RESUMO
In the present study, a nanoporous gold platform was applied for the amplified detection of Hepatitis B virus (HBV) by an electrochemical DNA biosensor. Ferrocene as a redox reporter was covalently attached to the DNA probe and its electrochemical signal was recorded as the biosensor response. For real samples, DNA was firstly extracted from blood of patients and then amplified by polymerase chain reaction (PCR) for 5cycles. Sensitivity of this biosensor was enhanced by using nanoporous gold electrode, therefore this sensor can discriminate the genome of HBV in real sample with low PCR cycles. By this strategy and signal amplification using nanoporous platform and covalently attached electroactive label, the biosensor can distinguish between healthy and HBV patients with limited PCR cycles. Moreover, the errors of PCR with large cycles can be disregarded. A linear dynamic range of 0.4 to 10nmol of mutant DNA was achieved, with reliable reproducibility (RSD) 8.9%.
Assuntos
Técnicas Biossensoriais/métodos , DNA Viral/análise , Ouro/química , Vírus da Hepatite B/isolamento & purificação , Nanoporos , Sequência de Bases , DNA Viral/genética , Eletroquímica , Eletrodos , Compostos Ferrosos/química , Vírus da Hepatite B/genética , Metalocenos , Mutação , Reação em Cadeia da Polimerase , Propriedades de SuperfícieRESUMO
The application of a nanoporous gold electrode (NPGE) in the fabrication of an electrochemical sensing system for the detection of single base mismatches (SBMs) using ferrocene-modified DNA probe has been investigated in the present manuscript. Ferrocene carboxylic acid is covalently attached to the amino-modified probe using EDC/NHS chemistry. By covalent attachment of the redox reporter molecules on the top of DNA, the direct oxidation of the ferrocene on the electrode surface is avoided. On the other hand, the electrochemical signals are amplified by anodizing the electrode surface and converting it to nanoporous form. By improving the sensitivity of the biosensor, the different SBMs including the thermodynamically stable G-A and G-T mismatches, can be easily distinguished. In this research, NPGE was prepared by anodization and chemical reduction of Au surface and used for signal amplification. Nanoporous electrode enhances the sensitivity of DNA biosensor and makes it capable to detect complementary target DNA in sub-nanomole scales.
Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/química , DNA/genética , Compostos Ferrosos/química , Nanoestruturas/química , Hibridização de Ácido Nucleico/métodos , Pareamento Incorreto de Bases , Sequência de Bases , DNA/química , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Metalocenos , Porosidade , Sensibilidade e Especificidade , TermodinâmicaRESUMO
In this manuscript, a strategy for the amplification of the responses of an electrochemical DNA hybridization biosensor using silver nanoparticles (Ag-NPs) as redox reporters and its capability for the detection of a single base mismatches (SBM) including thermodynamically stable ones, is described. In this assay, Ag-NPs are immobilized on the top of recognition layer and their oxidation signals are followed. Differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) are used to monitoring the electrode response. Only for complementary target sequence, electron can transfer between Electrode surface and nanoparticles via DNA and Ag-NPs can be oxidized. Therefore this DNA biosensor could differentiate between complementary target and one containing either SBM or thermodynamically stable G-A and G-T targets through oxidation signal of Ag-NPs. This biosensor is able to detect SBM by overcome the direct electron transfer of redox reporter with electrode surface and positioning of it before the mismatch position.
Assuntos
Pareamento Incorreto de Bases , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Espectroscopia Dielétrica , Técnicas Eletroquímicas , Nanopartículas Metálicas , Nanotecnologia , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/genética , Oxirredução , PrataRESUMO
Liquid-liquid-liquid microextraction (LLLME) in combination with HPLC and UV detection has been used as a sensitive method for the determination of ephedrine in urine samples. Extraction process was performed in a homemade total glass vial without using a Teflon ring, usually employed. Ephedrine was first extracted from 3.5 mL of urine sample (pH 12) into a microfilm of toluene/benzene (50:50). The analyte was subsequently back extracted into an acidic microdrop solution (pH 2) suspended in the organic phase. The extract was then injected into the HPLC system directly. An enrichment factor of 137 along with a good sample clean-up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 0.01-50 mg/L with regression coefficient corresponding to 0.998. The LODs and LOQs, based on a S/N of 3 and 10, were 5 and 10 microg/L, respectively. The method was eventually applied for the determination of ephedrine in urine sample after oral administration of 5 mg single dose of drug.
