Selection Of C-reactive Protein Special Aptamers Using Microfluidic Device

Aptamers are nucleic acid molecules, RNA or DNA, that bind to specific targetmolecules with high affinity and selectivity. They have been recognized as a usefultool in disease diagnostics, drug research, clinical treatment, proteomics as well asthe fields of gene expression and regulation mechanism research. For the moment, thebottleneck restraining the application of aptamers is the efficient screening technique.Usually, multiple rounds of selection (typically8～20rounds), or even30rounds arenecessary in order to isolate aptamers with sufficient affinity and specificity,requiring significant time and labor. So, to develop a new fast, efficient screeningmethod of the aptamer is in urgent need. In this work, a platform that integratesmicrobead-assisted SELEX with microfluidics technology has been developed andwas used for the rapid selection of C-reactive-protein special aptamers. The mainpoints of this thesis are summarized as follows:In order to shorten the time of the screening process, a simple microfluidic chipwithout external separation device was designed for SELEX based on the highseparation efficiency of microfluidic chip. The chip consists of four straight channels,and a contraction was designed at the middle section of each channel, to withholdcertain sized beads. For the chip fabrication requires good, stable etching conditionsand the etching rate in this condition, the composition of the etchant, etchingtemperature, etching speed were measured and optimized. Under optimum etchingconditions, different depth of channel was fabricated by controlling the etching time.Then, by etching twice, the channel was able to cut and keep certain sized bead.C-reactive protein was taken as a model of a protein for aptamer screening in themicrofluidic SELEX platform. First, C-reactive protein coating polystyrene beadswas fixed at the contraction, then the library was passed through the channel, and thesequences with high binding capacity for C-reactive protein would be retained on thebeads from the combination of the library separated from the screen before eachround is the microplate method of anti-screen operation.10rounds of screening werecarried out with BSA for the negative screening target molecules, TA cloning,sequencing and affinity, specificity analysis of the sixth round, the eighth round, thetenth round of the enriched library, a better affinity aptamer of C-reactive proteinwith dissociation constant of3.6nM was obtained. For the detection of C-reactive protein was often performed in blood with highlevels of HSA and IgG, addition of four rounds of selection from the fifth round wereperformed with HSA, IgG and BSA for the negative screening target molecules. Theeighth round enriched library were analysed, a C-reactive protein specific bindingaptamer with dissociation constant of104nM was obtained. The result showed thatthe platform can be used for fast and efficient screening of protein aptamer.