Sensors for toxicity of chemicals and oxidative stress based on electrochemical catalytic DNA oxidation

Films of DNA, enzymes, polyions, and catalytic redox polyions of nanometer thickness on electrodes can provide active elements for sensors for screening the toxicity of chemicals and their metabolites, and for oxidative stress. The unifying feature of this approach involves layer-by-layer electrostatic assembly of films designed to detect DNA damage. Films containing DNA and enzymes enable detection of structural damage to DNA as a basis for toxicity screening. These films bioactivate chemicals to their metabolites, which can then react with DNA, mimicking toxicity pathways in the human liver. Metallopolyions that catalyze DNA oxidation can be incorporated into DNA/enzyme films leading to "reagentless" sensors. These sensors are suitable for detecting relative DNA damage rates in <5 min of the enzyme reactions. Films of the osmium polymer [Os(bpy)(2)(PVP)(10)Cl](+) [poly(vinylpyridine), PVP] can be used to monitor DNA oxidation selectively. Such films may be applicable to determination of oxidized DNA as a clinical biomarker for oxidative stress. Inclusion of the analogous ruthenium metallopolymer in the sensor provides a monitor for oxidation of other nucleobases.     

Electrochemical detection of natural DNA damage induced by ferritin/ascobic acid/H2O2 system and amplification of DNA damage by endonuclease Fpg

Oppositely charged natural DNA and chitosan (CS) were assembled into (CS/DNA)_n layer-by-layer films on electrode surface, and Ru(bpy)₃²⁺ (bpy = bipyridyl) in solution was used as electroactive catalyst to detect damage of DNA in the films after incubation of the films in ferritin/AA/H₂O₂ solutions (AA = ascorbic acid). The mechanism of DNA damage caused by the ferritin/AA/H₂O₂ system was similar to that of Fenton reaction, where the reaction of ferritin with AA would release some Fe(II) ions from ferritin and the following reaction between Fe(II) ions and H₂O₂ would produce hydroxyl radical, which could induce DNA oxidative damage. This system provided an in vitro model to imitate the DNA damage indirectly induced by ferritin in real bio-systems. In addition, formamidopyrimidine DNA glycosylase (Fpg), a key endonuclease enzyme in repair of oxidatively damaged DNA, was used to amplify the DNA damage caused by ferritin/AA/H₂O₂ system through conversion of oxidative purine bases into single-strand breaks. The high sensitivity of electrocatalytic method with Ru(bpy)₃²⁺ as the catalyst in detection of DNA damage and the magnification function of Fpg may provide a novel idea to detect natural DNA lesion sensitively.     

A sensitive electrochemiluminescent sensor chip based on the ssDNA-Ru(II) complex and aptamer for the determination of thrombin

In this work, an electrochemiluminescence (ECL) sensor chip for sensitive detection of thrombin (TB) was prepared using a screen-printed electrode (SPE) as a working electrode and an aptamer as a specific recognition moiety. To produce an ECL sensor chip, a layer of pL-Cys was immobilized on the surface of the SPE using the cyclic voltammetry scanning method. A layer of gold nanoparticles (AuNPs) was assembled through an Au–S bond and hairpin DNA was further immobilized on the electrode surface. Ru(bpy)₂(mcpbpy)²⁺, as a luminescent reagent, was covalently bound to single-stranded DNA (ssDNA) to prepare a luminescence probe ssDNA-Ru. The probe was hybridized with TB aptamer to form a capture probe. In the presence of TB, the TB aptamer in the capture probe bound to TB, causing the release of ssDNA-Ru that could bind to hairpin DNA on the electrode surface. The Ru(II) complex as a luminescent reagent was assembled onto the electrode, and pL-Cys was used as a co-reactant to enhance the ECL efficiency. The ECL signal of the sensor chip generated based on the above principles had a linear relationship with log TB concentration at the range 10 fM to1 nM, and the detection limit was 0.2 fM. Finally, TB detection using this method was verified using real blood samples. This work provides a new method using an aptamer as a foundation and SPE as a material for the detection of biological substances.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

Amperometric biosensor based on enzymes immobilized in hybrid mesoporous membranes for the determination of acetylcholine

Acetylcholine sensor is successfully prepared by using immobilized enzymes, i.e., acetylcholinesterase and choline oxidase within separate hybrid mesoporous silica membranes with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide produced by two sequential enzyme reactions. The determination range and the response time are 6.0–800 μM and within approximately 3 min, respectively. The sensor is very stable compared to free enzymes and 80% of the initial response was maintained even after storage for 80 days. These results show that two enzymes are successfully immobilized and well stabilized, and at the same time, two sequential enzyme reactions efficiently proceed within the separate hybrid mesoporous membranes. Further, we studied the possible detection of organophosphorus pesticides in terms of the inhibition of acetylcholinesterase activity, i.e., the decrease of current response, and demonstrated that the nanomolar concentrations of pesticide (DZN-oxon) can be detected with our sensor.     

Product-assisted catalysis in base-excision DNA repair

Most spontaneous damage to bases in DNA is corrected through the action of the base-excision DNA repair pathway. Base excision repair is initiated by DNA glycosylases, lesion-specific enzymes that intercept aberrant bases in DNA and catalyze their excision. How such proteins accomplish the feat of catalyzing no fewer than five sequential reaction steps using a single active site has been unknown. To help answer this, we report the structure of a trapped catalytic intermediate in DNA repair by human 8-oxoguanine DNA glycosylase. This structure and supporting biochemical results reveal that the enzyme sequesters the excised lesion base and exploits it as a cofactor to participate in catalysis. To our knowledge, the present example represents the first documented case of product-assisted catalysis in an enzyme-catalyzed reaction.     

Electrochemiluminescent pH sensor measured by the emission potential of TiO2 nanocrystals and its biosensing application

This work reports for the first time a potential‐based nano‐electrochemiluminescent (ECL) pH sensor, using anatase TiO₂ nanocrystals (NCs) as the ECL probe. The first ECL peak potential of the TiO₂ NCs shifted negatively with increasing pH, showing a linear range from −0.47 V (vs Ag/AgCl) at pH 3 to −1.06 V at pH 10. This phenomenon was attributed to the absorption of ‘potential‐determining ions’ of OH⁻ on the surface of TiO₂ NCs, leading to larger impedance of the electron injection. Other common ‘potential‐determining ions’, such as phosphate, induced a slight potential shift of 0.03 V at a concentration of 0.1 M. Using urease as an enzyme model, a urea biosensor was developed by the simultaneous modification of urease and TiO₂ NCs on indium–tin oxide (ITO) electrodes. The biosensor, measured on the basis of the pH increase caused by the enzyme catalysis reaction, had a linear range of 0.01–2.0 mM, with a potential shift of 0.175 V. The as‐prepared pH sensor, which has simple construction procedures and acceptable sensitivity and selectivity, may provide new avenues for the construction of ECL bioanalytical methodologies. Copyright © 2014 John Wiley & Sons, Ltd.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

Optical mapping of DNA: single-molecule-based methods for mapping genomes

The technologies associated with DNA sequencing are rapidly evolving. Indeed, single-molecule DNA sequencing strategies are cheaper and faster than ever before. Despite this progress, every sequencing platform to date relies on reading the genome in small, abstract fragments, typically of less than 1000 bases in length. The overarching aim of the optical map is to complement the information derived from DNA sequencing by providing long-range context on which these short sequence reads can be built. This is typically done using an enzyme to target and modify at short DNA sequences of, say, six bases in length throughout the genome. By accurately placing these short pieces of sequence on long genomic DNA fragments, up to several millions of bases in length, a scaffold for sequence assembly can be obtained. This review focuses on three enzymatic approaches to optical mapping. Optical mapping was first developed using restriction enzymes to sequence-specifically cleave DNA that is immobilized on a surface. More recently, nicking enzymes have found application in the sequence-specific fluorescent labeling of DNA for optical mapping. Such covalent modification allows the DNA to be imaged in solution, and this, in combination with developing nanofluidic technologies, is enabling new high-throughput approaches to mapping. And, finally, this review will discuss the recent development of mapping with subdiffraction-limit precision using methyltransferase enzymes to label the DNA with an ultrahigh density.     

A comparison of the in vitro genotoxicity of anticancer drugs idarubicin and mitoxantrone

Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assaywe showed that the drugs at 0.01-10 microM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 microM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.     

Quantum dots-based multifunctional dendritic superstructure for amplified electrochemiluminescence detection of ATP

A novel multifunctional dendrimeric CdSe-CdS-Quantum dots (QDs) hybrid superstructure with highly intense electrochemiluminescence (ECL), fluorescence and excellent magnetic property is prepared for the first time, and successfully applied to amplified ECL assays of ATP using DNA cycle amplification technique. The magnetic nanoparticles (MNPs) were firstly assembled with unique dendrimer nanoclusters (NCs), then large numbers of QDs were labeled onto the dendrimer NCs, the superstructure exhibits highly enhanced ECL and fluorescence than the pure QDs. Remarkable ECL quenching of the nanocomposites by gold nanoparticles (GNPs) was observed, based on which a novel strategy for highly sensitive ATP detection was developed by cycle amplification technique. Furthermore, the nanocomposites with excellent magnetic properties can be easily labeled, separated and immobilized onto a magnetic electrode. In particular, all the procedures such as linking GNPs, sensing target and DNA cycle amplification were directly accomplished on the nanocomposites, which is more rapid, convenient, complete and has better reproducibility than the conventional methods on electrode. To the best of our knowledge, this is the first report on the multifunctional QDs superstructure with highly intense ECL, fluorescence, excellent magnetism and its ECL biosensing, which opens a new pathway for developing QD-based nanocomposites for broad applications in ECL bioassays and optical imaging.     

Sensitive detection of p53 tumor suppressor gene using an enzyme-based solid-state electrochemiluminescence sensing platform

An enzyme-based solid-state electrochemiluminescence (ECL) sensing platform for sensitive detection of a single point mutation is developed successfully using p53 tumor suppressor gene as a model analyte. A composite of multiwalled carbon nanotubes and Ruthenium (II) tris-(bipyridine) (MWNTs-Ru(bpy)(3)(2+)) was prepared and coated on an electrode surface, which was covered by polypyrrole (PPy) to immobilize ssDNA. Then, the ssDNA recognized the gold nanoparticle (AuNP)-labeled p53 tumor suppressor gene, and produced AuNP-dsDNA electrode with AuNP layer. The surface adsorbed the glucose-dehydrogenase (GDH) molecules for producing ECL signal. This system combined enzyme reaction with ECL detection, and it can recognize sequence-specific wild type p53 sequence (wtp53) and muted type p53 sequence (mtp53) with discrimination of up to 56.3%. The analytic results were sensitive and specific. It holds promise for the diagnosis and management of cancer.     

Cold plasma functionalized TeraHertz BioMEMS for enzyme reaction analysis

In this paper, we describe the development, functionalization and functionality testing of a TeraHertz (THz) Bio-MicroElectroMechanical System (BioMEMS) dedicated to enzyme reaction analysis. The microdevice was fabricated by mixing clean room microfabrication with cold plasma deposition. The first is used to build the microfluidic circuits and the THz sensor, while the later serves for the polymerization of allylamine using a homemade glow discharge plasma reactor for a subsequent immobilization of enzymatic biocatalysts. Thermal stability of the deposited plasma polymer has been investigated by infrared spectroscopy. Fluorescent detection confirmed the efficiency of the immobilization and the enzyme hydrolysis into the BioMEMS microchannels. For the first time, the progression of the hydrolysis reaction over time was monitored by the THz sensor connected to a vectorial network analyzer. Preliminary results showed that sub-THz transmission measurements are able to discriminate different solid films, various aqueous media and exhibit specific transmission behavior for the enzyme hydrolysis reaction in the spectral range 0.06–0.11 THz.     

Amperometric biosensor based on enzymes immobilized in hybrid mesoporous membranes for the determination of acetylcholine

Acetylcholine sensor is successfully prepared by using immobilized enzymes, i.e., acetylcholinesterase and choline oxidase within separate hybrid mesoporous silica membranes with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide produced by two sequential enzyme reactions. The determination range and the response time are 6.0–800 μM and within approximately 3 min, respectively. The sensor is very stable compared to free enzymes and 80% of the initial response was maintained even after storage for 80 days. These results show that two enzymes are successfully immobilized and well stabilized, and at the same time, two sequential enzyme reactions efficiently proceed within the separate hybrid mesoporous membranes. Further, we studied the possible detection of organophosphorus pesticides in terms of the inhibition of acetylcholinesterase activity, i.e., the decrease of current response, and demonstrated that the nanomolar concentrations of pesticide (DZN-oxon) can be detected with our sensor.     

A DNA sensor based on surface plasmon resonance for apoptosis-associated genes detection

A practical and simple DNA sensor based on surface plasmon resonance (SPR) had been developed to determine apoptosis-associated genes, bcl-2 and bax. This SPR sensor was designed on the basis of simultaneous multi-wavelength detection. The complementary sequences of bcl-2 or bax oligonucleotide labeled with biotin were used as the probes. Biotin-avidin system was used to immobilize the bio-DNA on the sensor surface. The assembling processes and conditions for the DNA sensor were examined. The SPR sensor could be used to monitor the process of the immobility of the bio-DNA and DNA hybridization in real-time. The determination range of bcl-2 and bax oligonucleotide (20 bases) were 50-400 ng/mL. The determination range of polymerase chain reaction (PCR) product of bcl-2 (405 bases) was 5-60 ng/mL and PCR product of bax (538 bases) was 5-40 ng/mL. The stability, reversibility and specificity of the DNA sensor were also investigated. It was found from the experiment that the sensor could be applied for a quite long time (about 90 times). The relative standard deviation (R.S.D.) for determination oligonucleotides and PCR products of bcl-2 were 1.2 and 1.3%, respectively. The interference of noncomplementary DNA sequence with the determination of DNA was examined and it was found that noncomplementary 20-mer and 21-mer DNA (p53 and p21) do not affect the determination of bcl-2 or bax. This device could be used to study apoptosis and signal transduction routine genes. The sensor was shown to be of simplicity, sensitivity, selectivity, rapid response and cost effectiveness.     

The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA

The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.     

Development of acetylcholine sensor using carbon fiber (amperometric determination)

An enzyme sensor is developed using carbon fiber to measure acetylcholine concentration. The mechanism is based on the detection of H₂O₂ which is a product of the sequential enzyme reactions of acetylcholinesterase and choline oxidase. The fabrication of the electrode is described. The sensor is polarized at 1.2 V. Enzymes are co-immobilized in polyvinyl alcohol containing styryl pyrydinium (photo-crosslinkable polymer). A fast response time of 0.8 min is obtained. A linear correlation is observed between 0.2 and 1.0 mM. Other optimal operational conditions with respect to pH, temperature and stability are discussed. The use of carbon fiber containing co-immobilized enzymes could offer several model advantages especially in neuroscience research. In conclusion, the aims of the present work are centered on carbon fiber electrode fabrication, immobilization electrochemical measurements.     

Fabrication of comb interdigitated electrodes array (IDA) for a microbead-based electrochemical assay system

This research is directed towards developing a more sensitive and rapid electrochemical sensor for enzyme labeled immunoassays by coupling redox cycling at interdigitated electrode arrays (IDA) with the enzyme label beta-galactosidase. Coplanar and comb IDA electrodes with a 2.4 microm gap were fabricated and their redox cycling currents were measured. ANSYS was used to model steady state currents for electrodes with different geometries. Comb IDA electrodes enhanced the signal about three times more than the coplanar IDAs, which agreed with the results of the simulation. Magnetic microbead-based enzyme assay, as a typical example of biochemical detection, was done using the comb and coplanar IDAs. The enzymes could be placed close to the sensing electrodes (approximately 10 microm for the comb IDAs) and detection took less than 1 min with a limit of detection of 70 amol of beta-galactosidase. We conclude that faster and more sensitive assays can be achieved with the comb IDA.