Doctor of Philosophy (PHD)
Mechanical Engineering (Engineering)
Date of Defense
First Committee Member
Second Committee Member
Third Committee Member
Fourth Committee Member
Chun-Yuh Charles Huang
Optical biosensors that utilize unmodified Gold nanoparticles (GNPs) and nucleic acid probes are one of the most popular biosensors, thanks to the unique colorimetric and fluorescent properties of GNPs. These biosensors are based on the interactions between GNPs, DNA probes, and target molecules. As a result, their performance is dependent on the relative binding strength between DNA probes, GNPs, and target molecules. However, there is no systematic study on the thermodynamics and kinetics of interactions between DNA and GNPs. Moreover, there is no accessible tool for biomedical researchers to quantitatively study the interactions between DNA probes and target molecules, which could be DNA or other molecules. The current work consists of experimental study of the interactions between DNA and GNPs, as well as computational study of the interactions between DNA and target molecules. Systematic investigations on both thermodynamics and kinetics of interactions between DNA molecules and GNPs have been conducted. In the thermodynamics study, we developed titration experiments based on critical coagulation concentration (c.c.c.). To study the sequence dependency of DNA molecules, we used nucleobases, ribonucleosides, deoxynucleosides, deoxynucleoside monophosphate, deoxynucleoside triphosphates, and homo-oligonucleotides with 15 nucleotides (nt). We found that DNA molecules with bases thymine (T) have the weakest binding strength to GNPs, which is due to the lack of amine groups in T as indicated by previous studies. To study the length dependency of DNA molecules, we used homo-oligonucleotides of different lengths, ranging from 5 nt to 100 nt. It was found that shorter DNAs generally bind to GNPs stronger compared to longer DNAs. To study the difference of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), we used dsDNA in different conformations. It was discovered that single-stranded DNA (ssDNA) binds to GNPs much stronger than double-stranded DNA (dsDNA). In addition, dsDNA with overhangs or mismatches bind to GNPs differently than completely complementary dsDNA. In kinetics study, we developed fluorescent experiments based on fluorescent quenching effect of gold surfaces on fluorophores. To study the kinetics and effect of salt on the interactions between DNA and GNPs, we used 15mer homo-oligonucleotides and two sets of completely complementary dsDNA. It was observed that the longer the incubation time and/or the higher the NaCl concentration, the more DNAs bind to GNPs. We also found that the binding kinetics and the effect of salt is sequence dependent. To provide a user-friendly tool to quantitatively study interactions between DNA and target molecules, a thermodynamics based computational model was implemented with two Microsoft® Excel spreadsheets that utilized macros and visual basic applications (VBA). One spreadsheet is for up to three DNA molecules and the other spreadsheet is for up to two DNA molecules and one non-nucleic acid molecule. We have extensively tested and verified the two spreadsheets under various situations. The results of this work could be used to optimize the design of biosensors based on GNPs and nucleic acid probes, thus improving on selectivity and sensitivity of these biosensors.
Biosensor; DNA; GNPs; Thermodynamics; Kinetics; c.c.c.
Zou, Jiaqi, "Study of Interactions between DNA and Gold Nanoparticles for Biosensor Applications" (2013). Open Access Dissertations. 1069.