Nucleic Acid Aptamer Functionalized Nano Channel For The Separation Of Biomolecules Analysis Research

Nanochannel is a kind of the pore or tube with nanometer structure, havingdiameter of0.1to100nm. In the current research，nanochannel mainly involves thenatural protein ion channel and synthetic solid nanochannel. As an important branchof the nanobiotechnology, nanochannel technology has obtained widely attentions inrecent years. Because of the structure effect and physical chemical properties,nanochannel shows the unique advantages in gene sequencing, single molecularanalysis, the biomimetic ion channels design and drug load. This thesis studied thepreparation and modification of nanochannel, the transportation mechanism andcharacteristics of biological molecules through nanochannels, the separation andanalysis of biological molecules.The nucleic acid aptamers, oligonucleotides (DNA or RNA ligands) selected bysystematic evolution of ligands by exponential enrichment (SELEX) in vitro, can bindprotein, small molecules or metal ions with high affinities and specificities.Researches on aptamers have achieved much progress in biosensors, biochips, diseasebiomarker discovery and target therapy. Generally, biological substances are oftenseparated and detected by gas chromatography-mass spectrometry, liquidchromatography-mass spectrometry, high performance liquid chromatography,capillary electrophoresis and capillary electro chromatography. Compared with thesetraditional separation and analysis methods, nanochannel modified with aptamers ismore effective and has better performance. However, there are few reports onbiological molecules separation and analysis by smart nanochannel technology.Details are presented in this research work as follows:(1) An electroless deposition method was used to deposit gold within pore wallsof the porous polycarbonate membrane template and gold nanochannel array withdifferent diameters was manufactured successfully. The mechanism of the preparationand modification of nanochannel was discussed. The factors affecting the depositionprocess were studied in detail. SEM, TEM and Zeta potential were used tocharacterize the pore diameter and feature. The size of the pore can be easily controlled with high reproducibility by changing the deposition time.(2) The gold nanochannels were modified with the aptamer of β-Estradiol bymolecular self-assembly. Then, they can be used as very well molecular nanofilters,which can discriminate and gate the transportation of different moleculars, andfacilitate the transportation of β-Estradiol through the membranes while the Estrone isnot easy to transport. To a certain extent, β-Estradiol and Estrone can be separatedbased on this unique selective nanochannel membrane.(3) The gold nanochannels were modified with the aptamer of Lysozyme bymolecular self-assembly, and they can select Lysozyme moleculars with high affinityand specificity. At the same time, The negatively charged nanochannel membranefacilitate the transportation of Lysozyme moleculars and impede the permeation ofBSA moleculars.The difference of transportation performance between Lysozyme andBSA was studied and analysed by mathematical modeling, which provide certaintheoretical basis on the separation and analysis of protein.(4) The gold nanochannels were modified with the aptamer of Lysozyme byself-assembly, and it can selects Lysozyme moleculars with high affinity andspecificity. Due to the adsorption and desorption of Lysozyme, the changes ofimpedance spectra and current blockade were measured, and the mechanism wasstudied by mathematical modeling. Based on the function of current decrease andconcentration of lysozyme, the lysozyme can be detected label-freely.In summary, we developed some new approaches and achieved some results inbiological molecules separation and detection technology by smart nanochannel incombination with traditional analysis methods. Some mechanisms and theories in thetransportation process were proposed and discussed. We greatly expect to providesome theoretical support for more widely practical application of nanaochannel inbiological molecules separation and analysis technology.