Electrochemical DNA biosensor for analysis of wastewater samples

The application of a disposable electrochemical DNA biosensor to wastewater samples is reported. The DNA biosensor is assembled by immobilising double-stranded calf thymus DNA on the surface of a disposable, carbon screen-printed electrode (SPE). The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal. The presence of compounds with affinity for DNA is measured by their effect on the guanine oxidation. The comparison of the results with a toxicity test based on bioluminescent bacteria has confirmed the applicability of the method to real samples.     

Electrochemical DNA biosensor as a screening tool for the detection of toxicants in water and wastewater samples

Applications of a disposable electrochemical DNA biosensor to standard solutions and to real samples are reported. The DNA biosensor is assembled by immobilising the double stranded calf thymus DNA on the surface of a disposable carbon screen-printed electrode. The immobilised ds-DNA interacts with the sample for 2 min; then is washed and immersed in a clean buffer where the analytical signal (the oxidation peak area of the guanine base) is obtained by a square-wave voltammetric scan. The results were compared with some currently used toxicity tests and in particular with a commercial luminescent bacteria test, Toxalert(R)100.     

Electrochemical DNA nano-biosensor for the detection of genotoxins in water samples简

In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing DNA on Au nanoparticles and a self-assembled monolayer of cysteamine modified Au electrode. The assembly processes of cysteamine, Au nanoparticles and DNA were characterized by cyclic voltammetry （CV）. The Au nanoparticles enhanced DNA immobilization resulting in an increased guanine signal. The interaction of the analyte with the immobilized DNA was measured through the variation of the electrochemical signal of guanine by square wave voltammetry （SWV）. The biosensor was able to detect the known genotoxic compounds： 2-anthramine, acridine orange and 2- naphthylamine with detection limits of 2, 3 and 50 nmol/L, respectively. The biosensor was also used to test actual water samples to evaluate the contamination level. Additionally, the comparison of results from the classical genotoxiciw bioassay has confirmed the applicability of the method for real samoles.     

A Ready‐to‐Use Electrochemical Kit Design for the Diagnosis of Single Nucleotide Polymorphisms

In this study, for the first time a model electrochemical kit was constructed for the detection of a functional polymorphism in catechol‐O‐methyl transferase (COMT) gene which is important for diagnosis of neuropsychiatric disorders as Alzheimer disease. The disposable pencil graphite electrode (PGE) is designed as a “kit” and the probe DNA covered PGE can detect single nucleotide polymorphisms (SNPs) from real samples based on the guanine oxidation signal even after 5 months of kit preparation (150 days durability).The detection limit (S/N=3) of the biosensor was calculated as 1.18 pmol of synthetic target sequence and 6.09×10⁵ molecules of real samples in 30 min detection time.     

Electrochemical DNA biosensor for polycyclic aromatic hydrocarbon detection

Four DNA electrochemical biosensors using four types of DNA (calf thymus ssDNA, calf thymus dsDNA, salmon testis ssDNA and salmon testis dsDNA) were constructed using graphite screen printed electrodes. These biosensors were exploited as analytical tool to detect polycyclic aromatic hydrocarbons-DNA interactions using benzo(a)anthracene and phenantrene as model analytes, the guanine oxidation peak variation being the signal revealing the interaction between PAHs and immobilized DNA. The salmon testis ssDNA biosensor resulted as the most promising device and was further evaluated for benzo(a)anthracene, fluorene, indeno(1,2,3-cd)pyrene, anthracene, and phenanthrene in 5–40 ng mL^?1 solutions, and for benzo(a)pyrene (5–50 ng mL^?1). A concentration dependent variation of the DNA guanine oxidation peak was observed for all compounds. The effect of benzo(a)pyrene ultraviolet (UV) activation on the benzo(a)pyrene (BaP)-DNA interaction was evaluated at concentration levels of 20 and 50 ng mL^?1, and a 3.5- and 2.7-fold increases of the guanine oxidation peak was measured respectively. The salmon testis ssDNA biosensor was examined with PAHs contaminated samples of Mytilus galloprovincialis. Upon UV irradiation of three sample extracts exceeding the BaP maximum level, a positive variation of the DNA guanine oxidation was obtained. An average 2.4-fold increase of the guanine oxidation peak was detected demonstrating that the sensor can be used to detect toxic degradation products of PAHs.     

Electrochemical biosensor for simultaneous determination of guanine and adenine based on dopamine-melanin colloidal nanospheres–graphene composites

Dopamine-melanin colloidal nanospheres (Dpa-melanin CNDs)–graphene composites-modified glassy carbon electrode (GCE) was prepared by a simple procedure and then successfully used to simultaneously determine guanine and adenine. Scanning electron microscopy (SEM) images and transmission electron microscopy (TEM) were used to characterize the morphology of the Dpa-melanin CNSs–graphene composite. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the electrode modifying process. Differential pulse voltammetry (DPV) was used to study the electrocatalytic activity toward the electrochemical oxidation of guanine and adenine. The modified electrode exhibited enhanced electrocatalytic behavior and good stability for the simultaneous determination of guanine and adenine compared with bare GCE. The electrochemical biosensor exhibited wide linear range of 0.5 to 150 μM with detection limit of 0.05 and 0.03 μM for guanine and adenine detection (S/N?=?3), respectively. Furthermore, the biosensor showed high sensitivity, good selectivity, good reproducibility, and long-term stability to guanine and adenine detection. At the same time, the fabricated electrode was successfully applied for the determination of guanine and adenine in denatured DNA samples with satisfying results. These results demonstrated that Dpa-melanin CNSs–graphene composite was a promising substrate for the development of high-performance electrochemical biosensor.     

Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence.The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised.The analysis of PCR samples (244 bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10 min hybridisation time allowed a full characterisation of each sample.     

DNA electrochemical biosensors

Disposable electrochemical DNA-based biosensors are reviewed; they have been used for the determination of low-molecular weight compounds with affinity for nucleic acids and for the detection of the hybridisation reaction. The first application is related to the molecular interaction between surface-linked DNA and the target pollutants or drugs, in order to develop a simple device for rapid screening of toxic or similar compounds. The determination of such compounds was measured by their effect on the oxidation signal of the guanine peak of calf thymus DNA immobilised on the electrode surface and investigated by chronopotentiometric analysis. The DNA biosensor is able to detect known intercalating compounds, such as daunomycin, polychlorinated biphenyls (PCBs), aflatoxin B1, and aromatic amines. Applicability to river and waste water samples is also demonstrated. Disposable electrochemical sensors for the detection of a specific sequence of DNA were realised by immobilising synthetic single-stranded oligonucleotides onto a graphite screen-printed electrode. The probes became hybridised with different concentrations of complementary sequences present in the sample. The hybrids formed on the electrode surface were evaluated by chronopotentiometric analysis using daunomycin as indicator of the hybridisation reaction. The hybridisation was also performed using real samples. Application to apolipoprotein E (ApoE) is described, in this case samples have to be amplified by PCR and then analysed by DNA biosensor. The extension of such procedures to samples of environmental interest or to contamination of food is discussed.     

Determination of Anti-Parkinson Drug Pramipexole Using a Label-free Biosensor and Evaluation of its Interaction with ds-DNA

In this study, we fabricated an effective and sensitive DNA biosensor based on flower-like Pt/NiCo₂O₄ modified carbon paste electrode (FL-Pt/NiCo₂O₄/CPE) for detection of pramipexole (PPX). Spectrophotometry, differential pulse voltammetry (DPV) and docking methods were employed to evaluate the interaction of DNA-PPX. Moreover, the DPV technique was chosen to monitor the electrochemical response of guanine on the DNA biosensor. The relationship between the concentration of PPX and the oxidation signal of guanine was linear in the range of 0.4 to 310.0 μM and a limit of detection (LOD) of 0.09 μM was calculated.     

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

In this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10^?13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> single-strand binding protein–DNA interactions on carbon nanotube-modified electrodes from a label-free electrochemical hybridization sensor

An electrochemical hybridization biosensor based on the intrinsic oxidation signals of nucleic acids and proteins has been designed, that makes use of the unique binding event between Escherichia coli single-strand binding protein (SSB) and single-stranded DNA (ssDNA). The voltammetric signal from guanine oxidation significantly decreased upon binding of SSB to single-stranded oligonucleotides (probe), anchored on a single-walled carbon nanotube (SWCNT) -modified screen-printed carbon electrode (SPE). Simultaneously, oxidation of the tyrosine (Tyr) and tryptophan (Trp) residues of the SSB protein increased upon binding of the SSB protein to ssDNA and ss-oligonucleotides. After the hybridization, SSB did not bind to the double helix form, and the guanine signal could be observed along with the disappearance of the oxidation signal of the protein. The amplification of intrinsic guanine and protein oxidation signals by SWCNT, and a washing step with sodium dodecylsulfate, enabled the specific detection of a point mutation. Monitoring the changes in the guanine and protein signals upon hybridization greatly simplified the detection procedure. The detection limit of 0.15 g/ml target DNA can be applied to genetic assays. To the best of our knowledge, this is the first work that utilizes the monitoring of SSB–DNA interactions on a solid transducer for the electrochemical detection of DNA hybridization by using intrinsic oxidation signals.     

Quantitative Biodetection of Anticancer Drug Rituxan with DNA Biosensor Modified PAMAM Dendrimer/Reduced Graphene Oxide Nanocomposite

PAMAM dendrimer/reduced graphene oxide nanocomposite modified pencil graphite electrode (PAMAM/RGO/PGE) was used to fabricate an electrochemical DNA biosensor for determination of Rituxan (RTX) at low concentrations, for the first time. The fabricated biosensor was characterized with FE‐SEM, EIS, and CV techniques. The ds‐DNA/PAMAM/RGO/PGE was used as a working electrode to study the interaction between the RTX and salmon sperm ds‐DNA by DPV technique. Because of the interaction between the drug and DNA leads to a decrease in the guanine oxidation peak current, it was used as an indicator for the determination of the RTX. Under the optimized experimental conditions, a wide linear relationship between RTX concentration and guanine signal was obtained within the range of 7.0 to 60.0 μmol L⁻¹ and 60.0 to 300.0 μmol L⁻¹ with a low detection limit (0.56 μmol L⁻¹). To clarify the interaction mechanism between the RTX and the ds‐DNA, DPV and UV‐Vis measurements were used. The reproducibility, stability, and performance of the constructed biosensor was examined by quantitative measuring RTX in pharmaceutical and human serum samples with good precision (RSD; 2.0–6.0 %) and acceptable recoveries (100.04–101.95 %).     

Resin and fatty-acid analysis by solid-phase extraction coupled to atmospheric pressure chemical ionization–mass spectrometry

Abstract

DNA biosensors are realised immobilising a DNA structure on a suitable transducer to obtain selective information. In this paper we show how the determination of low-molecular weight compounds with affinity for DNA was measured by their effect on the oxidation signal of the guanine peak of calf thymus DNA immobilised on the electrode sensor and investigated by chronopotentiometric analysis. The DNA biosensor is able to detect known intercalating and groove binding compounds. Applicability to river water samples was demostrated.

Moreover, a piezoelectric sensor coupled to a short oligonucleotide can be used as detector of the hybridisation reaction. We show as a model the detection of a specific mutation in apolipoprotein E (ApoE) gene.

Biotinylated 23-mer probes were immobilised on the streptavidin coated gold surface of a quartz crystal; the protein was covalently bound to the thiol/dextran modified gold surface. The device was able to distinguish different synthetic oligonucleotides. The hybridisation reaction was also performed using real samples of DNA extracted from human blood and amplified by Polymerase chain reaction PCR. The extension of such procedure to samples of environmental interest is discussed.     

A modified method for the analysis of organics in industrial wastewater as directed by their toxicity to Vibrio fischeri

A method for the identification of toxic compounds in industrial wastewater is presented, consisting of sequential solid phase extraction (SPE), fractionation by HPLC and GC-MS for compound identification. All analytical steps are accompanied by an automated detection of the aquatic toxicity by luminescence inhibition of Vibrio fischeri, which helps to reduce the large number of samples and subsamples that have to be processed by exluding those without toxic effects. The advantages of this procedure in comparison to previous methods of toxicity directed water analysis are discussed. The procedure was successfully applied to various samples of tannery wastewater, showing that benzothiazoles account for the major toxicity of tanyard wastewater. For very polar wastewater constituents, such as in beamhouse wastewaters, the use of LC-MS/MS for the final compound identification is suggested.     

Prostate Cancer Biomarker Detection with Carbon Nanotubes Modified Screen Printed Electrodes

DNA hypermethylation is an epigenetic alteration and a promising biomarker for early prostate cancer detection. Simple, sensitive, easy to handle and rapid detection methodologies are imperative for point of care diagnostics especially for cancer. Herein, we describe for the first time a regenerable and compatible electrochemical biosensor for detection of Glutathione S‐Transferase P‐1 (GSTP‐1) gene hypermethylation related to prostate cancer via DNA hybridization onto the disposable Carbon and Multi Walled Carbon Nanotubes (MWCNT) Screen Printed Electrodes (SPEs). In the study, capture probes were adsorbed onto the SPEs by simple passive adsorption and then hybridization was achieved by sending the complementary target onto the probe‐modified electrodes. The selectivity of the biosensor was proved by control studies. Differential Pulse Voltammetry (DPV) technique was used to detect hybridization via guanine oxidation signals changes. The total time of the optimized method was nearly 1h, measurements took for less than 1 min, and the biosensor response was stable up to 40 days of storage period at 4 °C. The main advantages of the biosensor are very low detection limit (picomolar range) and capability of reusing the biosensor for at least 3 times after very simple regeneration process that is a unique property to reduce the cost of the assay. In addition, this is the first study that demonstrates the detection of GSTP‐1 hypermethylation electrochemically by using SPEs in order to create point of care diagnostics. The optimum parameters for the biosensor, as well as its future prospects to enhance the performance of DNA biosensors were also presented.     

Label-free electrochemical detection of Avian Influenza Virus genotype utilizing multi-walled carbon nanotubes–cobalt phthalocyanine–PAMAM nanocomposite modified glassy carbon electrode

A novel DNA electrochemical biosensor for label-free determination of DNA sequence related to the Avian Influenza Virus (AIV) genotype was demonstrated in this paper. First, the multi-walled carbon nanotubes–cobalt phthalocyanine (MWNTs–CoPc) nanocomposite and poly (amidoamine) (PAMAM) dendrimer (generation 4.0) were modified on the glassy carbon electrode (GCE) sequentially. Then, DNA probes were successfully immobilized on the modified electrode with G4 PAMAM dendrimer acting as the coupling agent. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement based on the oxidation signals of guanine without any external labels. Under the optimal conditions, the difference in guanine oxidation signal of the probe modified GCE in the absence and presence of complementary target (ΔI_p) was linear with the logarithmic value of the complementary target concentration from 0.01 to 500 ng/ml with a correlation coefficient of 0.998 and a detection limit of 1.0 pg/ml.     

Experimental and theoretical investigation effect of flavonols antioxidants on DNA damage

A new electrochemical biosensor was developed to demonstrate the effect of Acridine Orange (AO) on DNA damage. Then, the biosensor was used to check the inhibitors effect of three flavonols antioxidants (myricetin, fisetin and kaempferol) on DNA damage. Acridine Orange (AO) was used as a damaging agent because it shows a high affinity to nucleic acid and stretch of the double helical structure of DNA. Decreasing on the oxidation signals of adenine and guanine (in the DNA) in the presence of AO were used as probes to study the antioxidants power, using DNA-modified screen printed graphene electrode (DNA/SPGE). The results of our study showed that the DNA-biosensor could be suitable biosensor to investigate the inhibitors ability of the flavonols antioxidants on the DNA damage. The linear dependency was detected in the two regions in the ranges of 1.0–15.0 and 15.0–500.0 pmol L⁻¹. The detection limit was found 0.5 pmol L⁻¹ and 0.6 pmol L⁻¹ for guanine and adenine, respectively. To confirm the electrochemical results, Uv–Vis and fluorescence spectroscopic methods were used too. Finally molecular dynamic (MD) simulation was performed on the structure of DNA in a water box to study any interaction between the antioxidant, AO and DNA.     

Comparison of three microbial assay procedures for measuring toxicity of chemical compounds: ToxAlert10, CellSense and Biolog MT2 microplates

Methods for measuring toxicity or respiratory activity of microbial cultures can be used as tools for assessing the impact of chemicals or waste streams on biological wastewater treatment plants. Easy-to-use and highly standardised toxicity tests are gaining wide acceptance due to their capability to make an assessment of the overall composite toxicity of the discharge. In this study we evaluate the relative sensitivity and performance of three microbial assay procedures for measuring toxicity of nine commonly used organic compounds in pharmaceutical processes: ToxAlert^® (using Vibrio fischeri), CellSense biosensors (using activated sludge, Pseudomonas putida and V. fischeri sensors) and Biolog MT2 microplates (using industrial activated sludge). Results were statistically compared and characteristics of the three procedures are discussed in terms of their sensitivity, reproducibility, representativity and ease of execution. All tests were found to be easy to perform. ToxAlert^® gave the best quantitative results, best reproducibility and repeatability followed by Biolog MT2. The uncertainty in the estimates for CellSense was found to be large, leading to wide confidence intervals. Although ToxAlert^® gave the best quantitative results the Biolog MT2 microplates which used industrial activated sludge were found to be more representative of the microflora present in industrial biological treatment plants. The microplate respiration tests are recommended for screening large number of toxicants or wastewater samples. CellSense gave poor reproducibility, however, it is a method with potential since it uses whole cell biosensors which allow different species to be easily and quickly tested.     

Electrochemical Monitoring of DNA Hybridization by Multiwalled Carbon Nanotube Based Screen Printed Electrodes

The application of multiwalled carbon nanotube (MWCNT) based screen printed graphite electrodes (SPEs) was explored in this study for the electrochemical monitoring of DNA hybridization related to specific sequences on Hepatitis B virus (HBV) DNA. After the microscopic characterization of bare MWCNT‐SPEs and DNA immobilized ones was performed, the optimization of assay has been studied. The development of screen printing process combined with nanomaterial based disposable sensor technology leads herein a great opportunity for DNA detection using differential pulse voltammetry (DPV) by measuring the guanine oxidation signal observed at +1.00 V in the presence of DNA hybridization between HBV probe and its complementary, target. The detection limit estimated for signal to noise ratios =3 corresponds to 96.33 nM target concentration in the 40 μL samples. The advantages of carbon nanotube based screen printed electrode used for electrochemical monitoring of DNA hybridization are discussed with sensitivity, selectivity and reproducibility in comparison with previous nanomaterial based electrochemical transducers developed for DNA or other biomolecular recognitions.