino groups on the surface (P(SA)-M and P(A)-M), were investigated. The activities of parent and oxidized HRP were compared with activities of the corresponding enzymes in solution. It has been found that whereas HRP is not suitable for the covalent immobilization on P(SA) latex and loses its activity after adsorption onto P(S) latex, HRP-OX can be adsorbed onto P(S) latex and is readily immobilized covalently onto the ethylenediamine modified P(SA) and P(A) latexes, retaining much of its former enzymatic reactivity.This work was supported by the KBN Grant 2 0624 91 01     

Backbone modification promotes peroxidase activity of G-quadruplex-based DNAzyme

In this work we studied how backbone chemical modifications, such as 2'-O-methyl, phosphorothioate, L-form nucleotides and locked nucleic acid, on G-quadruplex based DNAzymes would affect their peroxidase activity. Our results indicate that 2'-O-methyl modification facilitates the formation of a perfectly compacted parallel structure and significantly promotes peroxidase activity of G-quadruplex based DNAzymes.     

Combing DNAzyme with single-walled carbon nanotubes for detection of Pb(II) in water

A sensitive and simple assay for the detection of Pb(2+) in aqueous solutions is reported. It takes advantage of the high affinity between single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWCNT) as well as the capability of SWCNT in fluorescence quenching. Lead(II) catalyzes the cleavage of a fluorescently labeled DNA substrate by a DNAzyme, which releases the single-stranded product to be adsorbed onto a SWCNT. The decrease in fluorescence is proportional to the Pb(2+) concentration. Concentrations as low as 1 nM Pb(2+) in water could be detected and the detection range spans over 5 orders of magnitude. The unique combination of Pb-specific DNAzyme with SWCNT produces a universal, facile and cost-effective sensing platform for lead ions. The concept can be applied to the design of detection assays for other metal ions or small molecules.     

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.     

Peroxidase-like catalytic activity of aqueous- and immobilized-Mn(3+)-octabromo-porphyrins on ion-exchange resin supplied as mimetic of horseradish peroxidase

In order to explore the capability of metal porphyrins as an alternative of horseradish peroxidase (HRP), HRP-like activity of three manganese-porphyrins (Mn-Ps) and three Mn-octabromo-porphyrins (Mn-OBPs) was examined in both aqueous and immobilized states. It was found that Mn(3+)-octabromotetrakis(1-methyl-pyridinium-4yl)porphine (Mn-OBTMPyP) has an activity of at least 90% of HRP in an aqueous solution. Mn-OBTMPyP exhibited a catalytic activity even in the presence of hydrogen peroxide without suicide reaction. In addition, Mn-OBTMPyP was revealed to function as an alternative to HRP in the quantitative determination of serum uric acid. These results are of great interest because they indicate that metal-octabromo-porphyrins possibly include promising candidates of artificial enzyme capable of substituting for HRP.     

Amplified fluorescence detection of mercury(II) ions (Hg2+) using target-induced DNAzyme cascade with catalytic and molecular beacons

In this work, a fluorescent sensing strategy was developed for the detection of mercury(II) ions (Hg(2+)) in aqueous solution with excellent sensitivity and selectivity using a target-induced DNAzyme cascade with catalytic and molecular beacons (CAMB). In order to construct the biosensor, a Mg(2+)-dependent DNAzyme was elaborately designed and artificially split into two separate oligonucleotide fragments. In the presence of Hg(2+), the specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction induced the two fragments to produce the activated Mg(2+)-dependent DNAzyme, which would hybridize with a hairpin-structured MB substrate to form the CAMB system. Eventually, each target-induced activated DNAzyme could catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers. This would significantly enhance the sensitivity of the Hg(2+) sensing system and push the detection limit down to 0.2 nM within a 20 min assay time, much lower than those of most previously reported fluorescence assays. Owning to the strong coordination of Hg(2+) to the T-T mismatched pairs, this proposed sensing system exhibited excellent selectivity for Hg(2+) detection, even in the presence of 100 times of other interferential metal ions. Furthermore, the applicability of the biosensor for Hg(2+) detection in river water samples was demonstrated with satisfactory results. These advantages endow the sensing strategy with a great potential for the simple, rapid, sensitive, and specific detection of Hg(2+) from a wide range of real samples.     

Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection

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

Colorimetric sensing of trace UO2(2+) by using nanogold-seeded nucleation amplification and label-free DNAzyme cleavage reaction

In pH 4.4 HAC-NaAC buffer solution at 80 °C, nanogold particles (NG) strongly enhanced the slow, colored reaction of Ag(i)-gallic acid to form nanosilver particles, which exhibited a strong surface plasmon resonance (SPR) absorption peak at 460 nm, but the aggregated nanogold particles (ANG) exhibited a weak enhancement. The increased absorption value at 460 nm was linear to the NG concentration in the range of 3.6-72.5 ng mL(-1) Au. In pH 5.5 MES buffer solution at 80 °C, single-stranded substrate DNA and DNAzyme hybridize to form double-stranded DNA (dsDNA). The presence of uranyl (UO(2)(2+)) resulted in cleavage of the substrate DNA of dsDNA, releasing a short, single-stranded DNA that can be adsorbed onto the NG and protect them from aggregation; those un-adsorbed NG were aggregated to ANG. As the UO(2)(2+) concentration increased, more short, single-stranded DNA were released, and more NG were protected by the cleavage of substrate single-strand DNA, so the colored particle reaction and the absorption value at 460 nm enhanced linearly. On those grounds, 0.083-0.67 nmol L(-1) UO(2)(2+) can be detected rapidly by this colorimetric sensing assay, with a detection limit of 0.04 nmol L(-1).