Microbead-based affinity chromatography chip using RNA aptamer modified with photocleavable linker.
A microbead-based affinity chromatography chip (micro-BACC) controlling hundreds of nanoliters of reaction volume was developed to separate and analyze hepatitis C virus (HCV) RNA polymerase protein by immobilization of an RNA aptamer on beads. A photocleavable linker was conjugated in between the beads and the aptamer to elute the bound RNA polymerase from the RNA aptamer in one step by UV irradiation, resulting in an efficient method to elute and identify the target molecule bound on RNA using a mass spectrometer. This linker showed a cleavage activity over 70% upon UV irradiation at 1050 mW/cm2 for more than 5 min. The photoelution method could prevent the target molecule from contaminations in affinity chromatography caused by elution solutions of high salt concentration, extreme pH and detergent, respectively. In this chip, sample reagents up to 800 nL could be metered quantitatively into the bead chamber using a nanoliter dispenser working, based on surface-guided flow control and pneumatic control by external air pressure on the chip. RNA polymerase eluted after UV irradiation was successfully analyzed by trypsin treatment without additional purification. As a result, using the aptamer, we could detect RNA polymerase from 800 nL hepatitis C patient serum containing 96 fmol HCV RNA polymerase. The detection limit of this system was estimated to be 9.6 fmol HCV RNA polymerase.
Cho
Suhyung
S
Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Korea.
Lee
Sang-Ho
SH
Chung
Woo-Jae
WJ
Kim
Yong-Kweon
YK
Lee
Yoon-Sik
YS
Kim
Byung-Gee
BG
D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D003432:Cross-Linking Reagents / Q000528:radiation effects; D012321:DNA-Directed RNA Polymerases / Q000097:blood*; D016174:Hepacivirus / Q000235:genetics; D006526:Hepatitis C / Q000175:diagnosis*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D008863:Microspheres; D010782:Photolysis; D012313:RNA / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014764:Viral Proteins / Q000097:blood
D003432:Cross-Linking Reagents; D014764:Viral Proteins; D012313:RNA; D012321:DNA-Directed RNA Polymerases