Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

In this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through Au? S bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10^?13 to 1.0×10^?6 mol·L^?1 with a detection limit of 3.5×10^?14 mol·L^?1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.     

A Sensitive Nanoporous Gold-Based Electrochemical DNA Biosensor for Escherichia coli Detection

A sensitive electrochemical DNA biosensor based on a mixed monolayer structure self-assembled at nanoporous gold (NPG) electrode surface was prepared for Escherichia coli (E. coli) detection. The NPG was fabricated on gold electrode, onto which thiolated oligonucleotides (SH-DNA) and mercaptohexanol (MCH) were covalently linked forming a mixed self-assembled monolayer (SAM). The hybridization between the SH-DNA/MCH modified biosensor and E. coli DNA was monitored with differential pulse voltammetry measurement using methylene blue (MB) as the hybridization indicator. The biosensor can detect 1 × 10^?12 M DNA target and 50 cfu/μL E. coli without any nucleic acid amplification steps. The detection limit was lowered to 50 cfu/mL after 5.0 h of incubation.     

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.     

碳纳米管修饰金电极检测特定序列DNA

利用化学偶联法将末端修饰氨基的寡聚核苷酸固定在表面修饰有羧基化碳纳米管(CNTs-COOH)的金电极表面, 制备新型核酸探针, 可以特异性结合目标单链寡聚核苷酸. 以阿霉素作为嵌合指示剂, 利用示差脉冲法测定杂交的结果. 经过实验条件的优化, 测定DNA浓度在1.0×10^－6～1.0×10^－9 mol/L呈良好的线性关系. 检测限为: 2.54×10^－10 mol/L. 碳纳米管特有的纳米结构对检测结果的放大作用, 提高了该传感器的检测限和灵敏度.     

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 Biosensors Based on Palladium Nanoparticles Combined with Carbon Nanotubes

Palladium nanoparticles, in combination with multi‐walled carbon nanotubes (MWCNTs), were used to fabricate a sensitivity‐enhanced electrochemical DNA biosensor. MWCNTs and palladium nanoparticles were dispersed in Nafion, which were used to modify a glassy carbon electrode (GCE). Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. Due to the ability of carbon nanotubes to promote electron‐transfer and the high catalytic activities of palladium nanoparticles for electrochemical reaction of MB, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.2×10^?13 M.     

A new peptide nucleotide acid biosensor for electrochemical detection of single nucleotide polymorphism in duplex DNA via triplex structure formation

In this paper, we report a new PNA biosensor for electrochemical detection of point mutation or single nucleotide polymorphism (SNP) in p53 gene corresponding oligonucleotide based on PNA/ds-DNA triplex formation following hybridization of PNA probe with double-stranded DNA (ds-DNA) sample without denaturing the ds-DNA into single-stranded DNA (ss-DNA). As p53 gene is mutated in many human tumors, this research is useful for cancer therapy and genomic study. In this approach, methylene blue (MB) is used for electrochemical signal generation and the interaction between MB and oligonucleotides is studied by differential pulse voltammety (DPV). Probe-modified electrode is prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of Au electrode. A significant increase in the reduction signal of MB following hybridization of the probe with the complementary double-stranded oligonucleotide (ds-oligonucleotide) confirms the function of the biosensor. The selectivity of the PNA sensor is investigated by non-complementary ds-oligonucleotides and the results support the ability of the sensor to detect single-base mismatch directly on ds-oligonucleotide. The influence of probe and ds-DNA concentrations on the effective discrimination against complementary sequence and point mutation is studied and the concentration of 10^?6 M is selected as appropriate concentration. Diagnostic performance of the biosensor is described and the detection limit is found to be 4.15 × 10^?12 M.     

An Electrochemical DNA Sensor Based on Electrodepositing Aluminum Ion Films on Stearic Acid‐Modified Carbon Paste Electrode and Its Application for the Detection of Specific Sequences Related to Bar Gene and CP4 Epsps Gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid‐modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well‐defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single‐stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the specific sequence related to the target bar gene with the dynamic range comprised between 1.0×10^?7 mol/L to 1.0×10^?4 mol/L. A detection limit of 2.25×10^?8 mol/L of oligonucleotides can be estimated.     

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.     

Electrochemical DNA Biosensor Based on Partially Reduced Graphene Oxide Modified Carbon Ionic Liquid Electrode for the Detection of Transgenic Soybean A2704‐12 Gene Sequence

In this work a partially reduced graphene oxide (p‐RGO) modified carbon ionic liquid electrode (CILE) was prepared as the platform to fabricate an electrochemical DNA sensor, which was used for the sensitive detection of target ssDNA sequence related to transgenic soybean A2704‐12 sequence. The CILE was fabricated by using 1‐butylpyridinium hexafluorophosphate as the binder and then p‐RGO was deposited on the surface of CILE by controlling the electroreduction conditions. NH₂ modified ssDNA probe sequences were immobilized on the electrode surface via covalent bonds between the unreduced oxygen groups on the p‐RGO surface and the amine group at the 5′‐end of ssDNA, which was denoted as ssDNA/p‐RGO/CILE and further used to hybridize with the target ssDNA sequence. Methylene blue (MB) was used as electrochemical indicator to monitor the DNA hybridization. The reduction peak current of MB after hybridization was proportional to the concentration of target A2704‐12 ssDNA sequences in the range from 1.0×10^?12 to 1.0×10^?6 mol/L with a detection limit of 2.9×10^?13 mol/L (3σ). The electrochemical DNA biosensor was further used for the detection of PCR products of transgenic soybean with satisfactory results.     

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.     

Nonenzymatic determination of glucose on electrodes prepared by directed electrochemical nanowire assembly (DENA)

The analytical characteristics of gold nanowires prepared by direct electrochemical synthesis were studied with the use of the amperometric determination of glucose as an example. The applicability of the gold nanowires to the determination of glucose in a neutral medium (a phosphate buffer solution with pH 7.2) over a concentration range from 1 × 10^–4 to 5 × 10^–3 M at a detection potential of +0.35 V was shown. It was found that the sensitivity of a nonenzymatic sensor for the determination of glucose on the gold nanowires was high: 3.7 × 10^–4 A M^–1 m^–2. The limit of detection was 3.3 × 10^–5 M.     

New method for preparation of polyoxometalate-capped gold nanoparticles, and their assembly on an indium-doped tin oxide electrode

Functionalized gold nanoparticles capped with polyoxometalates were prepared by a simple photoreduction technique where phosphododecamolybdates serve as reducing reagents, photocatalysts, and as stabilizers. TEM images of the resulting gold nanoparticles show the particles to have a relative narrow size distribution. Monolayer and multilayer structures of the negatively charged capped gold nanoparticles were deposited on a poly(vinyl pyridine)-derivatized indium-doped tin oxide (ITO) electrode via the layer-by-layer technique. The surface plasmon resonance band of the gold nanoparticles displays a blue shift on the surface of the ITO electrode. This is due to the substrate-induced charge redistribution in the gold nanoparticles and a change in the electromagnetic coupling between the assembled nanoparticles. The modified electrode exhibits the characteristic electrochemical behavior of surface-confined phosphododecamolybdate and excellent electrocatalytic activity. The catalysis of the modified electrode towards the model compound iodate was systematically studied. The heterogeneous catalytic rate constant for the electrochemical reduction of iodate was determined by chronoamperometry to be ca. 1.34?×?10⁵ mol^?1·L·s^?1. The amperometric method gave a linear range from 2.5?×?10^?6 to 1.5?×?10^?3 M and a detection limit of 1.0?×?10^?6 M. We believe that the functionalized gold nanoparticles prepared by this photoreduction technique are advantageous in terms of fabrication of sensitive and stable redox electrodes.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Naphthalene Diimide Carrying Two Cysteine Termini at Both Imide Linkers as a Molecular Staple

Naphthalene diimide ( 1 ) carrying cysteines at the termini of amide substituents were synthesized to act as a molecular staple of double stranded DNA. Since 1 is able to bind to double stranded DNA with threading intercalation, the complex of 1 with double stranded DNA can be topologically immobilized on a gold surface through the S? Au linkage as confirmed by cyclic voltammetric experiment. Ferrocenyl‐double stranded 23‐mertic oligonucleotide, dsFcODN, was immobilized on gold electrode with 1.0×10¹² molecules cm^?2 when electrode was treated with 2.0 µM dsFcODN and 4.0 µM 1 for 1 h at room temperature. The coverage density was similar to that obtained for the terminal thiol‐modified oligonucleotide. Compound 1 was applied to detect the 321‐meric PCR product of P. gingivalis, which is important in the diagnosis of periodontal disease. This experiment, coupled with the use of ferrocenylnaphthalene diimide, FND as electrochemical indicator for double stranded DNA, resulted in quantitative detection of PCR product within the range of 10 pg µL^?1–10 ng µL^?1 (15 nM–15 µM). The 1 and FND established a simple and rapid detection method of double stranded PCR product with a detection limit of 10 pg µL^?1 (15 nM).     

Development of an Electrochemical Sensor for Selective Determination of Dopamine Based on Molecularly Imprinted Poly(p-aminothiophenol) Polymeric Film

In this study, a molecularly imprinted electrochemical sensor (MIP/DA) was investigated for selective and sensitive determination of dopamine (DA) by electrochemical polymerization of p-aminothiophenol in the presence of DA on gold electrode. According to electrochemical behaviour of the sensor, gained through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), MIP/DA sensor showed distinctive electron transfer characteristics in comparison to the non-imprinted (NIP/DA) sensor. Besides the MIP/DA sensor showed high selectivity for dopamine through its analyte specific cavities. The sensor had a broad working range of 5.0×10⁻⁸–2.0×10⁻⁷ M with a limit of detection (LOD) of 1.8×10⁻⁸ M and the developed sensor was successfully applied for determination of dopamine in pharmaceutical samples.     

Ultrasensitive electrochemical analysis of two analytes by using an autonomous DNA machine that works in a two-cycle mode

We report a novel autonomous DNA machine for amplified electrochemical analysis of two DNAs. The DNA machine operates in a two‐cycle working mode to amplify DNA recognition events; the working mode is assisted by two different nicking endonucleases (NEases). Two bio‐barcode probes, a ZnS nanoparticle (NP)–DNA probe and a CdS NP–DNA probe, were used to trace two target DNAs. The detection system was based on a sensitive differential pulse anodic stripping voltammetry (DPASV) method for the simultaneous detection of Zn^II and Cd^II tracers, which were obtained by dissolving the two probes. Under the optimised conditions, detection limits as low as 5.6×10^?17 (3σ) and 4.1×10^?17 M (3σ) for the two target DNAs were achieved. It has been proven that the DNA machine system can simultaneously amplify two target DNAs by more than four orders of magnitude within 30 min at room temperature. In addition, in combination with an aptamer recognition strategy, the DNA machine was further used in the aptamer‐based amplification analysis of adenosine triphosphate (ATP) and lysozyme. With the amplification of the DNA machine, detection limits as low as 5.6×10^?9 M (3σ) for ATP and 5.2×10^?13 M (3σ) for lysozyme were simultaneously obtained. The satisfactory determination of ATP and lysozyme in Ramos cells reveals the good selectivity and feasibility of this protocol. The DNA machine is a promising tool for ultrasensitive and simultaneous multianalysis because of its remarkable signal amplification and simple machine‐like operation.     

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.     

Determination of Oxytetracycline with a Gold Electrode Modified by Chitosan-Multiwalled Carbon Nanotube Multilayer Films and Gold Nanoparticles

A molecularly imprinted electrochemical sensor was fabricated based on a gold electrode modified by chitosan-multiwalled carbon nanotube composite (CS-MWCNTs) multilayer films and gold nanoparticles (AuNPs) for convenient and sensitive determination of oxytetracycline (OTC). The multilayer of CS-MWCNTs composites and AuNPs were used to augment electronic transmission and sensitivity. The molecularly imprinted polymers (MIPs) were synthesized using OTC as the template molecule and o-phenylenediamine (OPD) as the functional monomer. They were modified on a gold electrode by electropolymerization. The electrochemical behavior of OTC at the imprinted sensor was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), and amperometry. The molecularly imprinted sensor showed high selectivity and excellent stability toward OTC. The linear range was from 3.0 × 10^?8 to 8.0 × 10^?5 mol/L, with a limit of detection (LOD) of 2.7 × 10^?8 mol/L (S/N = 3). The developed sensor showed good recovery in spiked samples analysis.     

Incorporation of Tetrazolium Blue (TB)/Gold Nanoparticles (GNPs) into Carbon Paste Electrode: Application as an Electrochemical Sensor for the Sensitive and Selective Determination of Sotalol in Micellar Medium

The electrochemical oxidation of Sotalol (SOT) based on Tetrazolium Blue (TB)/gold nanoparticles (GNPs)‐modified carbon paste electrodes (CPE) have been studied in the presence of sodium lauryl sulphate (SLS). Cyclic voltammetry (CV), differential pulse voltammetry (DPV), chronoamperometry and electrochemical impedance spectroscopy (EIS) techniques have all been utilized within this study. GNPs and TB have a synergetic effect‐giving rise to highly improved electrochemical responses and provide an advantageous platform for the basis of an electrochemical sensor with excellent performance. The experimental parameters, electrodeposition time, pH and scan rate have all been examined and optimized. The sensing of SOT via DPV is found to exhibit a wide linear dynamic range of 1.0×10⁻⁷–7.5×10⁻⁴ M in pH 2. LOD and LOQ were calculated and found to correspond to 2.5×10⁻⁸ M and 8.3×10⁻⁸ M, respectively. The suggested sensor has been used successfully for SOT determination in pharmaceutical samples and human urine as real samples. Satisfactory recoveries of analyte from these samples are demonstrated indicating that the suggested sensor is highly suitable for clinical analysis, quality control and a routine determination of SOT in pharmaceutical formulations.