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Plasmid HIV p24 Gene Detection on Mercury Film Electrode using Osmium Labelling

According to the Joint United Nations Programme on HIV and AIDS, more than 35 million persons live with the human immunodeficiency virus (HIV), which causes immune deficiency disease (AIDS). It was estimated that since the start of the epidemics about 36 million people have died of the AIDS-related illnesses. Although the number of newly infected people is decreasing and was about 2.3 million in 2012, it still remains serious issue. A lot of methods has been developed for detection of HIV including enzyme immunoassay [1], enzyme-linked immunosorbent assay (ELISA) [2] and western blot test [3]. These antibody-based methods applied on blood samples are time-consuming, have long detection “window phase” (3 weeks to 6 months) and requires highly-trained workers, however, these are still gold standard for HIV detection. Polymerase chain reaction (PCR) based techniques have been also utilised for HIV detection and exhibited high sensitivity, but these are limited in distinguishing of single base mutations [4]. Electrochemical sensing is able to resolve these limitations and offers simple, portable, sensitive, inexpensive detection and it is useful in the detection of early HIV infection because of direct molecular recognition of HIV and its components [5]. Electrochemical detection of HIV protease, an enzyme essential to the assembly and maturation of HIV, based on the enzymatic cleavage of protease substrate from the gold electrode was suggested by Esseghaier et al. [6]. HIV reverse transcriptase and HIV p24, which appears at the earlier stage of HIV infection than antibodies, became another target molecule used for HIV detection [5,7,8]. There is also interest in the detection of short DNA sequences related to the HIV [9-11].

In this study, we designed a simple method for electrochemical detection of p24 gene. Osmium tetroxide complex with 2,2'-bipyridine (OsO4(bpy)) has been often used as a versatile chemical DNA probe [12-14]. We utilized the ability of OsO4(bpy) to react with pyrimidine moieties (mostly thymine) in single stranded DNA and created the electroactive oligonucleotide probe complementary to the p24 sequence [15]. The DNA-OsO4(bpy) adduct produces a catalytic signal (about -1.2 V) at mercury electrode and other smaller signals at less negative potentials [12]. Moreover, we used the oligonucleotide probe complementary to the 5’ end of p24 sequence bounded to a gold modified magnetic particle (AuMPs) via thiol group, which enables us to separate the anchored single stranded gene from the solution of non-targeted DNA and other molecules. The created construct was adsorbed at mercury surface of the mercury film (MFE) on glassy carbon electrode (GCE) [16]. The signal of OsO4(bpy) reporter molecule was analysed by differential pulse voltammetry (DPV).

Práce je spojená s projektem NanoBioMetalNet CZ.1.07/2.4.00/31.0023

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