SURFACE IMMOBILIZED DNA FOR USE IN BIOSENSING

Open Access
Author:
Brunker, Sarah Elizabeth
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
September 14, 2010
Committee Members:
  • Christine Dolan Keating, Dissertation Advisor
  • Christine Dolan Keating, Committee Chair
  • Philip C. Bevilacqua, Committee Member
  • Raymond Edward Schaak, Committee Member
  • James Hansell Adair, Committee Member
Keywords:
  • surfaces
  • encoded nanoparticles
  • polymerase chain reaction
  • ligase chain reaction
  • nucleic acids
  • biosensing
  • amplification
  • soft lithography
  • microcontact printing
  • general chemistry lab
Abstract:
The overall goal of this work was to understand the factors governing nucleic acid attachment, hybridization, and enzymatic extension in an immobilized format. Surface immobilized DNA is used in biosensing applications to capture an analyte of interest, which is then detected (in this case using fluorescence), as the detection of DNA sequences can be used to diagnose disease. Several encoding strategies are used to correlate the signal generated with the identity of the analyte, including spatial encoding incorporated into planar substrates, where a particular area on the surface corresponds to a particular analyte probe; and anisotropic encoding incorporated into particular substrates, where the particle itself codes for the analyte through its composition, etc. Both planar and particulate surfaces were analyzed in this work; the synthesis, functionalization, nucleic acid attachment and subsequent use as a probe in an assay, and signal transduction of bound analyte were all investigated. Barcoded nanowires serve as the encoded particle in this system, where the immobilized DNA sequence corresponds to the metallic pattern of the wire itself. Chapters 2 and 3 detail the use of polymerases and ligases, respectively, to perform enzymatic amplification reactions (polymerase chain reaction and ligase chain reaction) when the nucleic acid primer or probe is bound to a barcoded nanowire. Investigations into the reaction mechanism, such as attachment chemistry thermostability measurements and control experiments to determine fluorescence signal origin, were performed and a hypothesis to explain the behavior observed is proposed; these studies are applicable to surface phase amplification between differing systems. Chapter 4 uses microcontact printing, where a polydimethyl siloxane stamp is used to pattern planar substrates with DNA probe sequences. Surface functionalization was studied in order to optimize DNA pattern transfer. These studies pave the way for microcontact insertion printing, which can be used to pattern isolated DNA molecules for use in single molecule studies. Taken together, these investigations answer fundamental questions about the reactions that occur when DNA is immobilized onto a surface for use in a biological assay.