Preparation Of Nanoparticle Gene Probes And Their Application In Detection Of Hepatitis Virus

Sequence-specific methods for detecting polynucleotides are critical to the diagnosis of genetic and pathogenic diseases. Most detection systems make use of the hybridization of a target polynucleotide with oligo- or polynucleotide probes containing covalently linked reporter groups, which include radioactive labels, fluorescent labels and chemilumine- scence schemes, etc. Each of these strategies has advantages and disadvantages and no single method has gained supremacy. During the last decade, there has been an increasing interest in using nanoparticles in DNA detection. In the present paper, we prepared gold or gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes and using these probes we established a methology to detect HBV DNA molecule extracted from hepatitis B patient on nylon membrane by blot-hybridization or in liquid media by TEM. We have examined the feasibility of the detection of HBV DNA with gold-coated iron oxide nanoparticles and magnetic separator. And we further established a simple and rapid visual method based on either DNA microarray or TEM with nanogold supported probes for detecting HBV and HCV simultaneously.Au nanoparticles were produced via citrate reduction of tetrachloroauric acid (HAuCl4). Superparamagnetic iron oxide nanoparticles (SPION) were produced with chemical precipitation. The coating of Au on magnetic nanoparticles was performed via citrate reduction of HAuCl4 on the surface of SPION that served as seeds. Alkanethiol modified oligonucleotide was bound with self-made Au and Fe₃O₄(core)/Au(shell)nanoparticles to form nanoparticle HBV DNA gene probes through covanlt binding of Au-S. With a fluorescence-based method, for Au nanoparticles and Fe₃O₄(core)/Au(shell) nanoparticles, the maximal surface coverage of hexaethiol 30-mer oligonucleotides was (132±10) and (120±8) oligonucleotides per nanoparticle, and the maximal percentage of hybridization strands on nanoparticles was (22±3)% and (14±2)%, respectively. Based on a two-probe sandwich hybridization/ nanoparticle amplification/ silver staining enhancement method, Au nanoparticle and Fe₃O₄(core)/Au(shell) nanoparticle gene probes could detect as low as 10-11mol/L and 10-10mol/L composite HBV DNA molecules on nylon membrane, respectively, and the PCR products of HBV DNA visually. As evidenced by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules.HBV DNA and HCV RNA extracted from serum in patients with HBV and HCV coinfection can be specifically amplified simultaneously in one system by multi-primer PCR. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotide serving as detection probe, and oligonucleotide immobilized on nylon membrane surface acting as capturing probe, HBV and HCV PCR products were detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. As evidenced by transmission electron microscopy, large network aggregates assembled by nanoparticles identifying HBV and HCV simultaneously in serum in patients with HBV and HCV coinfection were seen clearly.Our results suggested that DNA modified nanoparticle probe can be used to detect virus nucleic acid. The established oligonucleotide array for the detection of HBV and HCV coinfection is convenient and efficient with high specificity. 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. It provides a nationale for the measurement of other viruses coinfection. Transmission electron microscopy can detect HBV or HBV and HCV simultaneously in serum in patients. Detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.