A Fundamental Study On Fabrication Method Of Single-molecule DNA Chip

The goal of this paper is to fabricate single-molecule DNA chip.First,silicon wafers were spin-coated with photoresist polymethylmethacrylate(PMMA),followed by fabrication of high density array of patterned nanowells through electron beam lithography(EBL).After streptavidin(STV)modification of the bottoms of the nanowells,Cy3-labelled biotinylated DNA molecules were immobilized in the nanowells through immuno-reaction between STV and biotin.It is anticipated to fabricate novel type of single-molecule DNA chip with higher molecule density than those fabricated through random deposition of DNA sample on the wafer surface.This paper achieved a fast and effective method to clean silicon wafers,gained the preliminary parameters for the preparation of 1STV-1DNA conjugates,and fabricated nanowell arrays through EBL.This paper investigated the following:1.Chemical modification of the silicon wafer and immobilization of DNA molecule on the wafer surface: It was shown that the method to clean wafer surface with chemical detergent and piranha solution to remove organic and inorganic contamination on the surface was simple and rapid as background fluorescence could hardly be observed under a fluorescence microscope.Silicon wafers cleaned with piranha solution were used to immobilize STV after modification with 3-aminopropyltriethoxysilane(APTES)and 1,4-Phenylene diisothiocyanate(PDITC).Biotinylated DNA molecules with Cy3 labelled on the opposite end were further immobilized on the wafer surface through STV and biotin interaction,followed by observation under total internal reflection fluorescence microscope(TIRFM).Results showed that this method could effectively immobilize DNA on the surface,providing an attempt for fabrication of single-molecule DNA nanoarray on silicon wafer.2.Preparation of 1STV-1DNA conjugates: A mixture of one STV with different number of biotinylated 500 bp double-stranded DNA molecules(from 1 to 4)was obtained through STV and biotin conjugation.The mixture product was investigated using atomic force microscope(AFM).Different molar ratios between STV and biotinylated DNA and incubation times were investigated through agar gel electrophoresis.Parameters for large quantity preparation of 1STV-1DNA conjugates were preliminarily achieved.3.Fabrication of nanowell array: High density of patterned nanowell arrays were successfully fabricated by using EBL processing on PMMA-coated silicon wafer as proved through characterization by scanning electron microscope(SEM).Fabrication of DNA chip was attempted through immobilization of biotinylated DNA molecules with Cy3 labelled on the opposite end after selective modification of the well bottoms although TIRFM observation showed there are many parameters still need to be optimized.