Preparation of Luminol-doped Nanoparticle and Its Application in DNA Hybridization Analysis

Introduction The analysis of DNA sequence and DNA mutant detection play fundamental roles in the rapid development of molecular diagnostics and in the anticancer drug screening. Therefor many detection techniques of DNA sequence have been developed in recent years. These techniques mainly depend on the nucleic acid hybridization1 and their sensitivities are related to the specific activity of the label linked to the DNA probe. The degree of hybridization of probe to its complementary DN…     

Nucleic Acid Biosensor for Detection of Human Immunodeficiency Virus Using Aquabis(1,10‐phenanthroline)copper(II) Perchlorate as Electrochemical Indicator

The electrochemical behavior of aquabis(1,10‐phenanthroline)copper(II) perchlorate [Cu(H₂O)(phen)₂]·2ClO₄, where phen=1,10‐phenanthroline, on binding to DNA at a glassy carbon electrode (GCE) and in solution, was described. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) results showed that [Cu(H₂O)(phen)₂]²⁺ had excellent electrochemical activity on the GCE with a couple of quasi‐reversible redox peaks. The interaction mode between [Cu(H₂O)(phen)₂]²⁺ and double‐strand DNA (dsDNA) was identified to be intercalative binding. An electrochemical DNA biosensor was developed with covalent immobilization of human immunodeficiency virus (HIV) probe for single‐strand DNA (ssDNA) on the modified GCE. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay. With this approach, a sequence of the HIV could be quantified over the range from 7.8×10^?9 to 3.1×10^?7 mol·L^?1 with a linear correlation of γ=0.9987 and a detection limit of 1.3×10^?9 mol·L^?1.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

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.     

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

In this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10^?13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.     

Indigo Carmine as New Label in PNA Biosensor for Detection of Short Sequence of p53 Tumor Suppressor Gene

A new electrochemical PNA hybridization biosensor for detection of a 15‐mer sequence unique to p53 using indigo carmine (IC) as an electrochemical detector is described in this work. This genosensor is based on the hybridization of target oligonucleotide with its complementary probe immobilized on the gold electrode by self‐assembled monolayer formation. Because this label is electroactive in acidic medium, the interaction between IC and short sequence of p53 is studied by differential pulse voltammety (DPV) in 0.1 M H₂SO₄. The results of electrochemical impedance spectroscopy and cyclic voltammetry in the solution of [Fe(CN)₆]^3?/4? shows no breakage in PNA‐DNA duplex. A decrease in the voltammetric peak currents of IC is observed upon hybridization of the probe with the target DNA. The influence of probe concentration on effective discrimination against non‐complementary oligonucleotides is investigated and a concentration of 10^?7 M is selected. The diagnostic performance of the PNA sensor is described and the detection limit is found to be 4.31×10^?12 M.     

Rolling‐Circle Amplification Detection of Thrombin Using Surface‐Enhanced Raman Spectroscopy with Core‐Shell Nanoparticle Probe

An ultrasensitive surface‐enhanced Raman spectroscopy (SERS) sensor based on rolling‐circle amplification (RCA)‐increased “hot‐spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO₂ core‐shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer‐by‐layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single‐stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10^?12 to 1.0×10^?8 M and a detection limit of 4.2×10^?13 M , and showed good performance in real serum samples.     

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.     

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

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

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.     

Determination of Electrochemical Interaction between 2‐(1H‐benzimidazol‐2‐yl) Phenol and DNA Sequences

A benzimidazole derivate, 2‐(1H‐benzimidazol‐2‐yl) phenol (2‐Bip) and its interaction mechanism with sequence specific DNA was examined with Differential Pulse Voltammetry (DPV). We, for the first time, investigated the effect of 2‐Bip on sequence specific DNA with electrochemical methods by evaluating both guanine and 2‐Bip oxidation signal changes. In the study, probe sequences were immobilized to the surface of the electrodes and then hybridization was achieved by sending the complementary target onto the probe modified electrodes. Following the hybridization, 2‐Bip solution was interacted with probe and hybrid sequences to see the effect of 2‐Bip on different DNA sequences. The binding constant (K), toxicity (S%) and thermodynamic parameters, i. e., Gibbs free energy (ΔG°) of 2‐Bip‐DNA complexes were evaluated. K was calculated as 5×10⁵ and the change in the ΔG° was found as ?32.50 kJ mol^?1, which are consistent well with the literature. Furthermore, S% showed that 2‐Bip is moderately toxic to single stranded DNA (ssDNA) and toxic to double stranded DNA (dsDNA). From our experimental data, we made four conclusions (i) 2‐Bip affects both ssDNA and dsDNA, (ii) 2‐Bip interaction mode with DNA could be non‐covalent interactions, (iii) 2‐Bip could be used as new DNA hybridization indicator due to its distinct effects on ssDNA and dsDNA, (iv) 2‐Bip could be used as a drug molecule for its DNA effect.     

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.     

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.     

Ultrasensitive Electrochemiluminescence Biosensor for mRNA Based on Polymerase Assisted Signal Amplification

A novel biosensor for ultra‐trace mRNA sensing was constructed based on isothermal circular strand‐replacement polymerization (CSRP) to amplify the electrochmeiluminescence (ECL) signal by combining quantum dots (CdTe) as luminophore. After the hairpin‐like capture DNA was opened by hybridization with target mRNA, the additive primer (DNA1) was able to get access to its complementary sequence which is partially belong to the stem part and triggered a polymerization of DNA strand, leading to the release of target mRNA and another polymerization cycle. The remaining sequence of the stem part continued to hybridize with QDs labeled DNA, accomplishing ECL signal amplification. Target mRNA could be specifically assayed with a linear relationship between the signal intensity and the logarithm of concentrations of target DNA in the range of 1.0×10⁻¹⁴∼5.0×10⁻¹⁰ M, with a low detection limit of 1.4×10⁻¹⁵ M. The signal could discriminate perfect matched target mRNA from 1‐base mismatch sequence. This proposed ECL biosensor exhibited an efficient performance in serum sample, opening new opportunities for genetic target analysis in diagnostic and clinic biomedical fields.     

A Sequence‐Selective Electrochemical DNA Biosensor Based on HRP‐Labeled Probe for Colorectal Cancer DNA Detection

Abstract

A highly‐sensitive sequence‐selective DNA sensor based on HRP‐labeled probe to detect specific K‐ras gene which is highly associated with colorectal cancer has been reported. Capture probe modified with–SH was first chemically adsorbed on the gold electrode through self‐assembly. Then, the hybridization of a complementary nucleic acid (target DNA:K‐ras gene) and HRP labeled oligonucleotide detection probe occurred in a sandwich way. Finally, H₂O₂ electroreduction current catalyzed by HRP was measured amperometrically in the presence of hydroquinon as mediator. The sequence selectivity is double guaranteed by the complementary hybridization of target DNA with capture and detection probes. The experimental conditions were optimized. The linear range is 1.17×10^?11～1.17×10^?7 mol l^?1 with a detection limit of 5.85×10^?12 mol l^?1. The electrode with capture probe can be reused after regeneration in boiling water.     

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.     

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

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

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.     

Employing Label‐free Electrochemical Biosensor Based on 3D‐Reduced Graphene Oxide and Polyaniline Nanofibers for Ultrasensitive Detection of Breast Cancer BRCA1 Biomarker

A label‐free DNA biosensor based on three‐dimensional reduced graphene oxide (3D‐rGO) and polyaniline (PANI) nanofibers modified glassy carbon electrode (GCE) was successfully developed for supersensitive detection of breast cancer BRCA1. The results demonstrated that 3D‐rGO and PANI nanofibers had synergic effects for reducing the charge transfer resistance (R_ct), meaning a huge enhancement in electrochemical activity of 3D‐rGO‐PANI/GCE. Probe DNA could be immobilized on 3D‐rGO‐PANI/GCE for special and sensitive recognition of target DNA (1.0×10^?15–1.0×10^?7 M) with a theoretical LOD of 3.01×10^?16 M (3S/m). Furthermore, this proposed nano‐biosensor could directly detect BRCA1 in real blood samples.     

Detection of DNA immobilized on bare gold electrodes and gold nanoparticle-modified electrodes via electrogenerated chemiluminescence using a ruthenium complex as a tag

Electrogenerated chemiluminescence (ECL) for DNA hybridization detection is demonstrated based on DNA that was self-assembled onto a bare gold electrode and onto a gold nanoparticles modified gold electrode. A ruthenium complex served as an ECL tag. Gold nanoparticles were self-assembled on a gold electrode associated with a 1,6-hexanedithiol monolayer. The surface density of single stranded DNA (ssDNA) on the gold nanoparticle modified gold electrode was 4.8?×?10¹⁴ molecules per square centimeter which was 12-fold higher than that on the bare gold electrode. Hybridization was induced by exposure of the target ssDNA gold electrode to the solution of ECL probe consisting of complementary ssDNA tagged with ruthenium complex. The detection limit of target ssDNA on a gold nanoparticle modified gold electrode (6.7?×?10^?12 mol L^?1) is much lower than that on a bare gold electrode (1.2?×?10^?10 mol L^?1). The method has been applied to the detection of the DNA sequence related to cystic fibrosis. This work demonstrates that employment of gold nanoparticles self-assembled on a gold electrode is a promising strategy for the enhancement of the sensitivity of ECL detection of DNA.