Electrochemical biosensor for detection of BCR/ABL fusion gene based on hairpin locked nucleic acids probe

This communication reports on a novel biosensor to study the hybridization specificity by using thiolated hairpin locked nucleic acids (LNA) as the capture probe. The LNA probe was immobilized on the gold electrode through sulfur–Au interaction and could selectively hybridize with its target DNA. Differential pulse voltammetry (DPV) was used to monitor the hybridization reaction on the probe electrode. The decrease of the peak current of methylene blue, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This LNA probe has been used for assay of fusion gene in Chronic Myelogenous Leukemia (CML) of the real sample with satisfactory result.     

基于锁核酸探针的传感器用于BCR/ABL融合基因检测的研究

基于慢性粒细胞白血病中BCR/ABL融合基因的碱基序列,设计了一种新型发夹结构锁核酸(locked nucleic acids, LNA)探针,把LNA探针通过Au-s键固定在金电极表面构建了特异的生物传感器.LNA探针与目标链DNA杂交,以自行合成的苯甲酸二聚铜配合物([Cu2(C7H5O2)4(C2H6O)2], 简称[Cu(R)2]2+)为杂交指示剂,应用差示脉冲伏安法进行检测,表现出良好的响应信号.该新型锁核酸传感器能较好的区分完全互补链DNA、单碱基错配链DNA.对互补链DNA检测的线性范围为1.0×10-8～1.0×10-6 mol•L-1,检出限为2.0×10-9 mol•L-1.     

Electrochemical sensing of DNA immobilization and hybridization based on carbon nanotubes/nano zinc oxide/chitosan composite film

A novel electrochemical DNA biosensor based on zinc oxide （ZnO） nanoparticles and multi-walled carbon nanotubes （MWNTs） for DNA immobilization and enhanced hybridization detection is presented. The MWNTs/nano ZnO/chitosan composite film modified glassy carbon electrode （MWNTs/ZnO/CHIT/GCE） was fabricated and DNA probes were immobilized on the electrode surface. The hybridization events were monitored by differential pulse voltammetry （DPV） using methylene blue （MB） as an indicator. The sensor can effectively discriminate different DNA sequences related to PAT gene in the transgenic corn, with a detection limit of 2.8× 10^-12 mol/L of target sequence.     

A Sandwich‐Type Electrochemical Biosensor for Detection of BCR/ABL Fusion Gene Using Locked Nucleic Acids on Gold Electrode

In this study, a sandwich‐type electrochemical enzyme‐based LNA‐modified DNA biosensor was developed to detect relative gene in chronic Myelogenous Leukemia first. This biosensor is based on a ‘sandwich’ detection strategy, which involves a pair of probes (a capture probe immobilized at the electrode surface and a reporter probe labeled biotin as an affinity tag for avidin‐HRP) modified LNA. Since biotin can be connected with avidin‐HRP, this biosensor offers an enzymatically amplified electrochemical current signal for the detection of target DNA. This new pattern exhibits high sensitivity and selectivity, and this biosensor has been used for an assay of PCR real sample with satisfactory result.     

Sensitive DNA-hybridization biosensors based on gold nanoparticles for testing DNA damage by Cd(II) ions

A DNA biosensor was constructed by immobilizing a 20-mer oligonucleotide probe and hybridizing it with its complementary oligomer on the surface of a glassy carbon electrode modified with gold nanoparticles. The properties of the biosensor and its capability of recognizing its complementary sequence were studied by electrochemical impedance spectroscopy. The oxidative stress caused by cadmium ions can be monitored by differential pulse voltammetry using the cobalt(III)tris(1,10-phenanthroline) complex and methylene blue as electrochemical indicators. The biosensor is capable of indicating damage caused by Cd(II) ions in pH 6.0 solution. The results showed that the biosensor can be used for rapid screening for DNA damage.

Ultrasensitive electrochemical supersandwich DNA biosensor using a glassy carbon electrode modified with gold particle-decorated sheets of graphene oxide

We describe a supersandwich type of electrochemical DNA biosensor based on the use of a glassy carbon electrode (GCE) modified with reduced graphene oxide (rGO) sheets that are decorated with gold nanoparticles (Au NPs). Thiolated capture DNA (probe DNA) was covalently linked to the Au NPs on the surface of the modified GCE via formation of Au-S bonds. In presence of target DNA, its 3′ terminus hybridizes with capture probe and the 5′ terminus hybridizes with signal probe labeled with Methylene Blue (MB). On increasing the concentration of target DNA, hybridization between signal probe and target DNA results in the formation of three different DNA sequences that form a supersandwich structure. The signal intensity of MB improves distinctly with increasing concentrations of target DNA in the sample solution. The assembling process on the surface of the electrode was studied by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Differential pulse voltammetry (DPV) was used to monitor the hybridization event by measuring the changes in the peak current for MB. Under optimal conditions, the peak currents in DPV for MB linearly increase with the logarithm of target DNA concentration in the range from 0.1 μM to1.0 fM, with a detection limit of 0.35 fM (at an signal/noise ratio of 3). This biosensor exhibits good selectivity, even over single-base mismatched target DNA.

DNA sensing on glassy carbon electrodes by using hemin as the electrochemical hybridization label

The electrochemical behavior of hemin, an iron complex of porphyrin, on binding to DNA at a glassy carbon electrode (GCE) and in solution, is described. Hemin, which interacts with covalently immobilized calf thymus DNA, was detected by use of a bare GCE, a double-stranded DNA-modified GCE (dsDNA-modified GCE), and a single-stranded DNA-modified GCE (ssDNA-modified GCE), in combination with differential pulse voltammetry (DPV). The structural conformation of DNA was determined from changes in the voltammetric signals acquired on reduction of hemin. As a result of its large steric structure and anionic substitution on its porphyrin plane, hemin intercalates between the base pairs of dsDNA. A scan-rate study for hemin and the dsDNA-hemin complex were also performed to determine the electrochemical behavior of the complex. The partition coefficient was obtained from the peak currents measured when different concentrations of hemin were in the presence of dsDNA. By observing the oxidation signals of guanine, damage to DNA after reaction with hemin at the GCE surface was also detected. The electrochemical detection of hybridization between the covalently immobilized probe and its target sequence was detected by use of hemin. These results demonstrate the use of DNA biosensors in conjunction with hemin for electrochemical detection of hybridization and damage to DNA.     

Reaction of Cd(II)-Morin with dsDNA for biosensing of ssDNA oligomers with complementary, GCE-immobilized ssDNA

In this work, the complex cadmium(II)-morin was synthesized and its interaction with double-stranded salmon sperm DNA was studied by electrochemical methods on glassy carbon electrode (GCE). It was shown that Cd(II)-Morin with high electrochemical activity can intercalate into the double-helix DNA, and the binding stoichiometry and equilibrium dissociation constant according to the Hill model for cooperative binding were calculated to be 1.761 and 2.5 x 10(-5) M, respectively. Using Cd(II)-Morin as a novel hybridization indicator, the hybridization between the probe and its complementary and mismatched sequence was investigated by differential pulse voltammetry (DPV), which was to access the selectivity of the developed electrochemical DNA biosensor. The complementary target ssDNA could be quantified over the range from 2.69 x 10(-8) M to 9.16 x 10(-7) M with a linear correlation of 0.9971 and a detection limit of 9.30 x 10(-9) M. These results demonstrated that the Cd(II)-Morin indicator provides great promise for the rapid and selective measurement of the target DNA.     

DNA sensor based on an Escherichia coli lac Z gene probe immobilization at self-assembled monolayers-modified gold electrodes

A novel approach to construct an electrochemical DNA sensor based on immobilization of a 25 base single-stranded probe, specific to E. coli lac Z gene, onto a gold disk electrode is described. The capture probe is covalently attached using a self-assembled monolayer of 3,3′-dithiodipropionic acid di(N-succinimidyl ester) (DTSP) and mercaptohexanol (MCH) as spacer. Hybridization of the immobilized probe with the target DNA at the electrode surface was monitored by square wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. Variables involved in the sensor performance, such as the DTSP concentration in the modification solution, the self-assembled monolayers (SAM) formation time, the DNA probe drying time atop the electrode surface and the amount of probe immobilized, were optimized.

A good stability of the single- and double-stranded oligonucleotides immobilized on the DTSP-modified electrode was demonstrated, and a target DNA detection limit of 45 nM was achieved without signal amplification. Hybridization specificity was checked with non-complementary and mismatch oligonucleotides. A single-base mismatch oligonucleotide gave a hybridization response only 7 ± 3%, higher than the signal obtained for the capture probe before hybridization. The possibility of reusing the electrochemical genosensor was also tested.     

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

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

基于直立碳纳米管大面积金粒子的DNA生物传感器用于早幼粒白血病/维甲酸受体α融合基因检测

基于直立碳纳米管上的大面积金粒子构建了新型的电化学DNA生物传感器,用于急性早幼粒细胞白血病PML/RARα融合基因的检测。首先在直立碳纳米管电极表面溅射金粒子,采用自组装方法将巯基修饰的单链DNA固定到电极上,将氨基修饰的单链DNA和羧基化的CdTe量子点通过酰胺缩合反应生成CdTe修饰的DNA探针,通过与目标DNA的双杂交反应形成三明治结构,利用差分脉冲阳极溶出伏安法检测电极表面捕获的CdTe量子点,从而对DNA进行定量分析。结果表明,电极上Cd2+峰电流与目标DNA浓度(1.0×10-12~1.0×10-8 mol/L)的对数值呈线性关系,线性方程为ipa(μA)=1.626+0.132lgC(mol/L)(R=0.996),检出限为4.0×10-13 mol/L(3σ)。传感器表现出良好的重现性和稳定性。     

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

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

Cadmium sulfide nanocluster-based electrochemical stripping detection of DNA hybridization

A novel, sensitive electrochemical DNA hybridization detection assay, using cadmium sulfide (CdS) nanoclusters as the oligonucleotide labeling tag, is described. The assay relies on the hybridization of the target DNA with the CdS nanocluster oligonucleotide DNA probe, followed by the dissolution of the CdS nanoclusters anchored on the hybrids and the indirect determination of the dissolved cadmium ions by sensitive anodic stripping voltammetry (ASV) at a mercury-coated glassy carbon electrode (GCE). The results showed that only a complementary sequence could form a double-stranded dsDNA-CdS with the DNA probe and give an obvious electrochemical response. A three-base mismatch sequence and non-complementary sequence had negligible response. The combination of the large number of cadmium ions released from each dsDNA hybrid with the remarkable sensitivity of the electrochemical stripping analysis for cadmium at mercury-film GCE allows detection at levels as low as 0.2 pmol L(-1) of the complementary sequence of DNA.     

Label-free electrochemical detection of Avian Influenza Virus genotype utilizing multi-walled carbon nanotubes–cobalt phthalocyanine–PAMAM nanocomposite modified glassy carbon electrode

A novel DNA electrochemical biosensor for label-free determination of DNA sequence related to the Avian Influenza Virus (AIV) genotype was demonstrated in this paper. First, the multi-walled carbon nanotubes–cobalt phthalocyanine (MWNTs–CoPc) nanocomposite and poly (amidoamine) (PAMAM) dendrimer (generation 4.0) were modified on the glassy carbon electrode (GCE) sequentially. Then, DNA probes were successfully immobilized on the modified electrode with G4 PAMAM dendrimer acting as the coupling agent. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement based on the oxidation signals of guanine without any external labels. Under the optimal conditions, the difference in guanine oxidation signal of the probe modified GCE in the absence and presence of complementary target (ΔI_p) was linear with the logarithmic value of the complementary target concentration from 0.01 to 500 ng/ml with a correlation coefficient of 0.998 and a detection limit of 1.0 pg/ml.     

基于硫化镉纳米团簇标记DNA电化学传感的研究

合成了表面具有自由羧基的硫化镉纳米团簇，以乙基-（3-二甲基丙基）碳二 亚胺盐酸盐为偶联活化剂，将其标记于人工合成的5'端氨基修饰的寡聚核苷酸片段 上，制备成CdS纳米团簇标记DNA探针，该寡聚核苷酸片段与大肠杆菌肠毒素基因相 关。在一定的条件下，使基与固定晨玻碳电极表面的待测DNA序列进行杂交反应， 利用阳极溶出示差脉冲伏安法（ASDPV）间接测定Cd的量，实现对互补、非互补 DNA片段的识别和电化学检测，从而对大肠杆菌肠毒素基因片段识别和检测。     

Hybridization biosensor using 2,9-dimethyl-1,10-phenantroline cobalt as electrochemical indicator for detection of hepatitis B virus DNA

A label-free biosensor for the detection of oligonucleotides related to hepatitis B virus sequence via the interactions of DNA with redox-active complex, 2,9-dimethyl-1,10-phenantroline cobalt [Co(dmp)(H2O)(NO3)2] is described. The study was carried out by the hybridization of 21-mer probe DNA modified on glassy carbon electrode (GCE) with target DNA, and [Co(dmp)(H2O)(NO3)2] whose sizes are comparable to those of the small groove of native double-helix DNA was used as an electrochemical indicator. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the [Co(dmp)(H2O)(NO3)2] was active. Under the optimum conditions, the electrical signal had a linear relationship with the concentration of target DNA ranging from 3.96 x 10(-7) to 1.32 x 10(-6) M, and the detection limit was 1.94 x 10(-8) M (S/N=3). The biosensor has good selectivity by detecting the three-base mismatch sequence ssDNA.     

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.     

Detection of Human Interleukine‐2 Gene Using a Label‐Free Electrochemical DNA Hybridization Biosensor on the Basis of a Non‐Inosine Substituted Probe

The human interleukine‐2 gene (hIL‐2) is detected with a label‐free DNA hybridization biosensor using a non‐inosine substituted probe. The sensor relies on the immobilization of a 20‐mer antisense single strand oligonucleotide (chIL‐2) related to the human interleukine‐2 gene on the pencil graphite electrode (PGE) as a probe. The guanine oxidation signal was monitored using anodic differential pulse voltammetry (ADPV). The electrochemical pretreatment of the polished PGE at 1.80 V for 5 min is suggested. Then, 5 min immobilization at 0.50 V was found as the optimum condition for immobilization of the probe. The electrochemical detection of hybridization between chIL‐2 and hIL‐2 as a target was accomplished. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Diagnostic performance of the biosensor is described and the detection limit is found 36 pg/μL.     

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

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

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.