Naphthalene Diimide Carrying Two Cysteine Termini at Both Imide Linkers as a Molecular Staple

Naphthalene diimide ( 1 ) carrying cysteines at the termini of amide substituents were synthesized to act as a molecular staple of double stranded DNA. Since 1 is able to bind to double stranded DNA with threading intercalation, the complex of 1 with double stranded DNA can be topologically immobilized on a gold surface through the S? Au linkage as confirmed by cyclic voltammetric experiment. Ferrocenyl‐double stranded 23‐mertic oligonucleotide, dsFcODN, was immobilized on gold electrode with 1.0×10¹² molecules cm^?2 when electrode was treated with 2.0 µM dsFcODN and 4.0 µM 1 for 1 h at room temperature. The coverage density was similar to that obtained for the terminal thiol‐modified oligonucleotide. Compound 1 was applied to detect the 321‐meric PCR product of P. gingivalis, which is important in the diagnosis of periodontal disease. This experiment, coupled with the use of ferrocenylnaphthalene diimide, FND as electrochemical indicator for double stranded DNA, resulted in quantitative detection of PCR product within the range of 10 pg µL^?1–10 ng µL^?1 (15 nM–15 µM). The 1 and FND established a simple and rapid detection method of double stranded PCR product with a detection limit of 10 pg µL^?1 (15 nM).     

Electrochemical Detection of Duplex DNA Using Intercalation‐Triggered Decomplexation of Ferrocene with β‐Cyclodextrin

The redox peak of ferrocenylnaphthalene diimide used as a threading intercalator shifted positively due to the formation of its complex with β‐cyclodextrin. This complex collapsed upon the addition of double‐stranded DNA, and its redox potential shifted negatively. This behavior was applied for the homogenous detection of a polymerase chain reaction (PCR) product from Porphyromonas gingivalis, which is important for the diagnosis of periodontal disease, and its quantitative detection was achieved with a detection limit of 2.7 nM.     

Electrochemical DNA Biosensor Based on Partially Reduced Graphene Oxide Modified Carbon Ionic Liquid Electrode for the Detection of Transgenic Soybean A2704‐12 Gene Sequence

In this work a partially reduced graphene oxide (p‐RGO) modified carbon ionic liquid electrode (CILE) was prepared as the platform to fabricate an electrochemical DNA sensor, which was used for the sensitive detection of target ssDNA sequence related to transgenic soybean A2704‐12 sequence. The CILE was fabricated by using 1‐butylpyridinium hexafluorophosphate as the binder and then p‐RGO was deposited on the surface of CILE by controlling the electroreduction conditions. NH₂ modified ssDNA probe sequences were immobilized on the electrode surface via covalent bonds between the unreduced oxygen groups on the p‐RGO surface and the amine group at the 5′‐end of ssDNA, which was denoted as ssDNA/p‐RGO/CILE and further used to hybridize with the target ssDNA sequence. Methylene blue (MB) was used as electrochemical indicator to monitor the DNA hybridization. The reduction peak current of MB after hybridization was proportional to the concentration of target A2704‐12 ssDNA sequences in the range from 1.0×10^?12 to 1.0×10^?6 mol/L with a detection limit of 2.9×10^?13 mol/L (3σ). The electrochemical DNA biosensor was further used for the detection of PCR products of transgenic soybean with satisfactory results.     

A Single‐Surface Electrochemical Biosensor for the Detection of DNA Triplet Repeat Expansion

Molecular diagnostics of inherited neurodegenerative disorders such as fragile X syndrome, myotonic dystrophy or Friedreich ataxia (FRDA) is based on analysis of the length of trinucleotide repetitive sequences in certain loci of genomic DNA. The current methods employ PCR and electrophoretic determination of the amplified DNA fragment size. We have recently shown that length of a triplet repetitive DNA sequence can be determined using a double‐surface electrochemical technique involving multiple hybridization of the expanded triplet repeat with short labeled reporter probe (spanning several trinucleotides). Here we propose a single‐surface sensor employing an analogous principle. Target DNA (tDNA) is adsorbed onto surface of a carbon (pyrolytic graphite or screen‐printed) electrode. Biotin‐labeled reporter probe (RP) is hybridized with the immobilized tDNA followed by binding of streptavidin‐alkaline phosphatase (ALP) conjugate. The ALP catalyzes production of an electroactive indicator (1‐naphthol) which is detected voltammetrically on the same electrode. Signal resulting from this electrochemical enzyme‐linked DNA hybridization assay is normalized to the amount of tDNA immobilized at the transducer surface either by measuring intrinsic tDNA voltammetric response, or using electrochemical labeling of the tDNA with osmium tetroxide 2,2′‐bipyridine complex. Detection of (GAA)_n?(TTC)_n triplet repeat expansion in nanogram quantities of PCR‐amplified tDNAs, including amplicons of patients' genomic DNA, is demonstrated. We show that our technique allow differentiation between normal and pathological alleles of X25 gene related to the FRDA.     

Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

In this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through Au? S bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10^?13 to 1.0×10^?6 mol·L^?1 with a detection limit of 3.5×10^?14 mol·L^?1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.     

Highly Sensitive Indicator‐Free Impedance Sensing of DNA Hybridization Based on Poly(m‐aminobenzenesulfonic acid)/TiO2 Nanosheet Membranes with Pulse Potentiostatic Method Preparation

A direct electrochemical detection procedure for DNA hybridization by using the electrochemical signal changes of conductive poly(m‐aminobenzenesulfonic) acid (PABSA)/TiO₂ nanosheet membranes, which were electropolymerized by using the pulse potentiostatic method, is reported. Due to the unique properties of TiO₂ nanoparticles, m‐aminobenzenesulfonic acid monomers tend to be adsorbed around the particles, and the electropolymerization efficiency is greatly improved. The combination of TiO₂ nanoparticles and PABSA resulted in a nanocomposite membrane with unique and novel nanosheet morphology that provides more activation sites and enhances the surface electron‐transfer rate. These characteristics were propitious for the magnification of PABSA electrochemical signals and the direct detection of DNA hybridization. Owing to the presence of abundant sulfonic acid groups, PABSA could overcome the drawbacks of polyaniline and be used to detect bioanalytes at physiological pH. DNA probes could be covalently attached to the sulfonic groups through the amines of DNA sequences by using an acyl chloride cross‐linking reaction. After immobilization of probe DNA, the electrochemical impedance value increased significantly compared to that of PABSA/TiO₂ nanosheet membranes, and then decreased dramatically after the hybridization reaction of the probe DNA with the complementary DNA sequence compared to that of the probe‐immobilized electrode. Electrochemical impedance spectroscopy was adopted for indicator‐free DNA biosensing, which had an eminent ability for the recognition between double‐base mismatched sequences or non‐complementary DNA sequences and complementary DNA sequences. A gene fragment, which is related to one of the screening genes for the transgenically modified plants, the cauliflower mosaic virus 35S gene was satisfactorily detected. This is the first report for the indicator‐free impedance DNA hybridization detection by using PABSA/TiO₂ membranes under neutral conditions.     

Utilization of Nucleotide Probes in PCR‐ELISA Procedure for the Quantitative Determination of Plasmodium Falciparum DNA in Malaria

Abstract

The research work reported herein is the development of a simple and specific quantitative procedure for the determination of P. falciparum DNA in malaria that involves the direct detection of the highly 42‐kDa conserved C‐terminal regiopn of P. falciparum merozoite surface protein gene (MSP1 ₄₂ gene). This procedure entails the amplification of the MSP1 ₄₂ gene by using the PCR technique in the presence of digoxigenin‐11‐dUTP and the synthesis of the specific biotin label nucleotide probes directed to the MSP1 ₄₂ gene. These specific probes are then used in the Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative determination of the MSP1 ₄₂ gene which leads to the quantitative determination of P. falciparum DNA in malaria for quantitative diagnostic purpose. The P. falciparum malaria diagnostic results obtained from a small number of 18 whole blood samples show that the present quantitative PCR‐ELISA procedure allows the quantitative determination of P. falciparum DNA in malaria with a sensitivity and specificity over to those of the current standard microscopic examination. This quantitative PCR‐ELISA procedure is not only important for quantitative P. falciparum malaria diagnosis but also useful for monitoring the efficacy of any existing anti‐malarial drug as well as for testing the efficacy of any malaria vaccine.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

Electrochemical DNA Biosensors Based on Palladium Nanoparticles Combined with Carbon Nanotubes

Palladium nanoparticles, in combination with multi‐walled carbon nanotubes (MWCNTs), were used to fabricate a sensitivity‐enhanced electrochemical DNA biosensor. MWCNTs and palladium nanoparticles were dispersed in Nafion, which were used to modify a glassy carbon electrode (GCE). Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. Due to the ability of carbon nanotubes to promote electron‐transfer and the high catalytic activities of palladium nanoparticles for electrochemical reaction of MB, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.2×10^?13 M.     

Tagging of Model Amphetamines with Sodium 1,2‐Naphthoquinone‐4‐sulfonate: Application to the Indirect Electrochemical Detection of Amphetamines in Oral (Saliva) Fluid

Proof‐of‐concept is shown for the indirect electrochemical detection of model amphetamines, D ‐amphetamine sulfate and pseudoephedrine, based on the labeling of the amphetamine models with sodium 1,2‐naphthoquinone‐4‐sulfonate (NQS). The presence of the amphetamine models is monitored via either the reduction in the magnitude of the voltammetric peak corresponding to the electrochemical reduction of the quinone functionality of the sodium 1,2‐naphthoquinone‐4‐sulfonate or via growth of a new voltammetric peak related to reaction between the amphetamine model and NQS, both of which are well resolved from one another. The protocol is shown to be successful in artificial saliva and authentic human oral (saliva) fluid. Such a protocol may be particularly attractive for roadside testing of amphetamines in drug drivers.     

Multi‐Walled Carbon Nanotubes (MWCNT)‐Ionic Liquid‐Modified Carbon Paste Electrode: Probing FurazolidoneDNA Interactions and DNA Determination

The interactions of furazolidone (Fu) with double‐stranded calf thymus DNA (dsDNA) on the multi‐walled carbon nanotubes‐ionic liquid‐modified carbon paste electrode (MWCNT‐IL‐CPE) have been studied by cyclic voltammetry. In the presence of DNA, the cathodic peak current of Fu decreased and the peak potential shifted to a positive potential, indicating the intercalative interaction of Fu with DNA. The binding constant of Fu with DNA and stoichiometric coefficient has been determined according to the Hill's model. This electrochemical method was further applied to the determination of DNA. Two linear calibration curves were obtained for DNA detection in the concentration ranges of 0.03–0.10 and 0.10–4.0 μg l^?1 with a detection limit of 0.027 μg l^?1. The method was successfully applied to analyze Fu in serum samples.     

Homogeneous Electrochemical Assay for Real‐time Monitoring of Exonuclease III Activity Based on a Graphene Monolayer

A homogeneous electrochemical assay based on a graphene monolayer electrode was developed for simple, sensitive, rapid and quantitative analysis of the exonuclease III (Exo III) activity. The method utilized a methylene blue (MB) tagged DNA substrate with hairpin structure, and a graphene monolayer attached on the working electrode. Before digestion, the hairpin structure prevents the adsorption of the DNA substrate to the graphene surface. Degradation of the substrate by the 3′–5′ Exo III, however, yields single‐stranded DNA (ssDNA), resulting in its subsequent binding to the graphene surface through π‐π stacking, which produces the voltammetric current from electrochemical reduction of the MB tag anchoring at the end of ssDNA. A direct quantification of the Exo III activity can be achieved by measuring the reductive peak current of the MB tag under easily attainable potential (∼ −0.1 V vs Ag/AgCl) range comparably sensitive to the conventional methods such as a gel‐based or fluorescence‐based assays. Our approach can be applied to measure various exonucleases activity by adjusting the structure of DNA substrate suggesting a new assay method in drug screening and basic research related to the enzymes.     

New Electroactive Hybridization Indicators 2‐Phthalimido‐N‐Substitutedphenylethanesulfonamide Derivatives for Biosensor Applications: Ring Substituent Effect on Interaction between Compound and DNA

In this work, an electrochemical DNA‐based sensor was developed for the detection of the interaction between the anticonvulsant compounds 2‐phthalimido‐N‐substituted phenylethanesulfonamides (PMPES‐derivatives) and 24‐mer short DNA sequences by using differential pulse voltammetry (DPV) based on both compound and guanine oxidation signals at the renewable carbon graphite electrode (CGE) surface. The influence of compounds on DNA showed differences depending on the nature and position of the substituent on the N‐phenyl ring. Compounds bearing 3‐methoxy, 4‐chloro and 2,6‐dimethyl substituents bind to single stranded probe DNA more strongly than the other derivatives of PMPES. Thus, these compounds were evaluated for use as an electrochemical hybridization label (indicator).     

An Electrochemical DNA Sensor Based on Electrodepositing Aluminum Ion Films on Stearic Acid‐Modified Carbon Paste Electrode and Its Application for the Detection of Specific Sequences Related to Bar Gene and CP4 Epsps Gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid‐modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well‐defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single‐stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the specific sequence related to the target bar gene with the dynamic range comprised between 1.0×10^?7 mol/L to 1.0×10^?4 mol/L. A detection limit of 2.25×10^?8 mol/L of oligonucleotides can be estimated.     

Electrochemical Behavior of o‐Nitrophenol at Hexagonal Mesoporous Silica Modified Carbon Paste Electrodes

A hexagonal mesoporous silica (HMS) modified carbon paste electrode (CPE) was fabricated and characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods (ferrocene as a probe). The electrochemical behavior of nitrophenol (o‐NP) at the HMS modified electrode (HMSCPE) was investigated. Compared with CPE, a well‐defined reduction peak and a remarkably peak current response was observed. It is indicated that mesoporous HMS exhibited remarkable enhancement effects on the electrochemical reduction of o‐NP. The electrochemical reduction mechanism was also discussed. Consequently, a simple and sensitive electrochemical method was proposed for the determination of o‐NP, which was used to determine o‐NP in waste water samples.     

Facile Fabrication of Graphene‐Containing Foam as a High‐Performance Anode for Microbial Fuel Cells

Facile fabrication of novel three‐dimensional anode materials to increase the bacterial loading capacity and improve substrate transport in microbial fuel cells (MFCs) is of great interest and importance. Herein, a novel graphene‐containing foam (GCF) was fabricated easily by freeze‐drying and pyrolysis of a graphene oxide–agarose gel. Owing to the involvement of graphene and stainless‐steel mesh in the GCF, the GCF shows high electrical conductivity, enabling the GCF to be a conductive electrode for MFC applications. With the aid of agarose, the GCF electrode possesses a supermacroporous structure with pore sizes ranging from 100–200 μm and a high surface area, which greatly increase the bacterial loading capacity. Cell viability measurements indicate that the GCF possesses excellent biocompatibility. The MFC, equipped with a 0.4 mm‐thick GCF anode, shows a maximum area power density of 786 mW m^?2, which is 4.1 times that of a MFC equipped with a commercial carbon cloth anode. The simple fabrication route in combination with the outstanding electrochemical performance of the GCF indicates a promising anode for MFC applications.     

An Electropolymerized Membrane Biosensor for Speciffic DNA Recognition

A sensitive electrochemical biosensor for detecting the sequence of short DNA oligomers is represented. The biosensor is based on a platinum electrode covered a polymerized membrane of conductive monomer N‐[6‐(thien‐3‐yl)acetoxy]‐pyrrolidine‐2, 5‐dione (TAPD). The membrane of TAPD immobilizes a probe DNA on the electrode. The hybridization of the probe with a sequence‐specific DNA in sample solutions is monitored by a self‐synthesized electroactive indicator, which specifically intercalates in the hybrids on the electrode surface. The current signal of the biosensor is proportional to the concentration of the target DNA in samples, and a very low detection limit of 5 × 10^?10 mol/L is found. The biosensor has been used to detect the short oligomers containing of HTV‐1 and mycobacterrium nucleotide sequences.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.     

Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen

An electrochemical biosensor for the specific detection of short DNA sequences from the E. coli pathogen is described. This hybridization device relies on the immobilization of a 25-mer oligonucleotide probe, from the E. coli lacZ gene, onto a screen-printed carbon electrode. Chronopotentiometric detection of the Co(bpy)₃⁺³ indicator is used for monitoring the hybridization event. Numerous variables of the assay protocol, including those of the probe immobilization step, the hybridization event, and the indicator association/detection, are characterized and optimized. Hybridization times of 2- and 30-min are sufficient for detecting 300- and 50 ng/mL, respectively, of the E. coli DNA target. Applicability to analysis of untreated environmental water samples is illustrated. Such single-use electrochemical sensors hold great promise for decentralized environmental and food testing for the E. coli pathogen.     

Electrochemical DNA Biosensor Based on Magnetite/Multiwalled Carbon Nanotubes/Chitosan Nanocomposite for Bacillus Cereus Detection of Potential Marker for Gold Prospecting

A label‐free DNA biosensor based on magnetite/multiwalled carbon nanotubes/chitosan (Fe₃O₄/MWCNTs‐COOH/CS) nanomaterial for detection of Bacillus cereus DNA sequences was fabricated. Negatively charged DNA was electrostatically adsorbed onto materials by protonation of positively charged chitosan under acidic conditions. The electrode surface and hybridization process were carried out by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Under optimal conditions, the biosensor showed a good linear relationship between peak currents difference (ΔI) and logarithm of the target DNA concentration (Log C) ranging from 2.0×10⁻¹³ to 2.0×10⁻⁶ M with a detection limit of 2.0×10⁻¹⁵ M (signal/noise ratio of 3). The biosensor also revealed an excellent selectivity to three‐base, completely mismatched and completely matched DNA. This is a simple, fast and friendly method with a low detection limit for the detection of Bacillus cereus specific DNA compared with previously reported electrochemical DNA biosensor. Furthermore, the DNA biosensor may lead to the development of a technology for gold prospecting in the wild.