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).
Cho
Yoon-Kyoung
YK
Digital Bio Laboratory, Samsung Advanced Institute of Technology, Suwon 440-600, South Korea. dnachip@samsung.com
Kim
Sunhee
S
Kim
Young A
YA
Lim
Hee Kyun
HK
Lee
Kyusang
K
Yoon
DaeSung
D
Lim
Geunbae
G
Pak
Y Eugene
YE
Ha
Tai Hwan
TH
Kim
Kwan
K
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