Carbon nanotubes/(pLys/dsDNA) n layer-by-layer multilayer films for electrochemical studies of DNA damage

Carboxylic group-functionalized carbon nanotubes (c-CNT) were modified on the surface of carbon paste electrode to obtain a conducting precursor film. Positively charged poly-l-lysine (pLys) and negatively charged double-stranded DNA (dsDNA) were alternately adsorbed on the c-CNT-modified electrode, forming (pLys/dsDNA) _n layer-by-layer (LBL) films. Cyclic voltammetry and electrochemical impedance spectroscopy of the electroactive probe [Fe(CN)₆]^3−/4− could give the valuable dynamic information of multilayer films growth. The oxidative DNA damage induced by cadmium ion (Cd²⁺) in the LBL multilayer films was studied by differential pulse voltammetry (DPV) with methylene violet (MV) as the intercalation redox probe. The electrochemical signals of MV on the multilayer films were effectively amplified via LBL technology. The specific intercalation of MV into dsDNA base pairs and the amplified electrochemical response of MV, combined with the unique feature of loading reversibility of MV in the DNA layer-by-layer films, made the difference in DPV response between the intact, and damaged dsDNA films become pronounced. This biosensor exhibited that the (pLys/dsDNA) _n films could be utilized for investigations of DNA damage.     

Sensitively Electrochemical Sensing for Sequence‐Specific Detection of Phosphinothricin Acetyltransferase Gene: Layer‐by‐Layer Films of Poly‐L‐Lysine and Au‐Carbon Nanotube Hybrid

An electrochemical DNA sensing film was constructed based on the multilayers comprising of poly‐L ‐lysine (pLys) and Au‐carbon nanotube (Au‐CNT) hybrid. A precursor film of mercaptopropionic acid (MPA) was firstly self‐assembled on the Au electrode surface. pLys and Au‐CNT hybrid layer‐by‐layer assembly films were fabricated by alternately immersing the MPA‐modified electrode into the pLys solution and Au‐CNT hybrid solution. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3?/4? and [Co(phen)₃]^3+/2+ as the redox indicators. The outer layer of the multilayer film was the positively charged pLys, on which the DNA probe was easily linked due to the strong electrostatic affinity. The hybridization detection of DNA was accomplished by using methylene blue (MB) as the indicator, which possesses different affinities to dsDNA and ssDNA. Differential pulse voltammetry was employed to record the signal response of MB and determine the amount of the target DNA sequence. The established biosensor has high sensitivity, a relatively wide linear range from 1.0×10^?10 mol/L to 1.0×10^?6 mol/L and the ability to discriminate the fully complementary target DNA from single or double base‐mismatched DNA. The sequence‐specific DNA related to phosphinothricin acetyltransferase gene from the transgenically modified plants was successfully detected.     

Electrochemical DNA Biosensor Based on Graphene and TiO2 Nanorods Composite Film for the Detection of Transgenic Soybean Gene Sequence of MON89788

Based on graphene (GR), TiO₂ nanorods, and chitosan (CTS) nanocomposite modified carbon ionic liquid electrode (CILE) as substrate electrode, a new electrochemical DNA biosensor was effectively fabricated for the detection of the transgenic soybean sequence of MON89788. By using methylene blue (MB) as hybridization indicator for monitoring the hybridization with different ssDNA sequences, the differential pulse voltammetric response of MB on DNA modified electrodes were recorded and compared. Due to the synergistic effects of TiO₂ nanorods and GR on the electrode surface, the electrochemical responses of MB were greatly increased. Under optimal conditions the differential pulse voltammetric response of the target ssDNA sequence could be detected in the range from 1.0×10^?12 to 1.0×10^?6 mol/L with a detection limit of 7.21×10^?13 mol/L (3σ). This electrochemical DNA biosensor was further applied to the polymerase chain reaction (PCR) product of transgenic soybeans with satisfactory results.     

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.     

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.     

A Nanocomposite Containing Carbon Nano-onions and Polyaniline Nanotubes as a Novel Electrode Material for Electrochemical Sensing of Daidzein

Glassy carbon electrode (GCE) were modified with nanocomposites containing conductive polyaniline nanotubes (PANI_nt) and carbon nano-onions (CNOs). Herein we report a simple and sensitive way for daidzein (DA) determination at concentrations between 1 and 10 μM by linear sweep voltammetry using GCE/PANI_nt/CNOs system. The DA electrochemical behavior was examined in two buffer environments (pH 7.5 and 4.5) using electrodes modified with the oxidized CNOs or their derivatives containing carboxyl and benzylamino functional groups. The direct electrooxidation of DA was observed at +0.65 V and +0.8 V at pH 7.5 and at +0.7 V and +1.1 V vs. Ag/AgCl at pH 4.5.     

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.     

Synergistic effects of nano-ZnO/multi-walled carbon nanotubes/chitosan nanocomposite membrane for the sensitive detection of sequence-specific of PAT gene and PCR amplification of NOS gene

The remarkable synergistic effects of the zinc oxide (ZnO) nanoparticles and multi-walled carbon nanotubes (MWNTs) were developed for the ssDNA probe immobilization and fabrication of the electrochemical DNA biosensor. The ZnO/MWNTs/chitosan nanocomposite membrane-modified glassy carbon electrode (ZnO/MWNTs/CHIT/GCE) was fabricated and the ssDNA probes were immobilized on the modified electrode surface. The preparation method is quite simple and inexpensive. The hybridization events were monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as an indicator. As compared with previous MWNTs-based DNA biosensors, this composite matrix combined the attractive biocompatibility of ZnO nanoparticles with the excellent electron-transfer ability of MWNTs and fine membrane-forming ability of CHIT increased the DNA attachment quantity and complementary DNA detection sensitivity. The approach described here can effectively discriminate complementary DNA sequence, noncomplementary sequence, single-base mismatched sequence and double-base mismatched sequence related to phosphinothricin acetyltransferase (PAT) gene in transgenic corn. Under optimal conditions, the dynamic detection range of the sensor to PAT gene complementary target sequence was from 1.0 × 10⁻¹¹ to 1.0 × 10⁻⁶ mol/L with the detection limit of 2.8 × 10⁻¹² mol/L. The polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from the real sample of one kind of transgenic soybeans was also satisfactorily detected with this electrochemical DNA biosensor, suggesting that the ZnO/MWNTs/CHIT nanocomposite hold great promises for sensitive electrochemical biosensor applications.     

Kinetic study of DNA/DNA hybridization with electrochemical impedance spectroscopy

The hybridization of immobilized oligonucleotides probe strands with solution phase targets is the underlying principle of microarray-based techniques for the analysis of DNA variation. To study the kinetics of DNA/DNA hybridization, target DNA is often prior labeled with markers. A label-free method of electrochemical impedance spectra (EIS) for study the hybridization in process was reported. The Langmuir model was used to determine the association rate constant (K_on), the dissociation rate constant (K_off) and the affinity rate constant (K_A), for perfect matched DNA hybridization. The results show that, EIS is a successful technique possessing high effectivity and sensitivity to study DNA/DNA hybridization kinetics. This work can provide another view on EIS for the studying of DNA/DNA hybridization.     

Label-free oligonucleotide detection method based on a new L-cysteine-dihydroartemisinin complex electroactive indicator

A novel base-mismatched oligonucleotide assay method based on label-free electrochemical biosensor was developed, in which the L-cysteine (Cys)-dihydroartemisinin (DHA) complex was used as a new electroactive indicator. In DNA sensor, Cys-DHA complex was initially formed on electrode surface by cathodic scanning, and target oligonucleotide was conjugated with Cys-terminated DHA indicator through electrostatic interaction under optimal pH. The subsequent sequence assay was responsive to hybridization recognition, which target oligonucleotide was captured by the surface-anchored DNA/Cys-DHA probe. The electrochemical signals of biosensor before and after hybridization were compared basing the measurements of semi-derivative linear scan voltammetry (SDLSV) and electrochemical impedance spectroscopy (EIS). On the basis of signal amplification of electroactive indicator and specific recognition of DNA probe, five target oligonucleotides with different mismatched bases were assayed, and a detection limit reached 0.3 nM. Furthermore, atomic force microscopy (AFM) was used to visually characterize specific recognition spots of biosensor at nanoscale. This study demonstrated a new electroactive molecule-based, biomolecule-involved electroactive indicator and its application in recognition and detection of complementary and base-mismatched oligonucleotide.     

A cyclodextrin host-guest recognition approach to a label-free electrochemical DNA hybridization biosensor

A novel label-free electrochemical DNA hybridization biosensor using a β-cyclodextrin/poly(N-acetylaniline)/carbon nanotube composite modified screen printed electrode (CD/PNAANI/CNT/SPE) has been developed. The proposed DNA hybridization biosensor relies on the intrinsic oxidation signals of guanine (G) and adenine (A) from single-stranded DNA entered into the cyclodextrin (CD) cavity. Due to the binding of G and A bases to complementary cytosine and thymine bases in dsDNA, the signals obtained for ssDNA were much higher than that of dsDNA. The synergistic effect of the multi-walled carbon nanotubes provides a significantly enhanced voltammetric signal, and the CD encapsulation effect makes anodic peaks of G and A shift to less positive potentials than that at the bare SPE. The peak heights of G and A signals are dependent on both the number of the respective bases in oligonucleotides and the concentration of the target DNA sequences. Hybridization of complementary strands was monitored through the measurements of oxidation signal of purine bases, which enabled the detection of target sequences from 0.01 to 1.02 nmol μl(-1) with the detection limit of target DNA as low as 5.0 pmol μl(-1) (S/N = 3). Implementation of label-free and homogeneous electrochemical hybridization detection constitutes an important step toward low-cost, simple, highly sensitive and accurate DNA assay. Discrimination between complementary, noncomplementary, and two-base mismatch targets was easily accomplished using the proposed electrode.     

Impedance DNA Biosensor Using Electropolymerized Polypyrrole/Multiwalled Carbon Nanotubes Modified Electrode

In this paper, we present an electrochemical impedance‐based DNA biosensor by using a composite material of polypyrrole (PPy) and multiwalled carbon nanotubes (MWNTs) to modify glassy carbon electrode (GCE). The polymer film was electropolymerized onto GCE by cyclic voltammetry (CV) in the presence of carboxylic groups ended MWNTs (MWNTs‐COOH). Such electrode modification method is new for DNA hybridization sensor. Amino group ended single‐stranded DNA (NH₂‐ssDNA) probe was linked onto the PPy/MWNTs‐COOH/GCE by using EDAC, a widely used water‐soluble carbodiimide for crosslinking amine and carboxylic acid group. The hybridization reaction of this ssDNA/PPy/MWNTs‐COOH/GCE resulted in a decreased impedance, which was attributed to the lower electronic transfer resistance of double‐stranded DNA than single‐stranded DNA. As the result of the PPy/MWNTs modification, the electrode obtained a good electronic transfer property and a large specific surface area. Consequently, the sensitivity and selectivity of this sensor for biosensing DNA hybridization were improved. Complementary DNA sequence as low as 5.0×10^?12 mol L^?1 can be detected without using hybridization marker or intercalator. Additionally, it was found that the electropolymerization scan rate was an important factor for DNA biosensor fabrication. It has been optimized at 20 mV s^?1.     

Label-free electrochemical DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles, polythionine, and graphene

We describe the fabrication of a sensitive label-free electrochemical biosensor for the determination of sequence-specific target DNA. It is based on a glassy carbon electrode (GCE) modified with graphene, gold nanoparticles (Au-NPs), and polythionine (pThion). Thionine was firstly electropolymerized on the surface of the GCE that was modified with graphene by cyclic voltammetry. The Au-NPs were subsequently deposited on the surface of the pThion/graphene composite film by adsorption. Scanning electron microscopy and electrochemical methods were used to investigate the assembly process. Differential pulse voltammetry was employed to monitor the hybridization of DNA by measuring the changes in the peak current of pThion. Under optimal conditions, the decline of the peak current is linearly related to the logarithm of the concentration of the target DNA in the range from 0.1 pM to 10 nM, with a detection limit of 35 fM (at an S/N of 3). The biosensor exhibits good selectivity, acceptable stability and reproducibility.

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.     

A novel electrochemical DNA biosensor construction based on layered CuS–graphene composite and Au nanoparticles

A novel CuS–graphene (CuS-Gr) composite was synthesized to achieve excellent electrochemical properties for application as a DNA electrochemical biosensor. CuS-Gr composite was prepared by a hydrothermal method, in which two-dimensional graphene served as a two-dimensional conductive skeleton to support CuS nanoparticles. A sensitive electrochemical DNA biosensor was fabricated by immobilizing single-stranded DNA (ss-DNA) labeled at the 5′ end using 6-mercapto-1-hexane (HS-ssDNA) on the surface of Au nanoparticles (AuNPs) to form ssDNA-S–AuNPs/CuS-Gr, and hybridization sensing was done in phosphate buffer. Cyclic voltammetry and electrochemical impedance spectroscopy were performed for the characterization of the modified electrodes. Differential pulse voltammetry was applied to monitor the DNA hybridization using an [Fe(CN)₆]^3?/4? solution as a probe. Under optimum conditions, the biosensor developed exhibited a good linear relationship between the current and the logarithm of the target DNA concentration ranging from 0.001 to 1 nM, with a low detection limit of 0.1 pM (3σ/S). The biosensor exhibited high selectivity to differentiate one-base-mismatched DNA and three-base-mismatched DNA. The results indicated that the sensing platform based on CuS-Gr provides a stable and conductive interface for electrochemical detection of DNA hybridization, and could easily be extended to the detection of other nucleic acids. Graphical abstracts

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Synergistic Effect of MWNTs/CeO2/CHIT Film for Detection of CdSe Nanoparticle Labeled Sequence‐Specific of 35S Promoter of Cauliflower Mosaic Virus Gene

A porous composite film was fabricated combining the advantages of multiwalled carbon nanotubes, CeO₂ and chitosan. The synergistic effect of the film improved the immobilization of probe ssDNA. The loaded probe ssDNA was used for detection of CdSe quantum dots labeled target DNA. The DNA hybridization reaction was detected by differential pulse anodic stripping voltammetry of Cd²⁺ after the oxidative release of labeled CdSe quantum dots. The established DNA biosensor can discriminate different target sequences associated with 35S promoter of cauliflower mosaic virus gene with relatively wide linear range and low detection limit (2.4×10^?13 mol/L).     

Impedimetric DNA‐Based Biosensor for Silver Ions Detection with Hemin/G‐Quadruplex Nanowire as Enhancer

A DNA‐based biosensor was reported for detection of silver ions (Ag⁺) by electrochemical impedance spectroscopy (EIS) with [Fe(CN)₆]^4?/3? as redox probe and hybridization chain reaction (HCR) induced hemin/G‐quadruplex nanowire as enhanced label. In the present of target Ag⁺, Ag⁺ interacted with cytosine‐cytosine (C? C) mismatch to form the stable C? Ag⁺? C complex with the aim of immobilizing the primer DNA on electrode, which thus triggered the HCR to form inert hemin/G‐quadruplex nanowire with an amplified EIS signal. As a result, the DNA biosensor showed a high sensitivity with the concentration range spanning from 0.1 nM to 100 µM and a detection limit of 0.05 nM.     

QCM-DNA biosensor based on plasma modified PT/TiO2 nanocomposites

A quartz crystal microbalance DNA biosensor based on plasma prepared polythiophene /titanium dioxide (PT/TiO₂) nanocomposite was developed for the detection of genetically modified organisms (GMOs). DNA hybridization was studied by quartz crystal microbalance (QCM) and cyclic voltammetry (CV) measurements. Single stranded DNA probes were immobilized on the PT/TiO₂ coated quartz crystal electrode and the hybridization between the immobilized probe and the target complementary sequence in solution was monitored. The developed QCM-DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of genetically modified organisms.     

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-G_NP) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G_NP was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G_NP were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3−/4− as the redox indicator. The (CA-G_NP/SPAN)_n films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-G_NP) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)₆]^3−/4− in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-G_NP/SPAN)_n films, the target DNA with 20-base could be detected up to 2.13 × 10⁻¹³ mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated.     

An electrochemical biosensor for direct detection of DNA using polystyrene-g-soya oil-g-imidazole graft copolymer

A label-free electrochemical DNA biosensor was developed through the attachment of polystyrene-g-soya oil-g-imidazole graft copolymer (PS-PSyIm) onto modified graphene oxide (GO) electrodeposited on glassy carbon electrode (GC). GC/GO electrode was initially functionalised via electrochemical reduction of 4-nitrobenzene diazonium salt, followed by the electrochemical reduction of NO₂ to NH₂. Subsequent to the electrochemical deposition of gold nanoparticles on modified surface, the attachment of the PS-PSyIm graft copolymer on the resulting electrode was achieved. The interaction of PS-PSyIm with DNA at the bare glassy carbon electrode was studied by cyclic voltammetry technique, and it was found that interaction predominantly takes place through intercalation mode. The selectivity of developed DNA biosensor was also explored by DPV on the basis of considering hybridisation event with non-complementary, one-base mismatched DNA and complementary target DNA sequence. Large decrease in the peak current was found upon the addition of complementary target DNA. The sensitivity of the developed DNA biosensor was also investigated, and detection limit was found to be 1.20 nmol L^?1.