Electrochemical DNA-based Biosensor Research Articles

Fabrication of an electrochemical DNA-based biosensor for Bacillus cereus detection in milk and infant formula

This paper describes fabrication of a DNA-based Au-nanoparticle modified pencil graphite electrode (PGE) biosensor for detection of Bacillus cereus, causative agent of two types of food-borne disease, i.e., emetic and diarrheal syndrome. The sensing element of the biosensor was comprised of gold nanoparticles (GNPs) self-assembled with single-stranded DNA (ssDNA) of nheA gene immobilized with thiol linker on the GNPs modified PGE. The size, shape and dispersion of the GNPs were characterized by field emission scanning electron microscope (FESEM). Detection of B. cereus was carried out based on an increase in the charge transfer resistance (Rct) of the biosensor due to hybridization of the ss-DNA with target DNA. An Atomic force microscope (AFM) was used to confirm the hybridization. The biosensor sensitivity in pure cultures of B. cereus was found to be 100 colony forming units per milliliter (CFU/mL) with a detection limit of 9.4×10−12molL−1. The biosensor could distinguish complementary from mismatch DNA sequence. The proposed biosensor exhibited a rapid detection, low cost, high sensitivity to bacterial contamination and could exclusively and specifically detect the target DNA sequence of B. cereus from other bacteria that can be found in dairy products. Moreover, the DNA biosensor exhibited high reproducibility and stability, thus it may be used as a suitable biosensor to detect B. cereus and to become a portable system for food quality control.

Molecular behavior of the aptamer HJ24 self-assembled on highly oriented pyrolytic graphite (HOPG)

Nucleic acid sensing analysis has been widely applied to the fields of food quality control, environmental monitoring, and medical diagnosis. A key step in developing effective DNA-based electrochemical biosensors is to obtain a biorecognition layer on the biosensor, which can be influenced by many factors. Hence, we constructed a series of HJ24 layers on highly oriented pyrolytic graphite to investigate the relation between the configuration of the adsorbed probe HJ24 and the redox property using atomic force microscopy and voltammetry. We used HJ24 for its diagnostic value as it specifically recognizes the SH2 domain-containing phosphatase, a critical contributor in many important signaling pathways. The results demonstrated that increasing K ions induced G-quartet oxidation peak occurrence/increase ([K+]￡400 mmol/L), and also resulted in the formation of more compact single strand sheets ([K+]￡300 mmol/L). Moreover, transitions of molecule configurations and redox currents of G-quartets were observed at low concentration [K+]=12 mmol/L, which may indicate the appearance of new configurations under this condition. Besides, by analyzing atomic force microscopy (AFM) images, it was found that the different adsorbed configurations are correlated with the HJ24 concentration, the basal configuration, and the linker group on the HJ24 sequence. This information may be useful for understanding the adsorption process of HJ24 as well as for the further development of practical applications for HJ24 films on DNA electrochemical sensors, and may ultimately help improve the diagnosis and treatment of patients with SHP2-related diseases.

An electrochemical DNA-based biosensor to study the effects of CdTe quantum dots on UV-induced damage of DNA

A DNA-based biosensor is presented that can be applied to the detection of DNA damage caused by UV-C radiation (254 nm) in the presence of CdTe quantum dots (QDs). The sensor is composed of a glassy carbon electrode whose surface was modified with a layer of dsDNA and an- other layer of CdTe QDs. The response of this sensor is based on (a) the intrinsic anodic signal of the guanine moiety in the DNA that is measured by square-wave voltammetry, and (b) thecyclicvoltammetricresponseoftheredoxindicatorsystem hexacyanoferrate(III/II). Depending on the size of the QDs, they exert a significant effect on the rate of the degradation of dsDNA by UV-C light, and even by visible light. Time- dependent structural changes of DNA include opening of the doublehelix (as indicated byanincrease inthe redox response of the guanine moiety due to easy electron exchange with the electrode when compared to the original helix state and by an increase in the voltammetric peak current of the hexacyanoferrate(III/II) anion after degradation of the nega- tively charged DNA backbone on the electrode). The effects of QDs were verified for salmon sperm DNA and calf thymus DNA,andfurthercorroboratedbyexperimentsinwhichDNA solutions were irradiated in the presence of QDs.

Electrochemical Biosensor Development for Detection of Botulinum Toxin A (BoNT/A)

Effective methods for the detection of biological toxins are vital for food safety, human medicine, environmental maintenance, and safeguarding against biological warfare. In this project, we are developing DNA‐based electrochemical biosensors to sensitively detect botulinum toxin A (BoNT/A), the target involved in the life‐threatening paralytic disease botulism. We are utilizing rationally designed and synthesized oligonucleotide biosensors to bind to this target, generating measurable electrochemical changes upon binding the toxin in a potentiometric analysis. These biosensors are based on BoNT/A binding aptamers discovered through modified SELEX procedures. They are uniquely able to capture these data in real‐time in complex fluids, making them advantageous over current detection techniques. This approach should enable the detection of minute levels of BoNT/A in media such as foodstuffs, blood, or water, improving both prevention and diagnosis of botulism disease. Together, development of a BoNT/A biosensor will allow for detection in safety, research, commercial, and/or at‐home applications.

A new electrochemical aptasensor based on electrocatalytic property of graphene toward ascorbic acid oxidation

Based on the superior electrocatalytic property of graphene (GN) toward ascorbic acid (AA) oxidation, a new electrochemical aptasensor has been developed. Here, adenosine triphosphate (ATP) is used as the model to demonstrate the performance of the developed aptasensor. Briefly, GN is attached to the thiolated ATP binding aptamer (ABA) modified gold electrode through π-π stacking interaction, resulting in a significant oxidation signal of AA. In the presence of ATP, the formation of ATP-ABA complex leads to the release of GN from sensing interface, resulting in a sharp decrease of the oxidation peak current of AA and an obviously positive shift of the related peak potential. Taking both the change values of the peak current and peak potential of AA oxidation as the response signals, ATP can be detected sensitively. This is the first time to demonstrate the application of GN as the nanocatalyst in an amplified aptasensor. It can be expected that GN, as nanocatalyst, should become the very promising amplifying-elements in DNA-based electrochemical biosensors.

A ratiometric electrochemical biosensor for sensitive detection of Hg2+ based on thymine–Hg2+–thymine structure

In this paper, a simple, selective and reusable electrochemical biosensor for the sensitive detection of mercury ions (Hg2+) has been developed based on thymine (T)-rich stem–loop (hairpin) DNA probe and a dual-signaling electrochemical ratiometric strategy. The assay strategy includes both “signal-on” and “signal-off” elements. The thiolated methylene blue (MB)-modified T-rich hairpin DNA capture probe (MB-P) firstly self-assembled on the gold electrode surface via Au–S bond. In the presence of Hg2+, the ferrocene (Fc)-labeled T-rich DNA probe (Fc-P) hybridized with MB-P via the Hg2+-mediated coordination of T–Hg2+–T base pairs. As a result, the hairpin MB-P was opened, the MB tags were away from the gold electrode surface and the Fc tags closed to the gold electrode surface. These conformation changes led to the decrease of the oxidation peak current of MB (IMB), accompanied with the increase of that of Fc (IFc). The logarithmic value of IFc/IMB is linear with the logarithm of Hg2+ concentration in the range from 0.5nM to 5000nM, and the detection limit of 0.08nM is much lower than 10nM (the US Environmental Protection Agency (EPA) limit of Hg2+ in drinking water). What is more, the developed DNA-based electrochemical biosensor could be regenerated by adding cysteine and Mg2+. This strategy provides a simple and rapid approach for the detection of Hg2+, and has promising application in the detection of Hg2+ in real environmental samples.

DNA-based biosensors for Hg(2+) determination by polythymine-methylene blue modified electrodes.

In this work we have developed a new electrochemical DNA-based biosensor for the selective determination of the Hg(2+) ion by the use of different electrodes modified with polythymine, bearing methylene blue, as redox probe, in 3' position. The determination of Hg(2+) can be employed with an excellent degree of selectivity by the use of DNA biosensors through the formation of the complex Thymine-Hg-Thymine (T-Hg-T): in fact, Hg(2+) tends to bind two thymines, generating a T-Hg-T complex with a formation constant higher than that one of the coupling Adenine-Thymine, which can be employed for a selective, fast and cost-effective Hg(2+) detection. The presence of the Hg(2+) in solution leads to the formation of T-Hg-T complex thus causing the "hairpin-like" folding of oligonucleotide, leading to an improved electronic exchange of methylene blue with the electrode surface due to the reduced distance and thus to an increase of the faradic current which is detected by means of square wave voltammetry (SWV). To test the feasibility of this kind of biosensor to be applied to the analysis of Hg(2+) we have developed several biosensors configuration by modifying the electrochemical sensor transducer: (a) Au electrode; (b) Au screen-printed electrode (SPE). The proposed system, allows the determination of Hg(2+) in the range 0.2-100 nM (0.05-20 ppb), with a sensitivity 0.327 µA/nM, LOD 0.1 nM (0.02 ppb), LOQ 0.2 nM (0.05 ppb) and RSD ≤4.3% when Au electrode is used as electrochemical transducer; on the other hand, in the case of Au SPE the linear range is 0.2-50 nM (0.05-10 ppb), with a sensitivity 0.285 µA/nM, while LOD and LOQ are the same as previously and RSD is ≤3.8%. This enabled the detection of mercury in real samples (waters and fishes) with good accuracy (recoveries 92-101% on waters and 92-107% on fishes, respectively) and reproducibility (RSD ≤9.6% for measurements on waters and ≤8.8% on fishes, respectively).

Redox targeting of DNA anchored to MWCNTs and TiO2 nanoparticles dispersed in poly dialyldimethylammonium chloride and chitosan

A key issue associated with electrochemical DNA-based biosensors is how to enhance DNA immobilization on the substrates. In order to improve the immobilization of DNA and to optimize DNA interaction efficiency, different kinds of strategies have been developed. In this regard, nanomaterials have attracted a great deal of attention in electrode surface modification for DNA biosensor fabrication. In this study, nanostructured films were deposited at the surface of a pencil graphite electrode (PGE) as a working electrode. For the present purpose, common polyelectrolytes are used for surface modification with double-stranded DNA. Two positively charged polyelectrolyte, namely poly dialyldimethylammonium chloride (PDDA) and chitosan, are initially compared for DNA immobilization at the surface of MWCNTs and TiO2 nanoparticles (TiO2NPs). In a second step, the basic electrochemical properties of the sensors are investigated using voltammetric methods. The modified electrodes are also characterized by scanning electron microscopy and electrochemical impedance measurements. It will be shown that electrode modification with DNA and the nanostructure that disperses in PDDA leads to an enhanced sensitivity of the DNA voltammetric detection mechanism. In a previous study, a comparison was done between MWCNTs and TiO2NPs for determining the effect of nanoparticle effect on DNA immobilization on the electrode surface. In order to compare the efficiency of the prepared DNA-based biosensors, methylene blue is chosen as an electroactive probe. It will be shown that the stability of the immobilized DNA within several days will be much higher when MWCNTs rather than TiO2NPs are used.

Ultra-sensitive biosensor for K-ras gene detection using enzyme capped gold nanoparticles conjugates for signal amplification

In this study, an ultra-sensitive hairpin DNA-based electrochemical DNA biosensor for K-ras gene detection is described. Gold nanoparticles (Au-NPs) and horseradish peroxidase (HRP)–streptavidin capped Au-NPs (HAS) conjugates are used for signal amplification. Initially, hairpin DNA dually labeled with thiol at its 5′ end and with biotin at its 3′ end is immobilized on the surface of Au-NPs modified electrode, and the hairpin DNA is in a “closed” state; hence, the HAS conjugates are shielded from being approached by the biotin due to steric hindrance. However, in the presence of target DNA, the target DNA hybridizes with the loop structure of hairpin DNA and causes conformational change, resulting in biotin forced away from the electrode surface, thereby becoming accessible for the HAS conjugates. Thus, the HAS conjugates are linked to the electrode surface via the specific interaction between biotin and streptavidin. Electrochemical detection was performed in phosphate-buffered saline (PBS) containing tetramethylbenzidine (TMB) and H2O2. Under optimal conditions, the peak current differences (ΔI) are linear with the target DNA in the range from 0.1fM to 1nM with a detection limit of 0.035fM. Furthermore, this biosensor also demonstrates its excellent specificity for single-base mismatched DNA.

DNA-based biosensors with external Nafion and chitosan membranes for the evaluation of the antioxidant activity of beer, coffee, and tea

Abstract Novel electrochemical DNA-based biosensors with outer-sphere Nafion and chitosan protective membranes were prepared for the evaluation of antioxidant properties of beverages (beer, coffee, and black tea) against prooxidant hydroxyl radicals. A carbon working electrode of a screen-printed three-electrode assembly was modified using a layer-by-layer deposition technique with low molecular weight double-stranded DNA and a Nafion or chitosan film. The membrane-covered DNA biosensors were initially tested with respect to their voltammetric and impedimetric response after the incubation of the beverage and the medium exchange for the solution of the redox indicator [Fe(CN)6]3−/4−. While the Nafion-protected biosensor proved to be suitable for beer and black tea extracts, the chitosan-protected biosensor was successfully used in a coffee extract. Afterwards, the applicability was successfully verified for these biosensors for the detection of a deep degradation of the surface-attached DNA at the incubation in the cleavage agent (hydroxyl radicals generated via Fenton reaction) and for the evaluation of antioxidant properties of coffee and black tea extracts against prooxidant hydroxyl radicals. The investigation of the novel biosensors with a protective membrane represents a significant contribution to the field of electrochemical DNA biosensors utilization.

DNA interaction of [Cu(dmp)(phen-dion)] (dmp = 4,7 and 2,9 dimethyl phenanthroline, phen-dion = 1,10-phenanthroline-5,6-dion) complexes and DNA-based electrochemical biosensor using chitosan–carbon nanotubes composite film

The interaction of two new water-soluble [Cu(4,7-dmp)(phen-dione)Cl]Cl (1) and [Cu(2,9-dmp)(phen-dione)Cl]Cl (2) which dmp is dimethyl-1,10-phenanthroline and phen-dion represents 1,10-phenanthroline-5,6-dion, with DNA in solution and immobilized DNA on a chitosan–carbon nanotubes composite modified glassy carbon electrode were investigated by cyclic voltammetry and UV–Vis spectroscopy techniques. In solution interactions, spectroscopic and electrochemical evidences indicate outside binding of these complexes. To clarify the binding mode of complexes, it was done competition studies with Hoechst and Neutral red as groove binder and intercalative probes, respectively. All these results indicating that, these two complexes (1) and (2) interact with DNA via groove binding and partially intercalative modes, respectively. The electrochemical characterization experiments showed that the nanocomposite film of chitosan–carbon nanotubes could effectively immobilize DNA and greatly improve the electron-transfer reactions of the electroactive molecules that latter finding is the result of strong interactions between captured DNA and Cu complexes. This result indicates that these complexes could be noble candidates as hybridization indicators in further studies. At the end, these new complexes showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cell lines.

Graphene and CdS nanocomposite: A facile interface for construction of DNA-based electrochemical biosensor and its application to the determination of phenformin

Graphene/cadmium sulphide (GR-CdS) nanocomposite was synthesized via a low temperature process in aqueous solution. The as-prepared nanocomposite was characterized by scanning electron microscopy, UV–visible spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. The impedance analysis indicated that GR-CdS nanocomposite possessed outstanding electrochemical performance for facile electron transfer. When DNA was immobilized on GR-CdS (DNA/GR-CdS) modified electrode, the electrochemical oxidation of guanine and adenine in DNA residue bases was significantly promoted. Due to the interaction of DNA with phenformin, the voltammetric current of guanine or adenine on the DNA/GR-CdS electrode was decreased when phenformin was present in the electrolytic solution. Under optimized conditions, the signal of guanine on DNA/GR-CdS electrode decreased linearly with increasing the concentration of phenformin in the range of 1.0×10−6molL−1 to 1.0×10−3molL−1. The proposed DNA-based electrochemical biosensor was successfully applied to the determination of phenformin in real samples.

Review: advances in electrochemical genosensors-based methods for monitoring blooms of toxic algae

Harmful algal blooms (HABs), which have expanded worldwide in their occurrence and frequency, are a serious menace to aquatic ecosystems and humans. The development of rapid, accurate and cost-effective detection systems for toxic algal monitoring in aquatic environments is urgently required. Although many efforts have been devoted to develop reliable tools to monitor the entire spectrum of existing toxic algae, a portable semi-automated system that enables HAB monitoring at a low cost is still not available for general purchase. This work reviews the challenges and opportunities in translating the remarkable progress of electrochemical genosensors-based methods towards practical in situ HAB monitoring applications. It is specifically focused on reviewing the optimised methods for a detection system based on a sandwich hybridisation assay (SHA) performed over transducer platforms of different materials, geometries and dimensions and presenting the diverse advantages and disadvantages among them. Probe design and specificity and optimisation of the genosensor in terms of hybridisation conditions and electrochemical signal are discussed as well as their long-term stability and storage and semi-automation attempts. With continuous innovation and attention to key challenges, we expect semi-automatic devices containing DNA-based electrochemical biosensors to have an important impact upon monitoring of serious HAB events.

DNA biomolecular-electronic encoder and decoder devices constructed by multiplex biosensors

We fabricated and tested encoders and decoders based on a multiplex, DNA-based electrochemical biosensor that uses electronic (electrochemical) signals as its readout. These devices use two or more sequence-specific DNA probes, with each being modified with a distinct redox reporter. These probes, when interrogated together, serve as encoders and decoders, converting patterns that are encoded and decoded by the presence or absence of specific DNA sequences into specific electronic outputs. We demonstrated these multifunctional, bio-electrochemical devices, for example, 4-to-2 and 8-to-3 encoders and 1-to-2 and 2-to-3 decoders. Accordingly, these devices bridge the division between DNA-based devices and silicon-based electronics. Fan Xia and co-workers have converted biomolecular information – the presence or absence of DNA strands – into electronic currents by assembling electrochemical sensors. Each sensor is composed of an electrode, a DNA strand and a redox reporter – such as anthraquinone, methylene blue or ferrocene – and responds to the presence of one particular DNA strand by a change in its redox current. A set of sensors were designed to respond to different DNA strands by emitting distinct currents, and combining these sensors in various ways led to a series of encoding and decoding devices. While systems previously developed had typically given fluorescent outputs, this electrochemical approach moves DNA-based devices a step closer to their silicon-based electronic counterparts, and could potentially become components in DNA-based data processors. We have fabricated and tested encoders and decoders based on a multiplex, DNA-based electrochemical biosensor that uses electronic (electrochemical) signals as its readout. We have demonstrated these multifunctional, bio-electrochemical devices, for example, 4-to-2 and 8-to-3 encoders and 1-to-2 and 2-to-3 decoders. In doing so, these devices bridge the barrier between DNA-based devices and silicon-based electronics.

Tetrahedron-structured DNA and functional oligonucleotide for construction of an electrochemical DNA-based biosensor

Tetrahedron-structured DNA (ts-DNA) in combination with a functionalized oligonucleotide was used to develop a "turn-on" biosensor for Hg(2+) ions. The ts-DNA provided an improved sensitivity and was used to block the active sites.

Voltammetric studies on the potent carcinogen, 7,12-dimethylbenz[ a]anthracene: Adsorptive stripping voltammetric determination in bulk aqueous forms and human urine samples and detection of DNA interaction on pencil graphite electrode

7,12-Dimethylbenz[ a]anthracene (DMBA), is a widely studied polycyclic aromatic hydrocarbon that has long been recognized as a very potent carcinogen. Initially, the electrochemical oxidation of DMBA at the glassy carbon and pencil graphite electrodes in non-aqueous media (dimethylsulphoxide with lithium perchlorate) was studied by cyclic voltammetry. DMBA was irreversibly oxidized in two steps at high positive potentials, resulting in the ill-resolved formation of a couple with a reduction and re-oxidation wave at much lower potentials. Special attention was given to the use of adsorptive stripping voltammetry together with a medium exchange procedure on disposable pencil graphite electrode in aqueous solutions over the pH range of 3.0–9.0. The response was characterized with respect to pH of the supporting electrolyte, pre-concentration time and accumulation potential. Using square-wave stripping mode, the compound yielded a well-defined voltammetric response in acetate buffer, pH 4.8 at +1.15 V (vs. Ag/AgCl) (a pre-concentration step being carried out at a fixed potential of +0.60 V for 360 s). The process could be used to determine DMBA concentrations in the range 2–10 nM, with an extremely low detection limit of 0.194 nM (49.7 ng L −1). The applicability to assay of spiked human urine samples was also illustrated. Finally, the interaction of DMBA with fish sperm double-stranded DNA based on decreasing of the oxidation signal of adenine base was studied electrochemically by using differential pulse voltammetry with a pencil graphite electrode at the surface and also in solution. The favorable signal-to-noise characteristics of biosensor resulted in low detection limit ( ca. 46 nM) following a 300-s interaction. These results displayed that the electrochemical DNA-based biosensor could be used for the sensitive, rapid, simple and cost effective detection of DMBA–DNA interaction.

DNA-based biosensor for the detection of strong damage to DNA by the quinazoline derivative as a potential anticancer agent

A disposable electrochemical DNA-based biosensor was developed and applied as a screening device to detect an effect of a synthetically prepared quinazoline derivative on the surface-attached double stranded calf thymus DNA. Screen-printed carbon electrodes without and with multi-walled carbon nanotubes interface served as the signal transducer. The quinazoline interaction with DNA was investigated voltammetrically using DNA-bound electrochemical indicators such as [Co(phen)3]3+, [Ru(bpy)3]2+, methylene blue, the K3[Fe(CN)6] complex present in the solution phase as well as by electrochemical impedance spectroscopy. A severe damage to DNA at the incubation of the biosensor in quinazoline solution was found which leads to the loss of DNA from the electrode surface. Agarose gel electrophoresis was used to verify the results.

Disposable Electrochemical Biosensor with Multiwalled Carbon Nanotubes-Chitosan Composite Layer for the Detection of Deep DNA Damage

A novel electrochemical DNA-based biosensor for the detection of deep DNA damage was designed employing the bionanocomposite layer of multiwalled carbon nanotubes (MWNT) in chitosan (CHIT) deposited on a screen printed carbon electrode (SPCE). The biocomponent represented by double-stranded (ds) herring sperm DNA was immobilized on this composite using layer-by-layer coverage to form a robust film. Individual and complex electrode modifiers are characterized by a differential pulse voltammetry (DPV) with the DNA redox marker [Co(phen)(3)](3+), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with [Fe(CN)(6)](3-) as a redox probe in a phosphate buffer solution (PBS). A good correlation between the CV and EIS parameters has been found, thus confirming a strong effect of MWNT on the enhancement of the electroconductivity of the electrode surface and that of CHIT on the MWNT distribution at the electrode surface. Differences between the CV and EIS signals of the electrodes without and with DNA are used to detect deep damage to DNA, advantageously using simple working procedures in the same experiment.

Electrochemical Biosensors for Sequence‐Specific DNA Detection

This review summarizes the most relevant work performed in the last years in the field of the DNA-based electrochemical biosensors for sequence-specific DNA detection. The approaches used for preparing the biosensing layer, as well as the schemes developed for the transduction of the hybridization event are also discussed.

DNA-based electrochemical biosensors for monitoring of bis-indoles as potential antitumoral agents, chemistry, X-ray crystallography

Facile and practical electrochemical DNA bioassay, X-ray diffraction analysis, synthesis and 1H and 13C NMR data of the 5,5′-disubstituted-3,3′-methanediyl- bis-indoles are reported. On the basis of electrochemical measurements we have hypothesized that the analyzed bis-indoles have an effect on human tumor cells due to DNA binding at adenine-thymidine deoxynucleotides rich region in a concentration/substituent dependent manner. Interesting N–H⋯π and hydrogen-bonding intermolecular interactions were observed which may differentiate their biological features. The 5,5′-dimethoxy-3,3′-methanediyl- bis-indole ( 2) was found to reduce considerably the growth of cancer cell lines HOP-92 (lung), A498 (renal) and MDA-MB-231/1TCC (breast). The results indicate that title compounds could be interesting as potential antitumoral chemotherapeutics.