Study on the electrochemiluminescence behavior of ABEI and its application in DNA hybridization analysis

The electrogenerated chemiluminescence (ECL) behavior of N-(4-aminobutyl)-N-ethylisoluminol (ABEI) was studied and it was found that ABEI could produce emission light when oxidized at a +1.0 V (vs. Ag/AgCl) potential in alkaline solution. The addition of H2O2 markedly improved the ECL sensitivity. The pH value of the solution as well as the H2O2 concentration and working potential all have influences on the ECL response. Under optimal conditions, ABEI can be detected in the range 1.3 x 10(-6)-6.5 x 10(-12) mol L(-1). A detection limit of 2.2 x 10(-12) mol L(-1) for ABEI was obtained at a signal-to-noise ratio of 3. ABEI was then used as a marker to label a known sequence oligonucleotide, which was used as a DNA probe for identifying a target ssDNA immobilized on a PPy modified electrode based on a specific hybridization reaction. The hybridization events were evaluated by the ECL measurements. The results showed that only a complementary sequence could form a double-stranded DNA with the DNA probe and give a strong ECL response. A three-base mismatch sequence and non-complementary sequence have no response. The intensity of the ECL was linearly related to the concentration of the complementary sequence in the range 9.6 x 10(-11)-9.6 x 10(-8) mol L(-1), the detection limit was 3.0 x 10(-11) mol L(-1).     

Detection of Chlamydia trachomatis by in situ hybridization with sulphonated total DNA

In situ nucleic acid hybridization was applied to the detection of Chlamydia trachomatis on microscope slides by use of sulphonated total DNA as a probe. Visualization of labelled DNA was obtained using a commercial enzyme-linked monoclonal antibody. A mixture of paraformaldehyde and glutaraldehyde was found to be the best fixative. With high probe concentration (10 μg/ml), intracellular inclusions were detected as early as 8 h after inoculating the cell culture. Extracellular elementary bodies could also be detected. Five genital specimens were tested by in situ hybridization; the results were in agreement with those observed by culture.     

Amplifying the electrical hybridization signals of DNA array by multilayer assembly of Au nanoparticle probes

This paper describes a versatile method for amplifying the signals of Au-nanoparticle-based DNA hybridization detecting systems. The Au nanoparticles usually serve as labels to enhance DNA hybridization signal. We further assembled several layers of nanoparticles to selectively increase the number of labelled nanoparticles. Through silver enhancement, the multilayer nanoparticles may produce significantly higher amounts of metal silver on the their surfaces than the monolayer nanoparticles did. This finally accounts for the greatly enhanced DNA hybridization signal. Particularly, the amplification of electrical detection system was demonstrated here. Electrical measuring results indicated that the current values were enhanced by approximately 3 orders of magnitude, and the single nucleotide mismatch discrimination ratio was enlarged to approximately 10(9):1.     

Detection of DNA damaging agents using layer-by-layer assembly

The layer-by-layer (LBL) electrostatic deposition method was used to fabricate multilayer films of pentaerythritol-based metallodendrimer with Ru^II terpyridine subunits (RuDen) that has a positive charge and ds-DNA (ds, double-stranded) that has a negative charge due to its phosphate backbone. Evidence of assembly was obtained by fabrication of (DNA | RuDen)_n on quartz treated with poly(diallyldimethylammonium chloride), PDDA. The absorbance at 263 nm varied linearly with n in the range 1-6. For electrochemical monitoring of damage by styrene oxide, the assembly was on a glassy carbon electrode that was coated with a monolayer of aminobenzoic acid. The measurement was based upon the RuDen-catalyzed oxidation of sites, e.g. guanine, that are exposed when ds-DNA is damaged. The peak current at 1.07 V versus Ag | AgCl in square wave voltammetry increases with incubation time for 30 min. The process was also monitored by the shift in the spectrum of a long period grating (LPG) fiber coated with (DNA | RuDen)₅. A typical shift, which is due to changes in the refractive index of the coating, was 0.3 and 1.8 nm for 5 and 30 min exposures, respectively, using an algorithm that can measure a shift of 10⁻⁴ nm.     

Electrochemical detection of DNA hybridization based on DNA-templated assembly of silver cluster

The growth of metals on DNA templates has generated considerable interest in connection to the design of metallic nanostructures. Here we exploit the DNA-induced generation of metal clusters for developing an electrical biosensing protocol. The new hybridization assay employs a probe-modified gold surface, and is based on the electrostatic ‘collection’ of silver cations along the DNA duplex, the reductive formation of silver nanoclusters along the DNA backbone, dissolution of the silver aggregate and stripping potentiometric detection of the dissolved silver at a thick-film carbon electrode. The new protocol thus combines the inherent signal amplification of stripping analysis with effective discrimination against nonhybridized DNA.     

Detection of enteric pathotypes of eEcherichia coli by hybridization using six DNA probes

A set of 6 DNA probes was tested to evaluate the incidence of various Escherichia coli pathotypes among 540 strains isolated in France from diarrhoeal stools of infants, children and adults. Enterotoxigenic E. coli were detected using 3 gene probes for enterotoxins LT, STaH and STaP. Enteroinvasive E. coli were detected using one DNA probe which specifically hybridizes with bacteria expressing the cell invasion phenotype ⪡INV⪢. They represented 1.5 % and 1.1 % of the total, respectively. An SLTI probe which contains the structural gene for the A subunit of Shiga-like toxin I was constructed to detect enterohaemorrhagic E. coli. Among the 5 strains detected, only 1 belonged to serotype O157:H7. An attempt was made to detect enteropathogenic E. coli (EPEC) using both an EPEC-adherence factor and the above mentioned SLTI probes. Under the experimental conditions, they did not appear to be efficient at detecting this pathotype.     

Detection of DNA hybridization on chemically modified graphene platforms

The increasing demand for simple, low-cost, rapid, sensitive and label-free methods for the detection of DNA sequences and the presence of single nucleotide polymorphisms (SNPs) has become an important issue in biomedical research. In this work, we studied the performances of several chemically modified graphene nanomaterials as sensing platforms by using the electrochemical impedance spectroscopy technique for the detection. We employed a hairpin DNA as a highly selective probe for the detection of SNP correlated to Alzheimer's disease. We believe that our findings may present a foundation for further research and development in graphene-based impedimetric biosensing.     

DNA sensor by using electrochemiluminescence of acridinium ester initiated by tripropylamine

It was found that tripropylamine (TPA) could be used as a coreactant to initiate the electrochemiluminescence (ECL) of acridinium NHS ester (AE NHS) labels attached to DNA. The radicals generated in the electro-oxidation process of TPA reacted with AE NHS to form the excited N-methylacridone, giving rise to light emission. The AE/TPA ECL system was for the first time used as the detection system for developing an ECL-based DNA sensor. In the protocol, streptavidin-modified gold nanoparticles were firstly immobilized onto a thiol-treated gold electrode. The streptavidin could specifically interact with the biontinylated capture DNA. Afterwards, the target DNA and the AE-labeled report DNA were conjugated onto the electrode step by step due to the hybridization reactions, and a sandwich-type sensor was fabricated. The ECL signals of the sensor were obtained under pulse potential condition in alkaline solution containing 50.0 mmol L⁻¹ TPA. Under optimized experimental conditions, the linear range of the DNA sensor for the determination of the target DNA was from 5.0 × 10⁻¹⁵ to 5.0 × 10⁻¹² mol L⁻¹. The detection limit (S/N = 3) was 3.0 × 10⁻¹⁵ mol L⁻¹. Moreover, the sensor could specifically recognize the target DNA against one base-pair mismatched sequences, two base-pair mismatched sequences, and the noncomplementary sequences. It is of great application potential in clinic analysis.     

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.     

Monitoring and modulating a catalytic hybridization circuit for self-adaptive bioorthogonal DNA assembly

Constructing artificial domino nanoarchitectures, especially dynamic DNA circuits associated with the actuation of biological functions inside live cells, represents a versatile and powerful strategy to regulate the behaviors and fate of various living entities. However, the stepwise operation of conventional DNA circuits always relies on freely diffusing reactants, which substantially slows down their operation rate and efficiency. Herein, a self-adaptive localized catalytic circuit (LCC) is developed to execute the self-sustained bioorthogonal assembly of DNA nanosponges within a crowded intracellular environment. The LCC-generated DNA scaffolds are utilized as versatile templates for realizing the proximity confinement of LCC reactants. Single-molecule-detecting fluorescence correlation spectroscopy (FCS) is used to explore the reaction acceleration of the catalytic circuit. This self-adaptive DNA circuit facilitates the bioorthogonal assembly of highly branched DNA networks for robust and accurate monitoring of miRNA targets. Based on its intriguing and modular design, the LCC system provides a pivotal molecular toolbox for future applications in early disease diagnosis.

A localized catalytic circuit, facilitating the self-assembly of DNA nanosponges, is developed for robust and accurate monitoring of miRNA targets in live cells and mice.     

电化学发光法测定发光标记试剂ABEI

ABEI-[N-(4-氨基丁基)-N-乙基]-氯基-2,3-二氢吩噻嗪-1,4-二酮是化学发光免疫分析法(简称LIA)中最常用的发光标记试剂,但有关电化学发光免疫分析目前尚未见报道。本文利用自制的电化学发光仪,对ABEI和ABEI标记兔抗HCG的电化学发光行为进行了研究。提出了利用电化学发光进行免疫分析的新途径。     

碱性水溶液中ABEI的电致化学发光的研究

研究了6-[N-(4-氨基丁基-N-乙基)-N-乙基]-氨基-2,3-二氢吩噻嗪1,4-二酮(ABEI)的电致化学发光的各种条件,发现最佳的电脉冲参数为:占空比0.45,脉冲周期20ms,脉冲幅值+1.6V(相对饱和甘汞电极);最佳介质是0.12mol/L KOH-0.080mol/L H_3BO_(3-)0.080 mol/L KCI(pH12.0).在这些条件下,ABEI的发光强度与浓度在1.0×10~(-8)～9.0×10~(-5)mol/L范围内呈线性关系.研究表明,溶液中的Cl~-先被氧化为CIO~-,CIO~-再与ABEI作用并使其发光.     

Detection of DNA damage by exploiting the distance dependence of the electrochemiluminescence energy transfer between quantum dots and gold nanoparticles

Microchimica Acta - We have developed a platform to detect DNA damage induced by perfluorooctanoic acid (PFOA) by measuring the electrochemiluminescence (ECL) of a layer-by-layer electrostatic...     

Detection of short oligonucleotide sequences using an electrochemical DNA hybridization biosensor

An electrochemical DNA hybridization biosensor was developed for the detection of DNA hybridization using MDB and proflavine as electrochemical labels. The biosensor was based on the interaction of 7-dimethyl-amino-1,2-benzophenoxazi-nium Meldola’s Blue (MDB) and proflavine with double stranded DNA (dsDNA) The electrochemical behaviour of MDB and proflavine as well as its interaction with double stranded (dsDNA) were investigated by cyclic (CV) and square wave voltammetry (SWV) and screen printed electrodes (ScPE). Furthermore, DNA-hybridization biosensors were developed for the detection of hybridization between oligonucleotides, which was detected by studying changes in the voltammetric peaks of MDB (reduction peak at −0.251 V) and proflavine (reduction peak at 0.075 V). MDB and proflavine were found to intercalate between the base pairs of dsDNA and oligonucleotides. Several factors affecting the dsDNA or oligonucleotides immobilization, hybridization and indicator preconcentration and interaction time, were investigated. As a result of the interaction of MDB with dsDNA and hybridized oligonucleotides, the voltammetric signals of MDB increased. Furthermore, guanine’s oxidation peak (at 0.901 V) was decreased as MDB’s concentration was increased. As a result of the interaction of proflavine with dsDNA and hybridized oligonucleotides, the voltammetric signals of proflavine decreased. These results were similar for carbon paste and screen printed electrodes. A comparison of the performance between CPE and ScPE was done. Our results showed that lower concentrations of MDB and proflavine were detected using screen printed electrodes. Moreover, reproducibility was better using screen printed electrodes and the detection was faster (regarding the experimental steps), but they are more cost effective.      

Detection of DNA hybridization by various spectroscopic methods using the copper tetraphenylporphyrin complex as a probe

We are presenting new and highly sensitive hybridization assays. They are based on various spectroscopic methods including resonance light scattering, circular dichroism, ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy, and relies on the interaction of the Cu(II), Ni(II), Mg(II), Co(II), Cd(II), and Zn(II) complexes, respectively, of tetraphenylporphyrin (TPP) with double-strand DNA (dsDNA) and single strand DNA (ssDNA). The interaction results in amplified resonance light scattering (RLS) signals and enables the detection of hybridization without the need for labeling DNA. The RLS signals are strongest in case of the Cu (II)-TPP complex which therefore was selected as the probe. The technique is simple, robust, accurate, and can be completed in less than one hour.

Detection of T4 DNA ligase using a solid-state electrochemiluminescence biosensing switch based on ferrocene-labeled molecular beacon

A solid-state electrochemiluminescence (ECL) biosensing switch based on special ferrocene-labeled molecular beacon (Fc-MB) has been successfully developed for T4 DNA ligase detection. Such special switch system consisted of two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Au nanoparticle and Ruthenium (II) tris-(bipyridine) (Ru(bpy)₃²⁺-AuNPs) onto Au electrode. A molecular beacon labeled by ferrocene as the ECL intensity switch. The molecular beacon is designed with special base sequence, which could combine with its target biomolecule via the reaction of the repair and recombination of nucleic acids by DNA ligase. During the reaction, the molecular beacon opened its stem-loop, and the labeled Fc was consequently kept away from the ECL substrate. Such structural change resulted in an obvious increment in ECL intensity due to the decreased Fc quenching effect to the ECL substrate. The analysis results are sensitive and specific.     

Control of DNA hybridization by photoswitchable molecular glue

Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.     

Chemomechanics of surface stresses induced by DNA hybridization

When biomolecular reactions occur on one surface of a microcantilever beam, changes in intermolecular forces create surface stresses that bend the cantilever. While this phenomenon has been exploited to create label-free biosensors and biomolecular actuators, the mechanisms through which chemical free energy is transduced to mechanical work in such hybrid systems are not fully understood. To gain insight into these mechanisms, we use DNA hybridization as a model reaction system. We first show that the surface grafting density of single-stranded probe DNA (sspDNA) on a surface is strongly correlated to its radius of gyration in solution, which in turn depends on its persistence length and the DNA chain length. Since the persistence length depends on ionic strength, the grafting density of sspDNA can be controlled by changing a solution's ionic strength. The surface stresses produced by the reaction of complementary single-stranded target DNA (sstDNA) to sspDNA depend on the length of DNA, the grafting density, and the hybridization efficiency. We, however, observe a remarkable trend: regardless of the length and grafting density of sspDNA, the surface stress follows an exponential scaling relation with the density of hybridized sspDNA.