[13p-PB7-1~14] English session: Asian Joint Symposium on Nanobiotechnology

▲ [13p-PB7-6] Comparative study of biomolecule immobilization chemistry on the solid surface for on-chip protein screening

〇(D)Subhashini RajKumal¹, Hiromi Kuramochi¹, Shingo Ueno¹, Takanori Ichiki¹ (1.Dept. of Bioeng, Univ. of Tokyo)

Keywords:Protein array,Biomolecule immobilization

Protein arrays are a powerful means of detecting proteins and investigating their functions. They are versatile tools for high-throughput protein interaction and stability screening in a time and cost effective manner. Screening of proteins such as antibodies for thermostability is conducted to generate mutants that retain activity even at extreme conditions and for longer storage stability. When screening protein on-chip under conditions of non-physiological temperature and pH, it is important that the physical or chemical bonding connecting the protein to the substrate surface is stable under the intended experimental conditions. In this work, we will present and compare the suitability of two different types of substrates, gold-coated substrate and (3-aminopropyl)-triethxoysilane (APTES) functionalized glass substrate, for use in on-chip protein screening. The gold-coated substrate was prepared by sputter-coating glass slides with chrome followed by gold. The APTES-functionalized substrate was prepared using a water based procedure that we had previously optimized. For demonstration purpose, thiolated Cy5-labeled DNA molecules were used as test molecules instead of protein molecules. Thiolated DNA molecules were directly adsorbed on to gold surface while N-(6-maleimidocaproyloxy)succinimide (EMCS) linker was used to connect the thiolated DNA molecules to APTES-functionalized substrate. Non-thiolated Cy5-labeled DNA molecules were used as negative control samples to determine the extent of non-specific binding to substrate. After binding probe molecules, unreacted surface on both the substrates were blocked with 6-mercaptohexanol (MCH) molecules. The substrates were imaged using fluorescence imager before and after exposure to a wide range of temperature and pH ranging from 25°C to 80°C, and pH 3 to pH 11 respectively for 1 hour. In both the substrates, extreme loss of probe DNA (more than 90%) was observed at 70°C and above, with a more severe loss observed for gold-coated substrate. Also, the efficiency of fluorescence based quantification using both the substrates was compared by hybridizing a Cy3-labeled target DNA molecules to the thiolated Cy5-labeled DNA immobilized on-chip. A standard fluorescence intensity curve was generated and used as reference.