Myoglobin-specific Aptamers Selection Based On Microfluidic Chips

Myoglobin is an oxygen-binding protein, in general, which could be found in thehuman muscle tissue. It has been often used as a specific marker of muscle injury,since muscle will release the myoglobin into the bloodstream only when muscle getsto be injured. Many diseases relate to the muscle injury in clinical, so the level ofmyoglobin concentration in blood is a very important biochemical index for manydiseases, such as AMI, Angina pectoris, Polymyositis. However, the traditionalmethods of myoglobin detection are encountered challenges in terms of specificityand sensitivity. Hence, it’s of grate significance to find the myoglobin-specificaptamers with high affinity and to improve the specificity and sensitivity ofmyoglobin detection. In addition, aptamers appear as a new ligand with thecharacteristics of a wide range targets, good stability, simple to produce, easymodification, which can even comparable with antibody in many aspects, bringingopportunities for overcoming those challenges above. On this occasion, selection ofaptamers become the focus of research in recent years. Meanwhile, microfluidic chipas a new type of platform in chemical, bio-analytical technology develops rapidly.Taking the advantages of fast, efficient, high-throughput, low cost, low samplevolume, miniaturization, integration, especially with the unique advantage in the fieldof separation, more and more selection of aptamer methods was developed based onthe microfluidic technology.In this thesis, the microfluidic chips with different structure were used for rapidselecting myoglobin-specific aptamers. In further, the sequences of selected aptamerswere optimized after analyzing their primary and secondary structure.(1) Myoglobin-specific aptamers selection based on microfluidic chip withimmobilized microbeadsFirstly, the microbeads which myoglobin modified previously were fixed into apinch of the microfluific channel. Secondly, the aptamers then separated from therandom pool by means of high separation efficiency of microfluidics. Thirdly, total6rounds selections were taken and the enrichment of each round collected productshave been monitored. After that the forth round enriched products were chosen forcloning and sequencing. Finally,3aptamers with high specificity and affinity tomyoglobin were obtained. (2) Myoglobin-specific aptamers selection based on positive and negativeselection units integrated microfluidic chipOn the basis of the first part of the work, in order to further reduce the overallselection time and obtain aptamers that are more suitable for the application, were-designed the structure of the chip and the sequence of original random pool. A chipwith two pinches was designed and fabricated, and then the myoglobin-coatedmicrobeads and negative proteins-coated microbeads were fixed on the one of thepinches, respectively. Positive selection and negative selection occuredsimultaneously after injecting the ssDNA pool. We designed two extracomplementary sequences for blocking two PCR primer termination of random poolsequences, this prevented the involvement of the primer-binding sequences in thetertiary folding of eventual aptamers. Total8rounds selections were taken and thesixth, seventh and eighth round enriched products were all cloned and sequenced.4aptamers in40nt with high specificity and affinity to myoglobin were obtain. Infurther, after analyzing the secondary structure，one sequence was further optimized.