Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Aunano-CNT/PANnano films

A polyaniline nanofibers (PAN_nano)/carbon paste electrode (CPE) was prepared via dopping PAN_nano in the carbon paste. The nanogold (Au_nano) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN_nano/CPE. The immobilization and hybridization of the DNA probe on the Au_nano-CNT/PAN_nano films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)₆]^3−/4− as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R_et) of the electrode surface increased after the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films and rose further after the hybridization of the probe DNA. The remarkable difference between the R_et value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au_nano-CNT/PAN_nano films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 × 10⁻¹² mol/L to 1.0 × 10⁻⁶ mol/L with a detection limit of 5.6 × 10⁻¹³ mol/L.     

Electrochemical impedance spectroscopy detection of lysozyme based on electrodeposited gold nanoparticles

A simple, highly sensitive, and label-free electrochemical impedance spectroscopy (EIS) aptasensor based on an anti-lysozyme-aptamer as a molecular recognition element, was developed for the detection of lysozyme. Improvement in sensitivity was achieved by utilizing gold nanoparticles (AuNPs), which were electrodeposited onto the surface of a gold electrode, as a platform for immobilization of the aptamer. To quantify the amount of lysozyme, changes in the interfacial electron transfer resistance (R_et) of the aptasensor were monitored using the redox couple of an [Fe(CN)₆]^3−/4− probe. The R_et increased with lysozyme concentration. The plot of R_et against the logarithm of lysozyme concentration is linear over the range from 0.1 pM to 500 pM with a detection limit of 0.01 pM. The aptasensor also showed good selectivity for lysozyme without being affected by the presence of other proteins.     

A DNA electrochemical sensor prepared by electrodepositing zirconia on composite films of single-walled carbon nanotubes and poly(2,6-pyridinedicarboxylic acid), and its application to detection of the PAT gene fragment

Carboxyl group-functionalized single-walled carbon nanotubes (SWNTs) and 2,6-pyridinedicarboxylic acid (PDC) were electropolymerized by cyclic voltammetry on a glassy-carbon electrode (GCE) surface to form composite films (SWNTs/PDC). Zirconia was then electrodeposited on the SWNTs/PDC/GCE from an aqueous electrolyte containing ZrOCl₂ and KCl by cycling the potential between −1.1 V and +0.7 V at a scan rate of 20 mV s⁻¹. DNA probes with a phosphate group at the 5′ end were easily immobilized on the zirconia thin films, because of the strong affinity between zirconia and phosphate groups. The sensors were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS was used for label-free detection of the target DNA by measuring the increase of the electron transfer resistance (R _et) of the electrode surface after the hybridization of the probe DNA with the target DNA. The PAT gene fragment and polymerase chain reaction (PCR) amplification of the NOS gene from transgenically modified beans were satisfactorily detected by use of this DNA electrochemical sensor. The dynamic range of detection of the sensor for the PAT gene fragment was from 1.0 × 10⁻¹¹ to 1.0 × 10⁻⁶ mol L⁻¹ and the detection limit was 1.38 × 10⁻¹² mol L⁻¹.     

Multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole DNA biosensor for label-free detection of genetically modified organisms by QCM and EIS

In this paper, we describe DNA electrochemical detection for genetically modified organism (GMO) based on multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole (PPy). DNA hybridization is studied by quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). An increase in DNA complementary target concentration results in a decrease in the faradic charge transfer resistance (R_ct) and signifying “signal-on” behavior of MWCNTs-PPy-DNA system. QCM and EIS data indicated that the electroanalytical MWCNTs-PPy films were highly sensitive (as low as 4 pM of target can be detected with QCM technique). In principle, this system can be suitable not only for DNA but also for protein biosensor construction.     

A label-free electrochemical biosensor based on a DNA aptamer against codeine

In order to develop a sensor for opium alkaloid codeine detection, DNA aptamers against codeine were generated by SELEX (systematic evolution of ligands by exponential enrichment) technique. An aptamer HL7-14, which is a 37-mer sequence with K_d values of 0.91 μM, was optimized by the truncation-mutation assay. The specificity investigation shows that HL7-14 exhibits high specificity to codeine over morphine, and almost cannot bind to other small molecule. With this new selected aptamer, a novel electrochemical label-free codeine aptamer biosensor based on Au-mesoporous silica nanoparticles (Au-MSN) as immobilized substrate has been proposed using [Fe(CN)₆]^3−/4− as electroactive redox probe. The linear range covered from 10 pM to 100 nM with correlation coefficient of 0.9979 and the detection limit was 3 pM. Our study demonstrates that the biosensor has good specificity, stability and well regeneration. It can be used to detect codeine.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

Simple and label-free electrochemical assay for signal-on DNA hybridization directly at undecorated graphene oxide

Exploring graphene oxide (GO), DNA hybridization detection usually relies on either GO decoration or DNA sequences labeling. The former endows GO with desired chemical, optical, and biological properties. The latter adopts labeled molecules to indicate hybridization. In the present work, we propose a simple, label-free DNA assay using undecorated GO directly as the sensing platform. GO is anchored on diazonium functionalized electrode through electrostatic attraction, hydrogen bonding or epoxy ring-opening. The π–π stacking interaction between hexagonal cells of GO and DNA base rings facilitates DNA immobilization. The adsorbed DNA sequence is specially designed with two parts, including immobilization sequence and probe sequence. In the absence of target, the two sequences lie nearly flat on GO platform. In the presence of target, probe hybridizes with it to form double helix DNA, which ‘stands’ on GO. While the immobilization sequence part remains ‘lying’ on GO surface. Hence, DNA hybridization induces GO interfacial property changes, including negative charge and conformational transition from ‘lying’ ssDNA to ‘standing’ dsDNA. These changes are monitored by electrochemical impedance spectroscopy and adopted as the analytical signal. This strategy eliminates the requirement for GO decoration or DNA labeling, representing a comparatively simple and effective way. Finally, the principle is applied to the detection of conserved sequence of the human immunodeficiency virus 1 pol gene fragment. The dynamic detection range is from 1.0 × 10⁻¹² to 1.0 × 10⁻⁶ M with detection limit of 1.1 × 10⁻¹³ M with 3σ. And the sequences with double- or four-base mismatched are readily distinguishable. In addition, this strategy may hold great promise for potential applications from DNA biosensing to nanostructure framework construction based on the versatile DNA self-assembly.     

Immobilization and hybridization of DNA based on magnesium ion modified 2,6-pyridinedicarboxylic acid polymer and its application for label-free PAT gene fragment detection by electrochemical impedance spectroscopy

A new approach for a simple electrochemical detection of PAT gene fragment is described. Poly(2,6-pyridinedicarboxylic acid) (PDC) modified glassy carbon electrode (GCE) was prepared by potential scan electropolymerization in an aqueous solution. Mg2 ions were incorporated by immer-sion of the modified electrode in 0.5 mol/L aqueous solution of MgCl2 to complete the preparation of a generic "activated" electrode ready for binding the probe DNA. The ssDNA was linked to the conduct-ing polymer by forming a bidentate complex between the carboxyl groups on the polymer and the phosphate groups of DNA via Mg2 . DNA immobilization and hybridization were characterized with dif-ferential pulse voltammetry (DPV) by using methylene blue (MB) as indicator and electrochemical im-pedance spectroscopy (EIS). The EIS was of higher sensitivity for DNA detection as compared with voltammetric methods in our strategy. The electron transfer resistance (Ret) of the electrode surface in EIS in [Fe(CN)6]3-/4- solution increased after the immobilization of the DNA probe on the Mg/PDC/GCE electrode. The hybridization of the DNA probe with complementary DNA (cDNA) made Ret increase further. The difference between the Ret at ssDNA/Mg/PDC/GCE and that at hybridization DNA modified electrode (dsDNA/Mg/PDC/GCE) was applied to determine the specific sequence related to the target PAT gene with the dynamic range comprised between 1.0 × 10-9 and 1.0 × 10_5 mol/L. A detection limit of 3.4 × 10-10 mol/L of oligonucleotides can be estimated.     

Electrochemical properties of niosomes modified Au electrode and DNA recognition

Non-ionic surfactant vesicles (NSVs), also referred to as niosomes, have been studied as an alternative to conventional liposomes. In this paper, electrochemical inspection of the interaction between Herring sperm DNA and niosomes has been investigated after a simple and novel method for the formation of niosomes on Au electrode. Each step of electrode modification has been confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The assembly of octadecanethiol (ODT) layer on the electrode surface generates a packed film that introduces a barrier to the interfacial electron transfer (R_et), and the subsequent immobilization of niosomes onto the self-assembled monolayer (SAM) layer results in a further increase of R_et, due to the formed bilayer almost blocked the redox probe to the electrode surface. When Herring sperm DNA was added, the R_et value decreased, indicating that the barrier of the redox probe to the surface was disrupted. The addition of DNA caused the formation of some transmembrane channels for the redox probe across the niosomes. A good linear relationship between R_et value and DNA concentration was found over the 0–0.05 mg mL⁻¹ concentration range.     

Microplate electrochemical DNA detection for phosphinothricin acetyltransferase gene sequence with cadmium sulfide nanoparticles

An electrochemical DNA detection method for the phosphinothricin acetyltransferase (PAT) gene sequence from the transgenetic plants was established by using a microplate hybridization assay with cadmium sulfide (CdS) nanoparticles as oligonucleotides label. The experiment included the following procedures. Firstly target PAT ssDNA sequences were immobilized on the polystyrene microplate by physical adsorption. Then CdS nanoparticle labeled oligonucleotide probes were added into the microplate and the hybridization reaction with target ssDNA sequences took place in the microplate. After washing the microplate for three times, certain amounts of HNO₃ were added into the microplate to dissolve the CdS nanoparticles anchored on the hybrids and a solution containing Cd²⁺ ion was obtained. At last differential pulse anodic stripping voltammetry (DPASV) was used for the sensitive detection of released Cd²⁺ ion. Based on this principle a sensitive electrochemical method for the PAT gene sequences detection was established. The voltammetric currents of Cd²⁺ were in linear range with the target ssDNA concentration from 5.0 × 10^− 13 to 1.0 × 10^− 10 mol/L and the detection limit was estimated to be 8.9 × 10^− 14 mol/L (3σ). The proposed method showed a good promise for the sensitive detection of specific gene sequences with good selectivity for the discrimination of the mismatched sequences.     

DNA impedance biosensor for detection of cancer, TP53 gene mutation,based on gold nanoparticles/aligned carbon nanotubes modified electrode

For the first time, a new platform based on electrochemical growth of Au nanoparticles on aligned multi-walled carbon nanotubes (A-MWCNT) was developed for sensitive lable-free DNA detection of the TP53 gene mutation, one of the most popular genes in cancer research. Electrochemical impedance spectroscopy (EIS) was used to monitor the sequence-specific DNA hybridization events related to TP53 gene. Compared to the bare Ta or MWCNT/Ta electrodes, the synergistic interactions of vertically aligned MWCNT array and gold nanoparticles at modified electrode could improve the density of the probe DNA attachment and resulting the sensitivity of the DNA sensor greatly. Using EIS, over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship in respect to the logarithm of the complementary oligonucleotides sequence concentrations in the wide range of 1.0 × 10⁻¹⁵ − 1.0 × 10⁻⁷ M, with a detection limit of 1.0 × 10⁻¹⁷ M (S/N = 3). The prepared sensor also showed good stability (14 days), reproducibility (RSD = 2.1%) and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining gold nanoparticles with the on-site fabricated aligned MWCNT array represents a promising platform for achieving sensitive biosensor for fast mutation screening related to most human cancer types.     

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

A sensitive and label-free impedimetric biosensor based on an adjunct probe

A highly sensitive and label-free impedimetric biosensor is achieved based on an adjunct probe attached nearby the capture probe. In this work, the adjunct probe was co-assembled on the surface of gold electrode with the capture probe hybridized with the reporter probe, and then 6-mercapto-1-hexanol was employed to block the nonspecific binding sites. When target DNA was added, the adjunct probe functioned as a fixer to immobilize the element of reporter probe displaced by the target DNA sequences and made the reporter probe approach the electrode surface, leading to effective inhibition of charge transfer. The increase in charge transfer resistance is related to the quantity of the target DNA in a wide range. The linear range for target DNA with specific sequences was from 0.1 nM to 0.5 μM with a good linearity (R = 0.9988) and a low detection limit of 6.3 pM. This impedimetric biosensor has the advantages of simplicity, sensitivity, good selectivity, and large dynamic range.     

A novel label-free electrochemical aptasensor for thrombin based on the {nano-Au/thionine}n multilayer films as redox probes

Herein, a novel label-free electrochemical aptasensor based on direct immobilization of the redox probes on an electrode surface was reported. Gold electrode coated Nafion was firstly modified with redox probe-thionine (Thi) through ion exchange adsorption. Then, with the help of chemisorption and electrostatic adsorption, negatively charged nano-Au and positively charged Thi were layer-by-layer (LBL) self-assembled onto the modified electrode surface, which formed {nano-Au/Thi⁺}_n multilayer films for improving the amount of redox probes and immobilizing thiolated thrombin aptamers (TBA). In the presence of target thrombin (TB), the TBA on the multilayer film could catch the TB onto the electrode surface, which resulted in a barrier for electro-transfer, leading to decrease of the current. The proposed method avoided the cubsome redox probe labeling process, increased the amount of redox probe and reduced the distance between the redox probe and electrode surface. Thus, the approach showed a high sensitivity and a wider linearity to TB in the range from 0.12 nM to 46 nM with a detection limit of 40 pM.     

Sensitive DNA biosensor improved by Luteolin copper(II) as indicator based on silver nanoparticles and carbon nanotubes modified electrode

A novel and sensitive electrochemical DNA biosensor has been developed for the detection of DNA hybridization. The biosensor was proposed by using copper(II) complex of Luteolin C₃₀H₁₈CuO₁₂ (CuL₂) as an electroactive indicator based on silver nanoparticles and multi-walled carbon nanotubes (Ag/MWCNTs) modified glassy carbon electrode (GCE). In this method, the 4-aminobenzoic acid (4-ABA) and Ag nanoparticles were covalently grafted on MWCNTs to form Ag/4-ABA/MWCNTs. The proposed method dramatically increased DNA attachment quantity and complementary ssDNA detection sensitivity for its large surface area and good charge-transport characteristics. DNA hybridization detection was performed using CuL₂ as an electroactive indicator. The CuL₂ was synthesized and characterized using elemental analysis (EA) and IR spectroscopy. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction between CuL₂ and ds-oligonucleotides (dsDNA). It was revealed that CuL₂ presented high electrochemical activity on GCE, and it could be intercalated into the double helices of dsDNA. The target ssDNA of the human hepatitis B virus (HBV) was quantified in a linear range from 3.23 × 10⁻¹² to 5.31 × 10⁻⁹ M (r = 0.9983). A detection limit of 6.46 × 10⁻¹³ M (3σ, n = 11) was achieved.     

A nano-porous CeO(2)/Chitosan composite film as the immobilization matrix for colorectal cancer DNA sequence-selective electrochemical biosensor

CeO₂/Chitosan (CHIT) composite matrix was firstly developed for the single-stranded DNA (ssDNA) probe immobilization and the fabrication of DNA biosensor related to the colorectal cancer gene. Such matrix combined the advantages of CeO₂ and chitosan, with good biocompatibility, nontoxicity and excellent electronic conductivity, showing the enhanced loading of ssDNA probe on the surface of electrode. The preparation method is quite simple and inexpensive. The hybridization detection was accomplished by using methylene blue (MB), an electroactive lable, as the indicator. The differential pulse voltammetry (DPV) was employed to record the signal response of MB and determine the amount of colorectal cancer target DNA sequence. The experimental conditions were optimized. The established biosensor has high detection sensitivity, a relatively wide linear range from 1.59 × 10⁻¹¹ to 1.16 × 10⁻⁷ mol L⁻¹ and the ability to discriminate completely complementary target sequence and four-base-mismatched sequence.     

Electrochemical DNA biosensor for the detection of short DNA species of Chronic Myelogenous Leukemia by using methylene blue

A novel assay for the voltammetric detection of 18-bases DNA sequences relating to Chronic Myelogenous Leukemia (CML, Type b3a2) using methylene blue (MB) as the hybridization indicator was reported. DNA was covalently attached onto a glassy carbon electrode (GCE) through amines of the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N′-ethyl carbodiimidehydrochloride (EDC). The covalently immobilized single-stranded DNA (ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. A significant increase of the peak current for methylene blue upon the hybridization of immobilized ssDNA with cDNA in the solution was observed. This peak current change was used to monitor the recognition of CML DNA sequence. This electrochemical approach is sequence specific as indicated by the control experiments in which no peak current change was observed if a non-complementary DNA sequence was used. Factors, such as DNA target concentration and hybridization conditions determining the sensitivity of the electrochemical assay were investigated. Under optimal conditions, this sensor has a good calibration range between 1.25 × 10⁻⁷ and 6.75 × 10⁻⁷ M, with CML DNA sequence detection limit of 5.9 × 10⁻⁸ M.     

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.     

Rolling cycle amplification based single-color quantum dots–ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA

In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen)₂(dppx)]²⁺ (dppx = 7,8-dimethyldipyrido [3,2-a:2′,3′-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen)₂(dppx)]²⁺ is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen)₂(dppx)]²⁺ through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover, this strategy applies QDs–Ru assembling dyads to the detection of single-strand DNA (ssDNA) without any functionalization and separation techniques.     

DNA/Single-Walled Carbon Nanotubes Based Fluorescence Detection of Hg2+

A new, highly selective, and sensitive technique has been developed for the detection of Hg²⁺ using singled-wall carbon nanotubes (SWNTs) and two kinds of oligonucleotides. The fluorescence of the thymine-rich single stranded DNA labeled with dye (the probe ssDNA) was effectively quenched by the SWNTs. In the presence of a target DNA (rich T-T mismatched with probe), the tightness of the DNA wrapping around the SWNTs was loosened. Since binding of Hg²⁺ turned the T-T mismatches to stable T-Hg²⁺-T base pairs, and the binding rate of DNA and the nanotube was lower than that of DNA hybridization, it induced the release of DNA molecules from the SWNTs, and this resulted in a remarkable increase of fluorescence compared to that of the DNA-SWNTs. The assay exhibited a dynamic response range for Hg²⁺ from 4.52 × 10^?8 M to 7.21 × 10^?7 M with a detection limit of 10 nM.