Label-Free Electrochemical Hybridization Genosensor for the Detection of Hepatitis B Virus Genotype on the Development of Lamivudine Resistance

Abstract

The resistance analysis related to the hepatitis B virus (HBV) genotyping and treatment procured key information for the study of infected patients. The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences related to the HBV genotype on the development of lamuvidine resistance by monitoring the oxidation signal of guanine. This new technique not only provides a rapid, cost-effective, simple analysis but also gives information concerning both genotyping and lamivudine resistance. Synthetic single-stranded oligonucleotides ("probe") including YMDD (HBV wild type) YVDD, or YIDD (mutations in the YMDD) variants have been immobilized onto pencil graphite electrodes with the adsorption at a controlled potential. The probes were hybridized with different concentrations of their complementary ("target") sequences such as synthetic complementary sequences, clonned PCR products, or real PCR samples. The formed synthetic hybrids on the electrode surface were evaluated by a differential pulse voltammetry technique using a label-free detection method. The oxidation signal of guanine was observed as a result of the specific hybridization between the probes and their synthetic targets and specific PCR products. The response of the hybridization of the probes with their single-base mismatch oligonucleotides at PGE was also detected. Control experiments using the noncomplementary oligonucleotides were performed to determine whether the DNA genosensor responds selectively. Numerous factors, affecting the probe immobilization, target hybridization, and nonspecific binding events, were optimized to maximize the sensitivity and reduce the assay time. Under the optimum conditions, 457 fmol/mL was found as the detection limit for target DNA. With the help of the appearance of the guanine signal, the new protocol is based on the electrochemical detection of HBV genotype for the development of lamuvidine resistance for the first time. Features of this protocol are discussed and optimized.

Detection of Sequence-Specific Gene by Multi-Channel Electrochemical DNA Chips

“…Using potassium ferricyanide as electrochemical indicator, the device could be used to recognize reliably not only synthetic sequence-specific DNA but also LAMP products. Though the detection limit of target DNA is larger than that obtained at solo DNA sensor as outlined in the references, [7][8][9][10]32 this can be developed by using some special indicators, such as Hoechst 33258, 4,33 [Ru(NH 3 ) 6 ] 3+ 20 and so on. In fact in this study we focus on many other attractive advantages of the electrochemical chip.…”

“…5,6 Therefore, a lot of achievements have been obtained in the detection of DNA by electrochemical methods. 5,[7][8][9][10][11][12][13] Over the past years, our research groups have also focused on the development of electrochemical DNA hybridization biosensors.…”

Synthesis and voltammetric detection of 1H-benzimidazole derivatives on the interaction with DNA

“…DGE's were pretreated by applying 1.4 V for 60 seconds in phosphate buffer solution (pH 7.4) for overoxidation. Calf thymus ds and ssDNA's were immobilized onto DGE surfaces via adsorption (34). Accumulation of 1H-benzimdazole derivates were performed at DNA modified DGE surfaces with various concentrations of their solutions by applying open circuit mode for 5 minutes (27,28).…”

Heat-transfer-based detection of SNPs in the PAH gene of PKU patients

“…This type of biosensor is often based on the direct oxidation or redox labeling of DNA, or uses intercalating or soluble redox indicators. [15][16][17][18] Single-nucleotide polymorphism (SNP) discrimination with oxidation-based biosensors is based on the difference in faradic charge or electron transfer through the π-stacking of double-stranded DNA (dsDNA) upon hybridization. The reason for this change is that π-stacking of DNA bases along the helix is interrupted in a mismatch, or heteroduplex, resulting in a decrease of electron-transfer efficiency.…”