Author: Sedighi, Abootaleb
In this thesis, we report the use of gold nanoparticles (AuNPs) to enhance the detection of single nucleotide polymorphism (SNPs) in the NanoBioArray (NBA) chip. A combination of gold nanoparticles (AuNPs) and nucleic acids has recently been used in many biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Our kinetic analysis shows that in the presence of AuNP-ssDNA interactions, mechanisms of DNA hybridization and dehybridization are altered. Our proposed mechanisms include a shift of the rate-limiting step of hybridization from mismatch-insensitive to the mismatch-sensitive zipping step. Furthermore, the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the mismatched (MM) duplex DNAs, destabilize the duplexes and accelerates the dehybridization process. We employ these alterations in mechanisms, both of which disfavor the formation of MM duplexes, to enhance the detection of SNPs in the NBA chip. In this technique, we load the target DNAs on the surface of AuNPs (i.e. AuNP targets) and then introduce them to the surface-immobilized probes for DNA hybridization. Our results show that AuNP targets, in contrast to the targets free in the solution (free targets), were able to discriminate between the perfectly matched (PM) probes and the mismatched (MM) ones. Using AuNP targets, we developed a room-temperature method for detection of SNPs in the KRAS gene codon 12 in the NBA chip. Then, a novel wash method based on AuNPs was developed to preserve the DNA hybridization signals in CD-NBA chip while discriminating MM duplexes from PM duplexes. In this method, AuNPs are suspended in the wash buffers to preferentially destabilize the MM duplexes, in presence of the PM duplexes. Enjoying this targeted mechanism, AuNP wash method enhances specificity without compromising signal intensity. This method is simple and compatible with multiplexed DNA hybridization settings. The findings in this thesis can be used to enhance the reliability of DNA biosensors (e.g. DNA microarrays) and might lead to new applications in DNA biosensing.
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Thesis advisor: Li, Paul C.H.
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