Coupling of a Reagentless Electrochemical DNA Biosensor with Conducting Polymer Film and Nanocomposite as Matrices for the Detection of the HIV DNA Sequences

Abstract

This paper describes a reagentless electrochemical DNA biosensor applied to the detection of human immunodeficiency virus (HIV) sequences based on electrochemical impedance spectroscopy (EIS). The novel DNA biosensor has been elaborated by means of an opposite‐charged adsorption Au‐Ag nanocomposite to a conductive polymer polypyrrole (PPy) modified platinum electrode (Pt) and self‐assembly the mercapto oligonucleotide probes onto the surface of modified electrode via the nanocomposite. The duplex formation was detected by measuring the electrochemical impedance signal of nucleic acids in phosphate buffer solution (PBS). Such response is based on the concomitant conductivity changes of the PPy film and nanocomposite. The reagentless scheme has been characterised using 21‐mer synthetic oligonucleotides as models: parameters affecting the hybridization assay were explored and optimized. The detection limit is 5.0×10^?10 M of target oligonucleotides at 3σ. The potential for development of reagentless DNA hybridization analysis in the clinical diagnosis is being pursued.     

Nano-ZnO/Chitosan Composite Film Modified Electrode for Voltammetric Detection of DNA Hybridization

Abstract

A sensitive electrochemical DNA biosensor based on nano-ZnO/chitosan composite matrix for DNA hybridization detection was developed. The Nano-ZnO was synthesized by the hydrothermal method and dispersed in chitosan, which was used to fabricate the modification of the glassy carbon electrode (GCE) surface. The ZnO/chitosan-modified electrode exhibited good biocompatibility and excellent electrochemical conductivity. The hybridization detection was monitored with differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. The established biosensor can effectively discriminate complementary target sequence and two-base-mismatched sequence, with a detection limit of 1.09 × 10^?11 mol L^?1 of complementary target.     

Electrochemical Determination of Aflatoxin B1 (AFB1) Using a Copper-Based Metal-Organic Framework (Cu-MOF) and Gold Nanoparticles (AuNPs) with Exonuclease III (Exo III) Assisted Recycling by Differential Pulse Voltammetry (DPV)

Abstract

A sensitive electrochemical biosensor was designed for determination of aflatoxin B1 (AFB1) using a copper-based metal-organic framework (Cu-MOF), which has strong electrochemical activity and exonuclease III (Exo III)-assisted recycling for dual signal amplification. Hairpin DNA (S1) was immobilized on the electrode. The AFB1 was recognized by aptamer DNA (S2) and complementary DNA (S3) was released. The S3 hybridized with the hairpin S1 to form the Exo III hydrolyzed double-stranded DNA, leaving a partial sequence of hairpin DNA (S1′) on the electrode and releasing S3 for the next cycle of the opening and digestion of hairpin S1. The amplified S1′ then was able to combine with more signal probes. Cu-MOF bond gold nanoparticles (AuNPs) by -NH₂ were immobilized to capture DNA (S4) to obtain Cu-MOF/AuNPs/S4. This signal probe Cu-MOF/AuNPs/S4 was able to hybridize with the electrode and generate an amplified electrochemical signal. Under the optimized conditions, this electrochemical biosensor for AFB1 exhibited a low detection limit of 6.7?×?10^?7?ng/mL at a signal-to-noise equal to 3 and a wide linear range from 10^?6 to 1?ng/mL. The biosensor was also used to analyze AFB1-spiked beer sample with recovery values between 96% and 103%. This method has the potential to become a valuable technology for detecting various toxins by the selection of the appropriate aptamer DNA.     

Label-Free Detection of DNA Hybridization Based on MnO2 Nanoparticles

Abstract

Nano-MnO₂/chitosan composite film modified glassy carbon electrode (MnO₂/CHIT/GCE) was fabricated and a DNA probe was immobilized on the electrode surface. The immobilization and hybridization events of DNA were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The EIS was applied to the label-free detection of the target DNA. The human immunodeficiency virus (HIV) gene fragment was successfully detected by this DNA electrochemical sensor. The dynamic detection range was from 2.0 × 10^?11 to 2.0 × 10^?6 mol/L, with a detection limit of 1.0 × 10^?12 mol/L.     

Electrochemical Studies of Adriamycin Interaction with Dna and Determination of Dna at a Ni/Gc Ion Implantation Modified Electrode

ABSTRACT

The electrochemical behavior of the reduction of adriamycin at Ni/GC modified electrode has been studied by linear sweep and cyclic voltammetry. The reaction of DNA with ADM formed an electrochemically nonactive complex, which resulted in a decrease in the ADM equilibrium concentration and its reduction current. The decrease in peak current was proportional to DNA concentration and can be used to determination DNA concentration. The experiments of AES and XPS showed that Ni is surely implanted into the surface of GCE and the depth distribution of Ni was in good agreement with Gooses normal distribution; the implanted Ni at GCE improved the electrocatalytic activity.     

Electrochemical DNA sensing based on gold nanoparticle amplification

A hybridization signal-amplified method based on a gold nanoparticle-supported DNA sequence for electrochemical DNA sensing has been investigated by cyclic voltammetry, differential-pulse voltammetry, and atomic-force microscopy (AFM). Quantitative analysis showed that the peak current increment (Ip) is linearly dependant on the concentration of the gold nanoparticle-supported DNA sequence Au2 over the range 0.51–8.58 pmol L^–1. AFM results indicated that the extent of surface hybridization was dependent on the concentration of the gold-nanoparticle-supported DNA sequence. Moreover, a new pair of peaks, which might arise from the special configuration of the gold-nanoparticle-supported DNA sequence, appeared in the cyclic voltammogram after hybridization. Although quite sensitive, this DNA sensing surface was not easily regenerated, so this kind of amplified method was suitable for disposable DNA sensors and chip-based gene diagnosis sensors.     

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.     

一个新的粘液酸锰(Ⅱ)配合物的合成、结构与性能研究

合成了1个以粘液酸为配体的锰(Ⅱ)配合物Mn[(muc)(H₂O)₃]·H₂O (1),并通过红外光谱,元素分析,热重分析和X-射线单晶衍射对其进行了表征。配合物1由一维链组成,其三维网络结构可以简化为dia拓扑。初步的电化学实验表明,配合物1在电化学储能上有应用潜力,可以作为氢氧化镍电极的添加剂。     

Electrochemical DNA biosensor for the detection of Listeria Monocytogenes using toluidine blue as a hybridization indicator

An electrochemical DNA biosensor was established for the determination of actin-assembly inducing protein (actA) gene sequences from Listeria monocytogenes and its polymerase chain reaction (PCR) product. The actA gene probe sequences were covalently immobilized on the surface of the mercaptoacetic acid self-assembled gold electrode with the help of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which was further used to hybridize with the target sequence. Toluidine blue (TB) was used as an effective electrochemical indicator for the discrimination of the hybridization reaction on the electrode surface, which had stronger interaction with double-stranded DNA (dsDNA) than single-stranded DNA (ssDNA). The electrochemical parameters of TB on DNA modified electrodes were carefully calculated. Based on the different electrochemical responses of TB on DNA modified electrodes, the actA gene sequences can be detected in the concentration range from 1.0 × 10^-7 to 8.0 × 10^-5 M. The PCR product of Listeria monocytogenes was successfully detected by the proposed electrochemical biosensor.     

Glutaraldehyde-modified electrode for nonlabeling voltammetric detection of <Emphasis Type="Italic">p16</Emphasis><Superscript><Emphasis Type="Italic">INK4A</Emphasis></Superscript> gene

A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 ^INK4A gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 ^INK4A gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.     

Cyclic voltammetry study of (5-ethoxycarbonylmethylidene-4-oxothiazolidin-2-ylidene)-N-phenylethanamide

As a continuation of our ongoing project on electrochemical properties of push-pull 5-substituted 2-alkylidene-4-oxothiazolidines (1a) differing in substituent R at C5-position and electron withdrawing group (EWG), we nave investigated the electrochemical behaviour of (5-etoxycarbonylmethylidene-4-oxothiazolidin-2-ylidene)-N-phenylethanamide 1a (R: =CHCO₂Et; EWG: CONHPh), consisting as a (2E,5Z)/(2Z,5Z) mixture, by cyclic voltammetry in polar as well as non-polar solvent (0.1 M TBAHFP in DMSO and CHCl₃, respectively). Cyclic voltammetry at stationary electrode was employed to characterize the electron transfer steps. Based on electrochemical criteria and correlation with the DigiSim simulations, an ECE mechanism, involving two electrochemical steps and one isomerisation step, was suggested. The article is published in the original.     

Label-Free DNA Biosensor for Electrochemical Detection of Short DNA Sequences Related to Human Papilloma Virus

Abstract

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of an electrochemical hybridization biosensor for the detection of short DNA fragments specific to the human papilloma virus (HPV). Unlabeled DNA probes have been immobilized by spontaneous coadsorption of thiolated single-stranded oligonucleotides (HS-ssDNA) onto the sensing surface of a screen-printed gold electrode (SPGE). The covalently immobilized single-stranded DNA probe (HS-ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. DNA is treated with acid (e.g., 0.5 M chloridric acid), and the acid-released purine bases are directly determined by square wave voltammetry (SWV).

Variables of the probe-immobilization and hybridization steps are optimized to offer convenient quantitation of HPV DNA target, in connection with a short hybridization time. Peak currents were found to increase in the following order: hybrid-modified SPGE, 11-base mismatched modified SPGE, 18-base mismatched SPGE, and the probe modified SPGE. Control experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. The effect of the target DNA concentration on the hybridization signal was also studied. Under optimal conditions, this sensor has a good calibration range with HPV DNA sequence detection limit of 2 pg · ml^?1 (S/N = 3).     

Preparation and characterization of graphene nanosheets dispersed pyrrole-chorobenzaldehyde-heptaldehyde conjugated terpolymer nanocomposites for DNA detection

A new conductive terpolymer/graphene nanosheet hybrid composite has been synthesized by polymerizing pyrrole, chlorobenzaldehyde, and heptaldehyde (PPyCB&;H), in the presence of graphene nanosheets (GNS), using p-toluene sulfonic acid as a catalyst. Fourier transform infrared spectra, proton nuclear magnetic resonance, transmission electron microscopy, and X-ray diffraction patterns confirm the formation of PPyCB&;H/GNS hybrid nanocomposites. Further, the resultant nanocomposite material is coated on ITO to construct an electrochemical sensor for the reliable detection of single-strand DNA (tDNA) which is cleaved from the genomic DNA of Escherichia coli. Under optimized conditions, linear detection of genomic DNA (tDNA) with concentration ranging from 1.3 × 10⁻¹² to 1.3 × 10⁻²³ M is observed and it is repeatable with a 1.3 × 10⁻²³ M lowest level detection limit. The present modified electrode of PPyCB&;H/GNS may show utility for constructing highly sensitive electrochemical sensors for the detection of E. coli.

Graphical abstract

     

Voltammetric detection of damage to DNA caused by nitro derivatives of fluorene using an electrochemical DNA biosensor

An electrochemical DNA biosensor based on the screen printed carbon paste electrode (SPCPE) with an immobilized layer of calf thymus double-stranded DNA has been used for in vitro investigation of the interaction between genotoxic nitro derivatives of fluorene (namely 2-nitrofluorene and 2,7-dinitrofluorene) and DNA. Two types of DNA damage have been detected at the DNA/SPCPE biosensor: first, that caused by direct association of the nitrofluorenes, for which an intercalation association has been found using the known DNA intercalators [Cu(phen)₂]²⁺ and [Co(phen)₃]³⁺ as competing agents, and, second, that caused by short-lived radicals generated by electrochemical reduction of the nitro group (observable under specific conditions only).     

A lipase-based electrochemical biosensor for target DNA

A lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The other end of the probe DNA is linked, via carboxy groups, to magnetic nanoparticles. The binding of target DNA transforms the hairpin structure of the probe DNA and causes the exposure of ester bonds. This results in the release of electro-active ferrocene after hydrolysis of the ester bonds, and in an observable electrochemical response. The quantity of target DNA in the concentration range between 1?×?10^?12 mol·L^?1 and 1?×?10^?8 mol·L^?1 can be determined by measuring the electrochemical current. The method can detect target DNA with rapid response (30 min) and low interference.

Electrochemical behavior and determination of L-tryptophan on carbon ionic liquid electrode

A carbon ionic liquid electrode (CILE) was fabricated by mixing N-butylpyridinium hexafluoro-phosphate (BPPF ₆ ) with graphite powder and further used for the investigation on the electrochemical behavior of L-tryptophan (Trp). The fabricated CILE showed good conductivity, inherent electrocatalytic ability and strong promotion to the electron transfer of Trp. On the CILE, an irreversible oxidation peak appeared at 0.948 V (vs. saturated calomel reference electrode). For 5.0 × 10⁻⁵ M Trp the oxidation peak current increased about 5 times and the oxidation peak potential decreased on 0.092 V compared to carbon paste electrode. The results indicated that an electrocatalytic reaction occurred on CILE. The conditions for the electrochemical detection were optimized and the electrochemical parameters of Trp on CILE were carefully investigated. Under the selected conditions, the oxidation peak current showed linear relationship with Trp concentration in the range of 8.0 × 10⁻⁶ ∼1.0 × 10⁻³ M for cyclic voltammetry and the detection limit was estimated as 4.8 × 10⁻⁶ M (3σ). The interferences of other amino acids or metal ions on the determination were tested and the proposed method was successfully applied to the synthetic sample analysis.     

Utilization of Pyronine B as Voltammetric Probe for the Determination of DNA

Abstract

The electrochemical behaviors of the interaction of pyronine B (PB) with DNA were investigated on the mercury drop working electrode. In pH 2.0 Britton‐Robinson (B‐R) buffer solution, PB can be easily reduced on the mercury electrode and had a well‐defined voltammetric reductive wave at ?0.86 V (vs. saturated calomelelectrode, SDE). On the addition of DNA into the PB solution, the reductive peak current of PB decreased with the positive movement of the peak potential and without the appearance of new peaks. The result showed that a new supramolecular complex was formed via intercalation of PB with DNA, which can't be reduced on the Hg electrode. The conditions of interaction and the electrochemical detection were carefully investigated. Under the optimal conditions the decrease of peak current was proportional to the concentration of DNA in the range of 1.0～30.0 mg/L with the linear regression equation as ΔIp″(nA)=51.84C (mg/L)–94.97 (n=13, γ=0.993) and the detection limit was 0.90 mg/L. The interaction mechanism was discussed with the aggregation of DNA‐PB supramolecular complex and the stoichiometry of the supramolecular complex was calculated with the binding number as 3 and the binding constant as 1.61×10¹⁵.     

Biosensor for Hepatitis B Virus DNA PCR Product and Electrochemical Study of the Interaction of Di(2,2′‐bipyridine)osmium(III) with DNA

The strategy for electrochemical detection of HBV DNA PCR product (181 bps) was designed by covalently immobilizing single‐stranded HBV DNA on preoxidized glassy carbon electrode surface. The immobilization of single stranded DNA was verified by AC impedance spectra. The following hybridization reaction on surface was evidenced by electrochemical methods using [Os(bpy)₂Cl₂]⁺ as an electroactive indicator. The interactions of [Os(bpy)₂Cl₂]⁺ with calf thymus single and double stranded DNA immobilized on preoxidized glassy carbon electrodes were studied. [Os(bpy)₂Cl₂]⁺ could bind preferentially to the duplex DNA by intercalating to base pairs. The intrinsic binding constant of [Os(bpy)₂Cl₂]⁺ with calf thymus DNA was calculated to be 1.21×10⁴ M^?1. Using [Os(bpy)₂Cl₂]⁺ as an electrochemical hybridization indicator, the HBV DNA sensor has been used to detect qualitatively target HBV DNA in solution with high sensitivity and selectivity.     

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.     

An Electrochemical Determination of Uric Acid in Sera by a Flow-Through Equipment with and Without Uricase

Abstract

A comparison of an enzymeless direct electrochemical oxidation procedure at a platinum electrode for the determination of uric acid, and an enzyme sensor with immobilized urate: oxygen oxidoreductase (uricase), was performed in flow stream systems. The uricase enzyme electrode is based on the H₂O₂ oxidation current. Both amperometric methods were related to the wall-known photometric uricase-catalase-procedure (UCM) as a reference method. The measured values of both methods are of the first derivatives of current change (dI/dt) due to the electrochemical or electrochemical enzymatic reaction, respectively. The analytical quality of the measurements is characterized by: precision s% within run < 2% day to day < 5% accuracy acceptable (control materials) correlation to reference method r >0.93 analysis rate 80 samples/hr