Study Of Free-lable Biology Sensors Based On Aptamer

Protein is a basic substance in living matter. The determination of protein hasa very important significance in the biological and medicine field. The aptamer, as anucleic acid molecule, can bind some specific molecules. Compared with theantibody, the aptamer with higher stability is easy to be modified on theelectrode.The aptamer is an ideal protein recognition substance. It has been widelyused in the identification and diagnosis in the living body. The sensitivity of aptamersensors is influenced by signal transfer matter. Compared with RNA, DNA is morestable and not susceptible to degradation of ribonuclease. DNA aptamer is morewidely used in biosensors.Lysozyme with a molecular weight of c.a14.4kDa contains129amino acidresidues, and its isoelectric point is11.0. By catalysing peptidoglycan N-acetylmuramic acid and N-acetyl glucosamine residue and hydrolysis of1,4-β chainbetween the shell dextrin N-acetyl glucosamine residues, lysozyme molecular candestruct bacteria cell wall. In humans, lysozyme widely exists in the cells secretionliquid, such as saliva, tears, and other body fluids. Lysozyme is also widely used in thepharmaceutical industry and the food industry because of its antibacterial properties.Due to its relatively small molecule weight, lysozyme is often used as the standardtarget detection material in the aptamer sensors fields.Adenosine is one of the nucleosides consisting parts of adenine and ribose (ribo-furanose). Triphosphate adenosine (ATP) is composed of adenosine and threephosphate groups, and its chemical formula is C10H16N5O13P3. The IUPAC name ofATP is [(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl(hydroxyphosphonooxyphosphoryl) hydrogen phosphate, and its molecularweight is507.18. ATP plays an important role in the field of biochemistry, e.g. it cantransfer energy.in adenosine triphosphate (ATP) or adenosine diphosphate (ADP).Sumio Iijima, a Japanese scientist, discovered carbon nanotubes in1991.According to the definition by M.Endo et al, if the the graphite hexagonal mesh plane(graphene sheets) rolled into seamless tubular to formation of defect-free "monolayer" tubular material or the "layers" of tubular material, those two kinds ofthe tubular material is called carbon nanotubes.In this paper, we prepared the electrochemical sensors for the determination ofprotein with the combination of aptamer and layer-by-layer self-assembly technique.The research ideas were outlined as follows:(1) aptamer with efficient identificationis used as a protein recognition element and prepared a high sensitive, stable andprotein sensor with good reproducibility, and used for the quantitative detection oflysozyme and adenosine triphosphate (2) a non-labeled protein sensor wis developedusing methylene blue, daunomycin and trivalent ruthenium ionthe as electrical signalsubstances.Detaila are presented in this thesis as follows:1Label-free electrochemical sensor based on aptamer for detection of lysozymeA simple signal-off electrochemical aptamer-based sensor for the detection oflysozyme was prepared. The gold nanoparticles were electrodeposited on the surfaceof a glassy carbon electrode. The aptamer was self-assembled through S-Au bondingon the surface of gold nanoparticles prior to blocking the open area with6-mercaptoethanol. The gold nanoparticles modified electrode was characterized byscanning electron microscope, and the electrochemical characteristics of theaptamer-modified electrode were investigated by electrochemical impedancespectroscopy (EIS) and cyclic voltammetry (CV). The peak current of differential pulsevoltammetry (DPV) decreased with increasing concentration of lysozyme. The sensorprovided a dynamic range from0.1nM to100nM of lysozyme with a detection limitof0.01nM (S/N=3) and a correlation coefficient of0.9876.2A Lysozyme electrochemical sensor based on aptamer competitionA simple signal-off electrochemical lysozyme aptamer based on the mechanismof competition was described. After the multiwalled carbon nanotubules (MWCNT)were modified on the surface of the glassy carbon electrode, aptamer dsDNA wasassembled through NH2-COOH on MWCNT surface. In the presence of the indicatordaunomycin, an electrical signal can be observed due to the insertion of daunomycininto dsDNA. The electrichemical signal decreased when lysozyme was added because of the competition between daunomycin and lysozyme resulted in the drop ofdaunomycin from dsDNA. Under optimum conditions, the biosensor exhibited alinear range from1nM to500nM of lysozyme with a detection limit of0.5nM (S/N=3) and a correlation coefficient of0.9995.3A adenosine triphosphate electrochemical Sensor Based on AptamerA simple signal-on electrochemical adenosine triphosphate aptamer sensor waspresented. The aptamer was self-assembled through S-Au bonding on gold diskelectrode（GDE）surface prior to blocking the surface with6-mercaptoethanol. Theaptamer DNA chain was linked to gold disk electrode surface by DNA hybridization.The electrochemical characteristics of the aptamer-modified electrode wasinvestigated by electrochemical impedance spectroscopy (EIS) and cyclicvoltammetry (CV). The differential pulse voltammetry (DPV) peak current increasedwith increasing concentration of adenosine triphosphate. The current is related tothe concentration of ATP in the range from20nM to500nM with a lower detectionlimit of5nM (S/N=3) and a correlation coefficient of0.9737.