DNA Hydrolysis and Voltammetric Determination of Guanine and Adenine Using Different Electrodes

Abstract

This work reports the development of a biosensor method for the label‐free detection of specific DNA sequences. In the initial phase, square wave voltammetry (SWV) was used in a comparative investigation into the electrochemical oxidation of purines (guanine and adenine) and DNA fragments at various electrode surfaces: carbon paste (CPE), glassy carbon electrode (GCE), and gold (AuE). Relative to the carbon electrodes, an approximate 4.0‐fold, 6.0‐fold, and 3.25‐fold increase in the anodic response was observed when guanine, adenine, and hydrolyzed DNA, respectively, were measured on the AuE. It was shown that the guanine and adenine bases could be successfully determined by use of SWV for a deoxyribonucleic acid sample following acid hydrolysis. This label‐free detection of hydrolyzed DNA on gold electrodes has significant advantages over methods using existing carbon electrode materials because of its higher sensitivity and the potential applicability of microfabrication techniques for the production of the requisite gold electrodes.

In another phase of development, the times and conditions for DNA hydrolysis and purine release were investigated. It was shown that under optimal conditions, trace levels of the purine bases could be readily detected following 20 min of hydrolysis at room temperature. The proposed method can be used to estimate the guanine and adenine contents in DNA with in a linear range of 5–30 ng ml^?1.

Finally, when appropriate probe sequences were first adsorbed on the surface of the screen‐printed gold electrode (SPGE), this electrochemical biosensor could be used to specifically detect sequences from ss corona virus aviair following hybridization and hydrolysis reactions on the sensor surface. No enhancement of the voltammetric response was observed when the sensor was challenged with a non‐complementary DNA sequence.     

Electrochemical Detection of Avian Influenza Virus Genotype Using Amino‐ssDNA Probe Modified Gold Electrodes

A DNA biosensor for the detection of specific oligonucleotide sequences of Avian Influenza Virus type H5N1 has been proposed. The NH₂‐ssDNA probe was deposited onto a gold electrode surface to form an amide bond between the carboxyl group of thioacid and the amino group from ssDNA probe. The signals generated as a result of hybridization were registered in square wave voltammetry and electrochemical impedance spectroscopy in the presence of [Fe(CN)₆]^3?/4? as a redox marker. The genosensor is capable to determine 20‐mer and 180‐bp (PCR products) oligonucleotides complementary sequences with detection limit in the fM range. The genosensor displays good selectivity and sensitivity. The 20‐mer as well as 180‐bp oligonucleotides without a complementary sequence generate very low signal.     

Fabrication of a Sensitive Impedance Biosensor of DNA Hybridization Based on Gold Nanoparticles Modified Gold Electrode

In this work, we report on the preparation of a simple, sensitive DNA impedance sensor. Firstly gold nanoparticles were electrodeposited on the surface of a gold electrode, and then probe DNA was immobilized on the surface of gold nanoparticles through a 5′‐thiol‐linker. Electrochemical impedance spectroscopy (EIS) was used to investigate probe DNA immobilization and hybridization. Compared to the bare gold electrode, the gold nanoparticles modified electrode could improve the density of probe DNA attachment and the sensitivity of DNA sensor greatly. The difference of electron transfer resistance (ΔR_et) was linear with the logarithm of complementary oligonucleotides sequence concentrations in the range of 2.0×10^?12 to 9.0×10^?8 M, and the detection limit was 6.7×10^?13 M. In addition, the DNA sensor showed a fairly good reproducibility and stability during repeated regeneration and hybridization cycles.     

A Simple Fabrication Method for Three‐Dimensional Gold Nanoparticle Electrodes and Their Application to the Study of the Direct Electrochemistry of Cytochrome c

A simple method for constructing gold nanoparticle‐modified electrodes with three‐dimensional nanostructures is demonstrated. The electrodes were prepared by casting citrate‐reduced AuNPs onto polycrystalline gold electrodes. The resultant electrodes had a large surface area‐to‐volume ratio, adequate for high protein loading and conferring high stability. The gold nanoparticle electrodes were covered with a self‐assembled monolayer of 11‐mercaptoundecanoic acid for electrostatic immobilization of cytochrome c (cyt c). At the electrode, direct, reversible electron transfer from cyt c was observed with remarkable stability. Moreover, an extremely high surface coverage of electrochemically active cyt c, 167 fully packed monolayers, was obtained through use of the electrode.     

Electrochemical biosensors for detection of point mutation based on surface ligation reaction and oligonucleotides modified gold nanoparticles

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.     

DNA Assisted Assembly of Multisegmented Nanowires

This work demonstrates a highly specific and selective assembly of multisegmented nanowires on prepatterned gold electrodes using DNA hybridization. Multisegmented Au/Pd/Au nanowires were synthesized using template‐directed electrodeposition. Two complementary single‐stranded DNAs modified with thiol tags adsorb on gold electrodes and gold segments of nanowires, and enable the nanowires to assemble across electrodes. The assembled nanowires show ohmic contact with minimum contact resistance. Using these nanowires, the temperature dependent electrical resistance and the sensing performance toward hydrogen were investigated. The temperature coefficient of resistance of nanowires was lower than bulk polycrystalline counterpart, because of higher electron scattering at the surface and grain boundaries of nanowires. The nanowires were sensitive toward hydrogen gas at room temperature with a detection limit of 0.5%.     

Voltammetric Behavior of Osmium‐Labeled DNA at Mercury Meniscus‐Modified Solid Amalgam Electrodes. Detecting DNA Hybridization

The complex of osmium tetroxide with 2,2′‐bipyridine has been utilized as a probe of DNA structure and an electroactive marker of DNA in DNA hybridization sensors. It produces several voltammetric signals, the most negative of them has been observed only at mercury electrodes. This signal is of catalytic nature affording a high sensitivity of DNA determination. The catalytic current due to evolution of hydrogen in voltammetry of DNA modified by complex of osmium tetroxide with 2,2′‐bipyridine (DNA‐Os,bipy) was studied. Solid amalgam electrodes (modified with mercury menisci) of silver (m‐AgSAE), copper (m‐CuSAE), gold, and of combined bismuth and silver, were used as possible substitutes for mercury electrodes. Besides the hanging mercury drop electrode (HMDE), the catalytic current was observed only on m‐AgSAE and m‐CuSAE. Electrodes of gold and bismuth amalgams did not give the catalytic current. The detection limit of DNA‐Os,bipy on HMDE was 0.1 ng mL^?1 (RSD=2.3 %, N=11), and on m‐AgSAE 0.2 ng mL^?1 (RSD=3.1%, N=11). The m‐AgSAE was successfully applied as a detection electrode in double‐surface DNA hybridization experiments offering highly specific discrimination between complementary (target) and nonspecific DNAs, as well as determination of the length of a repetitive DNA sequence. The m‐AgSAE has proved a convenient alternative to the HMDE or carbon electrodes used for similar purposes in previous work.     

Disposable Gold Electrodes with Reproducible Area Using Recordable CDs and Toner Masks

The fabrication and characterization of very cheap disposable gold disk electrodes with reproducible area is reported. The innovation of the proposed procedure is the use of toner masks to define reproducible areas on uniform gold surfaces obtained from recordable compact disks (CD‐R). Toner masks are drawn in a laser printer and heat transferred to gold surfaces, defining exactly the electrodes area. The electrochemical behavior of these disposable electrodes was investigated by cyclic voltammetry in Fe(CN)₆^4? solutions. The relative standard deviation for signals obtained from 10 different gold electrodes was below 1 %. The size of the disk electrodes can be easily controlled, as attested by voltammetric responses recorded by using electrodes with radii varying from 0.5 to 3.0 mm. The advantages of using this kind of electrode for analytical measurements of substances that strongly adsorb on the electrode surface such as cysteine are also addressed.     

Amplified Electrochemical DNA Sensor Based on Polyaniline Film and Gold Nanoparticles

In this work, an electrochemical DNA biosensor, based on a dual signal amplified strategy by employing a polyaniline film and gold nanoparticles as a sensor platform and enzyme‐linked as a label, for sensitive detection is presented. Firstly, polyaniline film and gold nanoparticles were progressively grown on graphite screen‐printed electrode surface via electropolymerization and electrochemical deposition, respectively. The sensor was characterized by scanning electron microscopy (SEM), cyclic voltammetry and impedance measurements. The polyaniline‐gold nanocomposite modified electrodes were firstly modified with a mixed monolayer of a 17‐mer thiol‐tethered DNA probe and a spacer thiol, 6‐mercapto‐1‐hexanol (MCH). An enzyme‐amplified detection scheme, based on the coupling of a streptavidin‐alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalyzed the hydrolysis of the electroinactive α‐naphthyl phosphate to α‐naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. In this way, the sensor coupled the unique electrical properties of polyaniline and gold nanoparticles (high surface area, fast heterogeneous electron transfer, chemical stability, and ease of miniaturisation) and enzymatic amplification. A linear response was obtained over a concentration range (0.2–10 nM). A detection limit of 0.1 nM was achieved.     

Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10^?12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Site-selective immobilization of gold nanoparticles functionalized with DNA oligomers

The organization of metal and semiconductor nanoparticles to form micro- and nanostructured assemblies is currently of tremendous interest. This communication reports on the utilization of DNA molecules as positioning elements for generating microstructured surface architecture from gold nanoparticles. Citrate-passivated 40 nm gold colloids were modified by chemisorptive coupling with a 5′-thiol-derivatized DNA oligomer. The nucleic acid was used as a molecular handle for the specific immobilization on solid supports, previously functionalized with capture DNA oligomers, complementary to the nanoparticle-bound DNA. As a consequence of the enormous specificity of nucleic acid hybridization, the DNA-directed immobilization (DDI) allows, to site-specifically target the hybrid nanoparticles to microlocations which contain the complementary oligomers. The site-selectivity of the surface adsorption is demonstrated by immobilizing the gold colloids on a DNA microarray on a glass cover slide. Moreover, scanning force microscopy (SFM) analysis, used to characterize the intermediate steps of the DDI on a gold substrate, provided initial insights into the specificity and efficiency of this technique. The application of the DDI to fabricate complex colloidal micro- and nanostructures is anticipated. Received: 26 July 2000/Accepted: 5 October 2000     

Lightweight ITO Electrodes Decorated with Gold Nanostructures for Electrochemical Applications

Gold nanostructures were fabricated on a transparent indium tin oxide (ITO) coated PET substrate by an electrodeposition technique from a potassium gold (III) chloride solution for two different types of applications. It was found that the optical transparency of lightweight ITO electrodes could be maintained by depositing isolated gold nanostructures while opening up the use of these electrodes for inner sphere electron reactions, such as hydroquinone oxidation, which are not possible at ITO electrodes. For practical applications the adhesion of gold to the ITO electrode was improved by modifying the ITO surface with 3‐mercaptopropyl‐trimethoxysilane (MPS). Compared to Au/ITO, the Au/MPS/ITO electrode showed vastly improved electrochemical activity toward various electron transfer reactions when subjected to mechanical stress. The biosensing properties of the Au/MPS/ITO electrode was also investigated by studying the detection of immobilized DNA on the Au/MPS/ITO electrode via electrochemical impedance spectroscopy (EIS).     

Electrochemical Properties and Range of Application of Mixed Gold Bismuth Electrodes

Electrochemical properties for mixed gold‐bismuth electrodes have been evaluated. Increased overpotential towards the hydrogen evolution reaction (HER) was found as a function of increased bismuth content in the gold‐bismuth mixture. Compared to a bare gold electrode, the overvoltage was increased by 80 mV for gold with 2% bismuth added (AuBi2), 170 mV for 6% bismuth added (AuBi6), and 580 mV for 25% bismuth added (AuBi25). Cyclic voltammetry (CV) in a NaOH solution was also carried out to study the formation of different oxide products on the surface. The practical use of such electrodes was demonstrated by differential pulse anodic stripping voltammetry (DPASV) for the detection of copper and mercury, and good linearity was found even for concentrations down to 0.25 μg/L. In addition to stability tests in NH₄Cl, the analytical use and stability over time were evaluated in purified scrubbing water and polluted river water.     

Disposable Gold Electrode Array for Simultaneous Electrochemical Studies

An efficient and inexpensive eight gold electrode array has been manufactured by a combination of screen printing and gold electrodeposition techniques. Gold electrodeposition was performed in potentiostatic and galvanostatic conditions. Different treatments, involving temperature and polishing control, led to electrodes with different roughness. The electrochemical behavior of the generated gold surface was studied by cyclic voltammetry showing the characteristic response of polycrystalline gold, in contrast with disposable gold electrodes fabricated by screen printing from gold inks. The electrodes were chemically modified through the adsorption of alkanethiols self‐assembled monolayers and the coupling of a model protein. Both reactions were followed by cyclic voltammetry and Electrochemical Impedance Spectroscopy (EIS). The electrodes have shown high reproducibility in their electrochemical behavior as well as in their modifications.     

Long Distance Electron Transfer Across >100 nm Thick Au Nanoparticle/Polyion Films to a Surface Redox Protein

Glutathione‐decorated 5 nm gold nanoparticles (AuNPs) and oppositely charged poly(allylamine hydrochloride) (PAH) were assembled into {PAH/AuNP}_n films fabricated layer‐by‐layer (LbL) on pyrolytic graphite (PG) electrodes. These AuNP/polyion films utilized the AuNPs as electron hopping relays to achieve direct electron transfer between underlying electrodes and redox proteins on the outer film surface across unprecedented distances >100 nm for the first time. As film thickness increased, voltammetric peak currents for surface myoglobin (Mb) on these films decreased but the electron transfer rate was relatively constant, consistent with a AuNP‐mediated electron hopping mechanism.     

Electrocatalytic Properties of a Gold Nanoseed Particle‐modified Indium Tin Oxide Electrode: Comparison of the Shape and Preparation Methods

Gold nanostructures are the most commonly used nanostructures for fabricating electrochemical sensors and biosensors. In this study, we compared the catalytic performances of three types of gold nanoseed particles having two different morphologies, upon attachment to an amino‐functionalized ITO electrode surface. The ITO electrode surface was modified with 3‐aminopropyltrimethoxysilane (APTMS) and (1) gold nanoseed spheres (AuNSS), prepared using the ion capture and successive reduction method (ICR), (2) commercially available 5 nm AuNSS, and (3) a newly synthesized gold nanoseed wire (AuNSW). The electrocatalytic properties of the three electrodes were evaluated. Among the three electrodes, the AuNSW/APTMS/ITO was found to be the electrode of choice and exhibited excellent electrocatalytic properties toward the biologically important analytes glucose, uric acid, and serotonin.     

Developing a Nano‐Biosensor for DNA Hybridization Using a New Electroactive Label

Development of electrochemical DNA hybridization biosensors based on carbon paste electrode (CPE) and gold nanoparticle modified carbon paste electrode (NGMCPE) as transducers and ethyl green (EG) as a new electroactive label is described. Electrochemical impedance spectroscopy and cyclic voltammetry techniques were applied for the investigation and comparison of bare CPE and NGMCPE surfaces. Our voltammetric and spectroscopic studies showed gold nanoparticles are enable to facilitate electron transfer between the accumulated label on DNA probe modified electrode and electrode surface and enhance the electrical signals and lead to an improved detection limit. The immobilization of a 15‐mer single strand oligonucleotide probe on the working electrodes and hybridization event between the probe and its complementary sequence as a target were investigated by differential pulse voltammetry (DPV) responses of the EG accumulated on the electrodes. The effects of some experimental variables on the performance of the biosensors were investigated and optimum conditions were suggested. The selectivity of the biosensors was studied using some non‐complementary oligonucleotides. Finally the detection limits were calculated as 1.35×10^?10 mol/L and 5.16×10^?11 mol/L on the CPE and NEGCPE, respectively. In addition, the biosensors exhibited a good selectivity, reproducibility and stability for the determination of DNA sequences.     

Self‐Assembled Multilayers of Polyethylenimine and DNA: Spectrophotometric and Electrochemical Characterization and Application for the Determination of Acridine Orange Interaction

This work deals with the study of the interaction between acridine orange (AO) and calf‐thymus double stranded DNA (dsDNA) present in supramolecular architectures built on gold electrodes modified with mercapto‐1‐propanesulfonic acid (MPS) by self‐assembling of polyethylenimine and dsDNA. The optimal conditions for building the supramolecular architecture were obtained from UV‐vis spectrophotometric experiments. The electrochemical studies were performed by adsorptive transfer square wave voltammetry from the evaluation of the oxidation signal of AO accumulated within the multistructure. The effect of the number of PEI‐dsDNA bilayers (Au/MPS/(PEI‐dsDNA)_n) on the accumulation and electrooxidation of AO is also discussed.     

Pulsed Amperometric Detection of Histamine at Glassy Carbon Electrodes Modified with Gold Nanoparticles

Gold nanocrystal‐modified glassy carbon electrodes (nAu‐GCE) were prepared and used for the determination of histamine by flow injection and high performance liquid chromatography using pulsed amperometric detection (PAD) as the detection mode. Experimental variables involved in the electrodeposition process of gold from a HAuCl₄ solution were optimized. A catalytic enhancement of the histamine voltammetric response was observed at the nAu‐GCE when compared with that obtained at a conventional Au disk electrode, as a consequence of the microdispersion of gold nanocrystals on the GC substrate. The morphological and electrochemical characteristics of the nAu‐GCE were evaluated by SEM and cyclic voltammetry. PAD using a very simple potential waveform consisting of an anodic potential (+700 mV for 500 ms) and a cathodic potential (?300 mV for 30 ms), was used to avoid the electrode surface fouling when histamine was detected under flowing conditions. Flow injection amperometric responses showed much higher I_p values and signal‐to‐noise ratios at the nAu‐GCE than at a conventional gold disk electrode. A limit of detection of 6×10^?7 mol L^?1 histamine was obtained. HPLC‐PAD at the nAu‐GCE was used for the determination of histamine in the presence of other biogenic amines and indole. Histamine was determined in sardine samples spiked at a 50 μg g^?1 concentration level, with good results. Furthermore, the chromatographic PAD method was also used for monitoring the formation of histamine during the decomposition process of sardine samples.     

Direct Detection of DNA and DNA‐Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (R_ct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm² ssDNA. The relative change in R_ct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)₃]²⁺) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)₃]²⁺ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance.