Oligonucleotide immobilization using 10-(carbomethoxy)decyl-dimethylchlorosilane for mRNA isolation and cDNA synthesis on a microfluidic chip

Fabrication of microfluidic systems capable of extracting and isolating nucleic acids from biological samples and preparing them for downstream applications within the same device is of interest as on-chip sample preparation reduces the time and effort expended on multi-step benchtop procedures. A microfluidic chip capable of cell lysis, nucleic acid extraction and immobilization has been developed in our laboratory. This report focuses on substrate development and chip integration of the nucleic acid immobilization platform. The immobilization region was initially developed using poly-adenylated mRNA released from normal human lymphoblastoid cells. After immobilization, the mRNA remains functional for amplification by the reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) gene expression analyses. Detailed surface functionalization, chemical modification, and covalent oligonucleotide immobilization methods are described. Vapor and liquid deposition techniques for Pyrex functionalization by 10-(carbomethoxy)decyl-dimethylchlorosilane (CMDCS) were developed and an optimal method was determined for microfluidic chip application. CMDCS substrate depositions and modification were characterized by atomic force microscopy and contact angle goniometry, respectively. Oligonucleotide immobilizations and mRNA bindings were determined by fluorescence microscopy. Functional, selective platform integration within the microfluidic chip was confirmed by standard RT-PCR and qPCR systems.