logo ban ban logo

Není hanbou nic nevědět, ale je hanbou, nechtít se ničemu učit.

mendel

Výzkum

Paramagnetic Particles Isolation of Influenza Oligonucleotide Labelled with CdS QDs

Influenza is an infectious disease caused by RNA viruses of the family Orthomyxoviridae. Influenza viruses can be found in the aerosols formed by sneezing and coughing and may cause acute infection of upper respiratory tract [1, 2]. Vaccine against influenza exists, however, it is effective for one year against selected subtypes. This is due to mutational changes in the structure of the virus, thus the reuse of the same vaccine in the following year has not a protected effect [3, 4]. It is clear that flu viruses are very susceptible to change antigenic equipment by drift and shift. These changes can easily create a new subtype, as in the case highly pathogenic avian influenza. Due to this, there is great research search for rapid and more reliable detection methods [1, 5, 6].

Nucleic acid hybridization on solid bases is widely used in biotechnology for the isolation of target DNA. Among numerous methods used in this field, many probe-target DNA assays use oligo-conjugated magnetic particles (MPs) for the isolation of nucleic acids of interest [7, 8]. The advantage of magnetic separation is in the possibility of modifying the surface of MPs, and thus the elimination of interfering adsorption of non-target biomolecules [9-11], because MPs are able to respond to external magnetic field, which is used for efficient separation. Both magnetic separation and modification of MPs surface are beneficial for DNA isolation [12-14]. Isolated DNA is usually detected by hybridization process using different materials such as biotin-avidin, protein (substrate)-enzyme, antigen-antibody and fluorescent dyes or other optical active substances as quantum dots [15, 16], because detection of hybridized DNA molecule is important for diagnosis of the presence of specific virus [17]. Application of optical detection methods on fluorescently active labels is widespread [18-20]. Recent advantages in synthesis and surface modifications of quantum dots (QDs) as new type of fluorescently active labels have resulted in various applications of these particles [21-25]. Wang et al. describe multi-target electrochemical DNA detection based on the use of different quantum dots tags. Multi-target electrical detection scheme developed by these authors incorporates the high sensitivity and selectivity advantages of nanoparticle-based electrical assays [17]. Based on the published promising results, the aim of this study was to isolate and detect of quantum dots labelled influenza oligonucleotide-SH (ODN-SH) H5N1. For our application, optical properties of QDs are beneficial, but the main interest is paid to electroactivity of metal part of QDs. Electrochemical methods are suitable not only for the detection of metal parts of the QDs, but also for detection of target oligonucleotide. Currently, electrochemistry is considered as a one of the most sensitive methods for both nucleic acids and metals determination [26-29], which is used in this study.

Conclusion
Nowadays, research is directed to finding methods for simultaneously detection of multiple DNA targets. The electrochemical coding technology is thus expected to open new opportunities for DNA diagnostics [17, 52]. In this study, it was suggested and optimized method for automated isolation of Cd labelled influenza oligonucleotide using automated pipetting station. It was observed the effect of hybridization temperature, hybridization time and concentration of ODN-SH-Cd complex on CA and Cd peak height, which were determined electrochemically using square wave voltammetry and differential pulse voltammetry. Our system can be used as electroanalytical tool for rapid detection of target oligonucleotide based on isolation by probe conjugated MPs.

Podpořeno projekty: CEITEC CZ.1.05/1.1.00/02.0068

Zemědělská 1/1665
613 00 Brno
Budova D		Tel.: +420 545 133 350
Fax.: +420 545 212 044