Fabrication of a PDMS microfluidic device for size selective DNA transport and single-stranded DNA generation for SELEX

Abstract

An innovative nanopore based microfluidic device for SELEX has been developed for single-stranded DNA generation and sizing. The objective of my research is to design, fabricate, test and model this device and make this device applicable for SELEX. The nanopores of the membrane controls fluidic transport between different planes in the device. It adds more functionality and flexibility to the microfluidic device. This device consists of two polydimethylsiloxane (PDMS) channels separated by a polycarbonate membrane. Channels were designed in a CAD software and a master was fabricated using rapid prototyping of PDMS. Multiple PDMS replicas were then cast from this master. The membrane was sandwiched between two channels. Oxygen plasma treatment was applied to bond two PDMS layers and glass substrates were used to support the whole device. Recovery of fluorescein across the membrane was compared with 10 and 80 nucleotide (nt) single stranded DNA(ssDNA) to characterize the device. Recovery of analytes improved with decreasing flow rate. Size selectivity was observed. Two mathematical models which were built based on conservation of mass and constitutive relationships described the process of DNA transportation in the microfluidic device. Trends in recovery measured at various flow rates were consistent with the trends predicted in the two models which support the premise that diffusion dominated the molecular transport in this device. In addition to that, Model 2 demonstrated recovery was affected by the ionic strength of the buffer as well. One application of this device was to automate the process to make double stranded DNA(dsDNA) single stranded which can be integrated into an automatic SELEX system. Streptavidin-coated polystyrene beads were immobilized with dual-biotin labeled dsDNA and alkaline treatment was adopted to denature dsDNA and release the non-biotinylated ssDNA. 25mM sodium hydroxide (NaOH) was optimized to achieve the best purity. 95.7% of the strands collected across the membrane were the non-biotinylated ssDNA. Capillary Electrophoresis (CE) results confirmed that the non-biotinylated ssDNA was the major component across the membrane.