Surface Enzyme Chemistries for the On-Chip Synthesis of DNA, RNA, and Protein Microarrays for in vitro Surface Plasmon Resonance Biosensing

This dissertation described the development of a unique set of methodologies for the multiplexed in vitro detection of biomarkers at very low concentration (1000 molecule level). This is accomplished through the combined use of surface enzyme chemistries, microfluidics and microarrays techniques, and novel surface biosensing methods. Of particular importance in these strategies will be the use of surface enzyme chemistries that including RNase H hydrolysis, T4 RNA ligation, PCR, surface in vitro transcription and translation. In addition to these surface chemistries, I implement microfluidic and microarray technologies, and detection techniques such as SPRI that will impart even higher sensitivity for in vitro biomarker detection. Chapter 2 described a four chamber microfluidic biochip that is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The observed detection limit is 10 fM or 30 zeptomoles (18,000 molecules) within only 200 seconds in the microliter volume format. In chapter 3, protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. In chapter 4, I demonstrate how plasmid microarrays can be directly PCR amplified on-chip and then used in conjunction with on-chip IVTT to create a protein microarray within one single microchip format. This microarray can then be used in three applications: i) to directly detect protein by fluorescence microscope; ii) to fabricate nanoparticles or beads with various protein from corresponding protein microarray elements; iii) to directly print in a one to one conversion process and results multiple copies of the protein microarray on gold microarray elements with a His-tag capture chemistry. This method can be used with solutions containing as little as 1000 plasmids. This PCR-IVTT conjugation method simplifies the protein microarray fabrication, and permit the rapid fabrication from stable DNA plasmid microarrays.