| Background and objectivesAcquired Immune Deficiency Syndrome (AIDS) is a serious infectious immunodeficiency disease caused by the human immunodeficiency virus infection. It is epidemic in some areas serious and specific populations, and gradually spread to the general population from high risk groups.Prevention and control situation is very serious. According to the different serological respond, HIV can be divided into two types of HIV-1and HIV-2.HIV-2limited to some areas of West Africa, while HIV-1popular in large parts of the world (including China). Since there is no effective treatment, prevention of infection as the primary means of controlling HIV spread. The laboratory diagnosis of HIV infection is an important part of AIDS prevention and control work, and therefore the establishment of sensitive and practical detection methods for monitoring, diagnostics, blood screening, it is particularly important to control the AIDS epidemic.The most common clinical application of HIV blood screening method is based on the color reaction in ELISA, it is mainly detected in the blood protein (antigen or antibody).Due to the limited sensitivity of the chromogenic reaction and existence of "window period", the ELISA method obviously does not meet the needs of the present stage of AIDS early screening.The viral nucleic acid after HIV infection, the earliest detection of indicators, with the application of molecular biology techniques, nucleic acid detection method has become an important means of HIV infection early detection. Commonly used method for the determination of the reverse transcription-PCR experiments (RT-PCR), nucleic acid amplification test (NASBA), branched DNA hybridization experiments (bDNA). Testing instruments, reagents, expensive and complicated to operate, operator requirements, it is difficult to promote in the general laboratory and do not apply to the rapid detection of a large number of patients, so the same is not suitable for a wide range of clinical applications. Therefore, it is necessary to improve the detection sensitivity, specificity, and to shorten the window period, but also simple, rapid and cost reduction has become the technical requirements of the development of HIV nucleic acid detection and direction.Biosensors has become a hot research field of life sciences.It uses various types of transducers to converted to electricity, sound, light and other physical signals can be detected, in order to achieve the detection of target biomolecules in the sample. Nucleic acid hybridization-based gene sensor is one of the fastest growing nucleic acid detection method for the detection of nucleic acid molecules provides a new worthy of the trust is quick and cheap way. For the detection of gene sensor technologies, including fluorescence techniques, quartz crystal microbalance, electrochemiluminescence surface plasmon resonance spectroscopy and electrochemical methods. In these methods, electrochemical methods because of less environmental interference signal measurement is simple, low cost instrument caused widespread concern.Electrochemical gene sensors based on nucleic acid sensitive components or detection of an object, signals generated in the specific recognition of nucleic acid molecules through an electrochemical transducer into electrical signals, in order to achieve qualitative or quantitative detection of nucleic acids. Recognition element (nucleic acid probe) and the electrochemical transducer (working electrode) is the two major components; one of the nucleic acid probe the nature of the recognition specificity of the sensor, the electronic conductivity of the working electrode to determine its sensitivity. Combine the high sensitivity of the detection process of the electrochemical method and a high degree of sequence specificity of nucleic acid hybridization, the electrochemical gene sensor is expected to become a simple, rapid, accurate and inexpensive means of genetic determination, and in line with the HIV nucleic acid detection technology requirements of the development and direction of HIV early screening.In recent years, a variety of HIV electrochemical gene sensors have been reported, mostly from the traditional micro-electrode (such as micro-graphite electrode, gold splash electrodes) on the fixed and HIV nucleic acid fragments complementary single-stranded linear DNA probe molecules build from. Traditional electrode limited conduction properties, and the detection specificity and sensitivity of linear single-stranded DNA probe molecules is not good.If you want electrochemical gene sensor for composition of complex clinical blood sample testing, you need to improve the binding affinity of the recognition element, to identify the specificity and sensitivity, and further optimization of the electrode conduction properties, and to establish a highly specific, ultra-sensitive electrical chemical genetic sensing detection system, and lay a solid foundation for the application of electrochemical sensors in clinical HIV testing.The structure of the recognition element is an important factor to affect its recognition specificity and sensitivity. In addition to the traditional linear structure, the most common probe structure was undoubtedly stem ring structure. People are most familiar with the stem circular probe is a molecular beacon.However, due to the optical detection system is bulky, expensive, difficult to maintain, the need for specialized personnel to operate, MBs have been introduced to the construction of the electrochemical method, known as electrochemical molecular beacons (E-MBs). In order to extend the MBs with high signal to noise ratio in the solution-free separation of the advantages of real-time monitoring, we intend to build a similar E-dimer-monomer fluorescence detection switch detection mode MBs to obtain such new E-MBs the key is to find a suitable electrochemical active molecules, marked on both ends of the sequence of the E-MBs, with the conformational change of the E-MBs with different electrochemical properties.Carminic acid (CA) that is an anthraquinone compound, the monomer form of CA due to the presence of two phenol groups Kuining Ji showed electrochemical activity (ON state), while the dimeric form of power chemical activity will disappear (OFF state), the presence or absence of the CA molecule electrochemical signal only depends on its existence. CA that is, we are able to the development of analog fluorescence MB of electrochemical active molecules to be built new electrochemical molecular letter nominal electrochemical activity-non-active switch beacon molecules of its two side are marked with the CA, it can also be called carminic acid-molecular beacons (CAs-MB).The electronic conductivity of the working electrode determines the sensitivity of the sensor. Modification of electrode is the main way to improve the performance of electrode conductivity. In recent years, the Modification of electrode mainly rely on nano-materials.Graphene is two-dimensional materials with only one carbon atom thick, it is currently the world’s thinnest but the most hard nano-materials,with low resistivity, which makes graphene and its derivatives for its excellent conductive properties, large surface area and good adsorption capacity.lt has been used in super-capacitors, field-effect transistors and chemical sensors. Nafion is a cation exchanger with excellent chemical stability, the increase of graphene soluble, stable and fixed on the electrode surface, the formation of the complex film of Nafion-of graphene. Thereby enabling the modification of graphene is more uniform, better conductive properties. This Nafion-graphene complex membrane modified screen printing microelectrode (SPCEs) constitute the ultra-sensitive electrochemical microelectrode:Nafion-graphene/SPCEs as the working electrode of the project. Combined with the new recognition element——CAs-MB, constructed for electrochemical gene sensing system of HIV-1, and its method of evaluation, and lay a solid electrochemical sensors in clinical HIV-1nucleic acid detection basis, but also for other types of infectious diseases, rapid nucleic acid diagnostic to provide a common platform.Around the above problem, the work of this thesis is divided into two parts:(1) The construction and confirmation of HIV-1electrochemically active-inactive switching molecular beacon.(2) The construction and evaluation of HIV-1electrochemical gene sensing system based on Nafion-graphene/SPCEs.Methods1. The construction and confirmation of HIV-1electrochemically active-inactive switching molecular beacon.After the synthesis and purification of CAs-MB target to HIV-1gag gene conserved sequence, cyclic voltammetry (CV) and Fourier transform infrared spectroscopy (FT-IR) were been used to verify its structure, differential pulse voltammetry (DPV) was been used to evaluate recognition function of CAs-MB. 2. The construction and evaluation of HIV-1electrochemical gene sensing system based on Nafion-graphene/SPCEs.In the first part, we were successed to build and validate CAs-MB against the conserved region of HIV-1gag gene. Graphene as the two-dimensional carbon nanomaterials have the advantages of conductivity performance, large specific surface area and adsorption capacity. Complex membrane made of Nafion-of graphene can increase the soluble of graphene, of,and stable characteristics of the electrode surface. This complex membrane modified screen printing microelectrode (SPCEs) constitute the ultra-sensitive electrochemical microelectrode: Nafion-graphene/SPCEs as the working electrode of the project. Combined with the new recognition element-CAs in the MB, constructed for electrochemical gene sensing system of HIV-1, and its method of evaluation.Results1, Changes in the monomer and CAs in the carminic acid (CA)-MB of the FT-IR diagram prove that CA has marked the end of MB, CV diagram to prove the existence of CAs-MB of the electrochemical activity of ON and OFF state. DPV results confirm that the electrochemical platform to achieve the target sequence-specific recognition, and can distinguish a single base difference.2, HIV-1electrochemical gene sensing system was successfully constructed based Nafion-graphene/SPCEs. CV and EIS results of Nafion-graphene/SPCEs in the5mM Fe (CN)63-/4-(1:1) solution contain0.1MKCl show:electicitic conduction efficiency Nafion-graphene/SPCEs as the working electrode significantly increased, and the DPV response signal of the CA monomer is much larger than the bare SPCE. Constructed on this basis, the value of40nmol/L～2.56μmol/L, within the purpose of concentration of nucleic acid sequence-based Nafion-graphene/SPCEs of HIV-1electrochemical gene sensing system, the oxidation peak current changes with the concentration of target nucleic acid sequence a linear relationship between the logarithm linear equation was Ⅰ (10-6A)=8.302log10(ctarget)-13.070, the correlation coefficient was0.982. Detection limit is15nmol/L.ConclusionsThis study demonstrated that the CAs-MB as a specific recognition element of HIV-1gag gene conserved region is expected to be used to build the new HIV-1electrochemical gene sensor. HIV-1electrochemical gene sensing system based on ultra-sensitive electrochemical micro-electrode:Nafion-graphene/SPCEs constructed as the working electrode combined with a new recognition element CAs-MB lay a solid foundation for electrochemical sensors in clinical of HIV-1nucleic acid detection, and provide a rapid nucleic acid diagnostics common platform for other types of infectious diseases. |
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