Electrochemical behavior and detection of hepatitis B virus DNA PCR production at gold electrode

Sequence-known short-stranded hepatitis B virus (HBV) DNA fragment (181 bps) was obtained by PCR method. The strategy for its electrochemical detection was designed by covalently immobilizing single-stranded HBV DNA on gold electrode surface via carboxylate ester as a linkage between 3′-hydroxy end of DNA and carboxyl group of thioglycolic acid (TGA) self-assembled monolayer. The hybridization reaction on surface was evidenced by electrochemical methods using ferrocenium hexafluorophosphate (FcPF₆) as an electroactive indicator. The interactions of Fc⁺ with single-stranded (ss) and double-stranded (ds) HBV DNA immobilized on TGA monolayer were studied. The difference between the responses of Fc⁺ at ss- and ds-DNA/Au electrodes suggested that this hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. AC impedance and XPS techniques have been employed to characterize the immobilization of ss-DNA on the gold surface.     

Electrochemical genosensors for biomedical applications based on gold nanoparticles

Two gold nanoparticles-based genomagnetic sensors designs for detection of DNA hybridization are described. Both assays are based on a magnetically induced direct electrochemical detection of gold tags on magnetic graphite-epoxy composite electrodes. The first design is a two strands assay format that consists of the hybridization between a capture DNA strand which is linked with paramagnetic beads and another DNA strand related to BRCA1 breast cancer gene used as a target which is coupled with streptavidin-gold nanoparticles. The second genomagnetic sensor design is a sandwich assay format with more application possibilities. A cystic fibrosis related DNA strand is used as a target and sandwiched between two complementary DNA probes: the first one linked with paramagnetic beads and a second one modified with gold nanoparticles via biotin-streptavidin complexation reactions. The electrochemical detection of gold nanoparticles by differential pulse voltammetry was performed in both cases. The developed genomagnetic sensors provide a reliable discrimination against noncomplementary DNA as well against one and three-base mismatches. Optimization parameters affecting the hybridization and analytical performance of the developed genosensors are shown for genomagnetic assays of DNA sequences related with the breast cancer and cystic fibrosis genes.     

Electrochemical biosensor for the detection of cauliflower mosaic virus 35 S gene sequences using lead sulfide nanoparticles as oligonucleotide labels

Lead sulfide (PbS) nanoparticles were synthesized in aqueous solution and used as oligonucleotide labels for electrochemical detection of the 35 S promoter from cauliflower mosaic virus (CaMV) sequence. The PbS nanoparticles were modified with mercaptoacetic acid and could easily be linked with CaMV 35 S oligonucleotide probe. Target DNA sequences were covalently linked on a mercaptoacetic acid self-assembled gold electrode, and DNA hybridization of target DNA with probe DNA was completed on the electrode surface. PbS nanoparticles anchored on the hybrids were dissolved in the solution by oxidation of HNO₃ and detected using a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used to monitor the hybridization reaction. The CaMV 35 S target sequence was satisfactorily detected with the detection limit as 4.38 × 10⁻¹² mol/L (3σ). The established method extends nanoparticle-labeled electrochemical DNA analysis to specific sequences from genetically modified organisms with higher sensitivity and selectivity.     

Integration pattern of hepatitis B virus DNA sequences in human hepatoma cell lines.

Four human hepatoma cell lines established from primary hepatocellular carcinomas were examined for the presence of hepatitis B virus DNA sequences. Reassociation kinetic analysis indicated that the cell lines HEp-3B 217, HEp-3B 14, HEp-3B F1, and PLC/PRF/5 contained two, one, one, and four genome equivalents per cell, respectively. Southern blot hybridization analysis demonstrated that hepatitis B virus DNA was integrated into the cellular DNAs of these cell lines. Further liquid hybridization studies with 32P-labeled HincII restriction fragments of hepatitis B virus DNA established that DNA sequences from all regions of the HBV genome were represented in the integrated viral sequences. Although the three HEp-3B cell lines were derived from the same tumor, they differed significantly in their patterns of integration of hepatitis B virus DNA, the number of copies of viral DNA per cell, and their ability to produce the virus-coded surface antigen.     

Label-free electrochemical detection of short sequences related to the hepatitis B virus using 4,4'-diaminoazobenzene based on multiwalled carbon nanotube-modified GCE

A novel label-free electrochemical DNA biosensor based on 4,4'-diaminoazobenzene (4,4'-DAAB) and multiwalled carbon nanotube (MWNT)-modified glassy carbon electrode (GCE) for short DNA sequences related to the hepatitis B virus (HBV) hybridization detection was presented. Differential pulse voltammetry (DPV) was used to investigate hybridization event. The decrease in the peak current of 4,4'-DAAB was observed on hybridization of probe with the target. This electrochemical approach was sequence specific as indicated by the control experiments, in which no peak current change was observed when a noncomplementary DNA sequence was used. Numerous factors affecting the target hybridization were optimized to maximize the sensitivity. Under optimal conditions, this sensor showed a good calibration range between 7.94 x 10(-8) M and 1.58 x 10(-6) M, with HBV DNA sequence detection limit of 1.1 x 10(-8) M.     

Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences

Microarrays based on DNA-DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of KD=13+/-4 nM at 295 K.     

Identification of a polymeric β-cyclodextrin-binding peptide from a phage-displayed peptide library

In this work, a phage-displayed peptide library was applied to identification of β-cyclodextrin (CD)-binding peptide tag, capable of being combined to target peptides or proteins in a homogeneous way by established methods such as peptide synthesis and the recombinant DNA technique. Four enriched sequences were obtained after five rounds of biopanning against polymeric β-CD beads. One of the sequences showed high binding affinity to β-CD beads with a dissociation constant of approximately 7 × 10^–6 M. The β-CD-binding sequence was used for immobilization of a hepatitis C virus (HCV) antigenic peptide on β-CD beads. The functionalized β-CD beads were successfully used for immunoassay of anti-HCV antibody with a detection limit of 1 ng. These results demonstrate that the identified peptide sequence has the potential of being used as an affinity tag to β-CD-containing surfaces.     

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection.     

New DNA viruses identified in patients with acute viral infection syndrome

A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-kappa2-NN'] dibromicoppers and DNA

A new Cu(II) complex CuL(2)Br(2) (L = azino-di(5,6-azafluorene)-kappa(2)-NN') was synthesized, and a new method of electrochemical probe has been proposed for the determination of hepatitis B virus (HBV) based on its interaction with [CuL(2)](2+). This ligand, containing functional groups, as well as planar aromatic domains, is capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Emphasis has been placed on the elucidation of the nature of the interaction by electrochemical techniques. The electroactive [CuL(2)](2+) could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a probe DNA sequence from HBV onto glassy carbon electrode (GCE). After hybridization with the complementary target sequence, [CuL(2)](2+) was accumulated within the dsDNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range. Using this approach, complementary target sequences of HBV can be quantified over the range of 1.74 x 10(-9) to 3.45 x 10(-7) M, with a detection limit of 8.32 x 10(-10) M and a linear correlation coefficient of 0.9936.In addition, this approach is capable of detecting hybridization of complementary sequences containing one or three mismatched bases.     

Hybridization biosensor using di(2,2'-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3(')-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy)(2)Cl(2)](+) as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy)(2)Cl(2)](+) in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy)(2)Cl(2)](+) at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.     

Detection of hepatitis B virus DNA in mononuclear blood cells.

The Southern transfer hybridisation technique was used to test mononuclear blood cells for hepatitis B virus DNA. Viral DNA sequences were detected in mononuclear cells of 10 out of 16 patients with hepatitis B virus infection and in none of 21 normal controls. Blood contamination was excluded by the absence of hepatitis B virus DNA in the corresponding serum samples in all cases. Free monomeric hepatitis B virus DNA was found in three patients positive for hepatitis Be antigen (HBeAg) and one positive for anti-HBe, and integrated hepatitis B virus DNA was present in four patients positive for anti-HBe. In two other patients the small size of the samples did not allow a distinction between free and integrated viral DNA. The state of the virus in the mononuclear cells seemed to correlate with the HBeAg or anti-HBe state, as has been noted in the liver. These results indicate that hepatitis B virus may infect mononuclear blood cells, thereby expanding the tissue specificity of this agent beyond the liver, as has been reported for pancreatic, kidney, and skin tissue. They also suggest that hepatitis B virus infection of mononuclear cells might be related to immunological abnormalities observed in carriers of the virus.     

Digestion of restriction enzyme for the detection of single-base mismatch in DNA

DNA hybridization and enzymatic digestion for the detection of mutation was investigated on the gold nanoparticles-calf thymus DNA (AuNPs-ctDNA) modified glassy carbon electrode (GCE). The thiol modified probe oligonucleotides (SH-ssDNA) were assembled on the surface of AuNPs-ctDNA modified GCE. The electrochemical response of the electrode was measured by differential pulse voltammetry and cyclic voltammetry. Methylene blue (MB) was used as the electroactive indicator. AuNPs were then dispersed effectively on the GCE surface in the presence of ct-DNA. When hybridization occurred, a decrease in the signal of MB current was observed. The modified electrode was used for the detection of mutations during the enzymatic digestion reaction in DNA. During this reaction, an increase in the signal of MB current was observed. So, the modified SH-ssDNA had a higher electrochemical response on the AuNPs-ctDNA/GCE because of the strong affinity of MB for guanine residues in it. The electrochemical detection of restriction enzyme digestion can provide a simple and practical method for observing single-base mismatches that can help in distinguishing mismatch sequences of DNA from the complementary ones.     

Nucleotide sequence of a cloned woodchuck hepatitis virus genome: comparison with the hepatitis B virus sequence.

The complete nucleotide sequence of a woodchuck hepatitis virus genome cloned in Escherichia coli was determined by the method of Maxam and Gilbert. This sequence was found to be 3,308 nucleotides long. Potential ATG initiator triplets and nonsense codons were identified and used to locate regions with a substantial coding capacity. A striking similarity was observed between the organization of human hepatitis B virus and woodchuck hepatitis virus. Nucleotide sequences of these open regions in the woodchuck virus were compared with corresponding regions present in hepatitis B virus. This allowed the location of four viral genes on the L strand and indicated the absence of protein coded by the S strand. Evolution rates of the various parts of the genome as well as of the four different proteins coded by hepatitis B virus and woodchuck hepatitis virus were compared. These results indicated that: (i) the core protein has evolved slightly less rapidly than the other proteins; and (ii) when a region of DNA codes for two different proteins, there is less freedom for the DNA to evolve and, moreover, one of the proteins can evolve more rapidly than the other. A hairpin structure, very well conserved in the two genomes, was located in the only region devoid of coding function, suggesting the location of the origin of replication of the viral DNA.     

Facile fabrication of magnetic gold electrode for magnetic beads-based electrochemical immunoassay: application to the diagnosis of Japanese encephalitis virus

A novel magnetic beads-based electrochemical immunoassay strategy has been developed for the detection of Japanese encephalitis virus (JEV). The magnetic gold electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. The proteins (e.g., antibodies or immunocomplexes) attached on the surface of magnetic beads were found to induce a significant decline in their electric conductivity. Multiwalled carbon nanotubes were introduced to improve sensitivity of the assay. The envelope (E) protein, a major immunogenic protein of JEV, was utilized to optimize the assay parameters. Under the optimal conditions, the linear response range of E protein was 0.84 to 11,200 ng/mL with a detection limit of 0.56 ng/mL. When applied for detection of JEV, the proposed method generated a linear response range between 2×10(3) and 5×10(5) PFU/mL. The detection limit for JEV was 2.0×10(3) PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. This method was also successfully applied to detect JEV in clinical specimens.     

Hepatitis B virus DNA and e antigen in serum from blood donors in the United Kingdom positive for hepatitis B surface antigen.

Serum samples from 214 blood donors in the United Kingdom who were carriers of hepatitis B surface antigen (HBsAg) were examined for hepatitis B virus deoxyribonucleic acid (DNA) by DNA:DNA hybridisation and for hepatitis B e antigen (HBeAg) and its antibody. One fifth of the donors carried infectious virus in their circulation. The presence of hepatitis B virus DNA correlated well with that of HBeAg, although hepatitis B virus DNA was found in five serum samples that were negative for HBeAg. It is concluded that analysis of serum samples for hepatitis B virus DNA by hybridisation should be the method of choice for determining whether carriers of HBsAg are infectious.     

乙肝治疗耐药基因突变的焦磷酸测序技术诊断

目的:建立焦磷酸测序技术检测拉米夫定和阿德福韦酯治疗乙肝所致乙肝病毒基因耐药突变的定量检测方法,为临床乙肝耐药诊断和治疗提供依据。方法:针对乙肝病毒DNA聚合酶基因序列上4个常见基因突变位点的6种突变形式,分别克隆构建野生型和突变型质粒作为标准品,应用生物信息学手段设计目标基因通用PCR引物和各突变点的焦磷酸测序引物,建立焦磷酸测序的突变检测方法。对接受拉米夫定、阿德福韦酯治疗的慢性乙型肝炎患者血清标本进行检测。结果:构建了乙肝病毒四种常见耐药性突变的标准株和变异株克隆,建立了分别或同时检测拉米夫定、阿德福韦酯耐药突变的焦磷酸测序方法,对68例临床耐药或疑似耐药的患者血清标本进行检测,双脱氧测序验证,检出拉米夫定耐药突变32例,阿德福韦酯耐药突变5例,其中焦磷酸测序检出20例为混合突变,而双脱氧测序显示为6例。结论:成功建立了焦磷酸测序定量检测拉米夫定、阿德福韦酯耐药基因突变的方法,构建了乙肝病毒耐药基因突变的标准质粒,为临床动态监测乙肝病毒变异病毒株、指导合理用药奠定了基础。     

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.