Characterization of DNA immobilization and subsequent hybridization using in situ quartz crystal microbalance, fluorescence spectroscopy, and surface plasmon resonance.
We have characterized the immobilization of thiol-modified oligomers on Au surfaces and subsequent hybridization with a perfectly matched or single-base mismatched target using a quartz crystal microbalance (QCM) and fluorescence spectroscopy. The surface density of immobilized probe molecules and the hybridization efficiency depending on the type of buffer and salt concentration were investigated. We observed some ambiguities in surface coverage deduced from QCM measurement and adopted a complementary fluorescence displacement method. Direct comparison of surface coverage deduced from frequency change in QCM measurement and determined by the fluorescence exchange reaction revealed that QCM results are highly overestimated and the amount of overestimation strongly depends on the type of buffer and the structure of the film. Discrimination capability of the surface attached 15-mer probe was also examined using a single-base mismatched target at various hybridization temperatures. Hybridization efficiency depending on the type of single base mismatch was investigated using surface plasmon resonance (SPR).
Digital Bio Laboratory, Samsung Advanced Institute of Technology, Suwon 440-600, South Korea. dnachip@samsung.com
Yoon-Kyoung
YK
Cho
Sunhee
S
Kim
Young A
YA
Kim
Hee Kyun
HK
Lim
Kyusang
K
Lee
DaeSung
D
Yoon
Geunbae
G
Lim
Y Eugene
YE
Pak
Tai Hwan
TH
Ha
Kwan
K
Kim
D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004566:Electrodes; D019793:Fluorescein / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D000441:Hexanols / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry; D011791:Quartz / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013499:Surface Properties
C503488:6-mercapto-1-hexanol; D000441:Hexanols; D009841:Oligonucleotides; D013438:Sulfhydryl Compounds; D011791:Quartz; D006046:Gold; D004247:DNA; D019793:Fluorescein