Aptamers are OLIGONUCLEOTIDE ligands of synthetic single-stranded DNA/RNAs (ssDNA/RNA) with specific conformations which were created by in-vitro selection from a large random sequence pool, based on the affinity between OLIGONUCLEOTIDEs and the specific target molecules. This screening method is called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Aptamers' unique characteristics in protein binding have made them highly valuable tools for different diagnostic applications, downstream purification processes, analysis, new drug development and drug delivery. One of the important and valuable therapeutic plasma proteins is human coagulation factor VIII (FVIII). In this investigation, FVIII was prepared from plasma source or recombinant production media and considered as a model protein for designing and development of relevant OLIGONUCLEOTIDE aptamers. As the first step, random OLIGONUCLEOTIDE library and its specific primers were designed and then chemically synthesized. After induction of 3D conformation by a specific temperature treatment, the random library was incubated with plasma derived FVIII. Gel filtration method was then employed as a partitioning step. During SELEX process, for the elimination of nonspecific cross interactions by other plasma proteins, two steps of negative selections were run. Finally, SELEX process was stopped when no more increase in the affinity constant (Kd) was observed and therefore the enriched pool was cloned. As the final step, protein affinity of enriched pools was determined using fluorescence method. It was found that the calculated Kd for enriched pools were 0.5 to 60nM and that they are comparable to the other specific ligands of the target protein.

Background: The microarray technology is in needed of cost-effective, low background noise and stable substrates for successful hybridization and analysis.Methods: In this research, we developed a three-dimentional stable and mechanically reliable microarray substrates by coating of two polymeric layers on standard microscope glass slides. For fabrication of these substrates, a thin film of oxidized agarose was prepared on the Poly-L-Lysine (PLL) coated glass slides. Unmodified OLIGONUCLEOTIDE probes were spotted and immobilized on these double layered thin films by adsorption on the porous structure of the agarose film. Some of the aldehyde groups of the activated agarose linked covalently to PLL amine groups, on the other side, they bound to amino groups of adsorbed tail of biomolecules. These linkages were fixed by UV irradiation at 254nm using a CL-1000 UV. These prepared substrates were compared to only agarose-coated and PLL-coated slides.Results: Atomic Force Microscope (AFM) results demonstrated that agarose provided three-dimensional surface which had higher loading and bindig capacity for biomolecules than PLL-coated surface which had two-dimensional surface. The nano-indentation tests demonstrated the prepared double coating was more reliable and flexible for mechanical robotic spotting. In addition, the repeated indentation on different substrates showed uniformity of coatings.The stability of novel coating was sufficient for hybridization process. The signal-to-noise ratio in hybridization reactions performed on the agarose-PLL coated substrates increased two fold and four fold compared to agarose and PLL coated substrates, respectively.Conclusion: Finally, the agarose-PLL microarrays had the highest signal (2920) and lowest background signal (205) in hybridization, suggesting that the prepared slides are suitable in analyzing wide concentration range of analytes.