The detection of HBV DNA with gold nanoparticle gene probes

Abstract

Objective Gold nanoparticle Hepatitis B virus(HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Methods Alkanethiol modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA gene probes, through covalent binding of Au-S. By using a fluorescence-based method, the number of thiolderivatized, single-stranded oligonucleotides and their hybridization efficiency with complementary oligonucleotides in solution was determined. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotides serving as detection probes, and oligonucleotides im-mobilized on a nylon membrane surface acting as capturing probes, HBV DNA was detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. The modified nanoparticle HBV DNA gene probes were also used to detect the HBV DNA extracted from serum in patients with hepatitis B. Results Compared with bare Au nanoparticles, oligonucleotide modified nanoparticles had a higher stability in NaCl solution or under high temperature environment and the absorbance peak of modified Au nanoparticles shifted from 520 nm to 524 nm. For Au nanoparticles, the maximal oligonucleotide surface coverage of hexaethiol 30-mer oligonucleotide was(132 ± 10) oligonucleotides per nanoparticle, and the percentage of hybridization strands on nanoparticles was(22 ± 3%). Based on a two-probe sandwich hybridization/nanoparticle amplification/silver staining enhancement method, Au nanoparticle gene probes could detect as low as 10 −11 mol/L composite HBV DNA molecules on a nylon membrane and the PCR products of HBV DNA visually. As made evident by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules in liquid. Conclusion Our results showed that successfully prepared Au nanoparticle HBV DNA gene probes could be used to detect HBV DNA directly. The detection-visuallized method has many advantages, including high sensitivity, simple operation and low cost. This technique has potential applications in many fields, especially in multi-gene detection chips.